U.S. patent application number 09/751561 was filed with the patent office on 2001-07-12 for method and apparatus for identifying, classifying, or quantifying dna sequences in a sample without sequencing.
This patent application is currently assigned to CuraGen Corporation. Invention is credited to Deem, Michael W., Rothberg, Jonathan Marc, Simpson, John W..
Application Number | 20010007985 09/751561 |
Document ID | / |
Family ID | 27406222 |
Filed Date | 2001-07-12 |
United States Patent
Application |
20010007985 |
Kind Code |
A1 |
Rothberg, Jonathan Marc ; et
al. |
July 12, 2001 |
Method and apparatus for identifying, classifying, or quantifying
DNA sequences in a sample without sequencing
Abstract
This invention provides methods by which biologically derived
DNA sequences in a mixed sample or in an arrayed single sequence
clone can be determined and classified without sequencing. The
methods make use of information on the presence of carefully chosen
target subsequences, typically of length from 4 to 8 base pairs,
and preferably the length between target subsequences in a sample
DNA sequence together with DNA sequence databases containing lists
of sequences likely to be present in the sample to determine a
sample sequence. The preferred method uses restriction
endonucleases to recognize target subsequences and cut the sample
sequence. Then carefully chosen recognition moieties are ligated to
the cut fragments, the fragments amplified, and the experimental
observation made. Polymerase chain reaction (PCR) is the preferred
method of amplification. Another embodiment of the invention uses
information on the presence or absence of carefully chosen target
subsequences in a single sequence clone together with DNA sequence
databases to determine the clone sequence. Computer implemented
methods are provided to analyze the experimental results and to
determine the sample sequences in question and to carefully choose
target subsequences in order that experiments yield a maximum
amount of information.
Inventors: |
Rothberg, Jonathan Marc;
(Branford, CT) ; Deem, Michael W.; (Cambridge,
MA) ; Simpson, John W.; (Madison, CT) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
CuraGen Corporation
|
Family ID: |
27406222 |
Appl. No.: |
09/751561 |
Filed: |
December 29, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09751561 |
Dec 29, 2000 |
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09724385 |
Nov 28, 2000 |
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09724385 |
Nov 28, 2000 |
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09322617 |
May 28, 1999 |
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6231812 |
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09322617 |
May 28, 1999 |
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08942406 |
Oct 1, 1997 |
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6141657 |
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08942406 |
Oct 1, 1997 |
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08547214 |
Oct 24, 1995 |
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5871697 |
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Current U.S.
Class: |
1/1 ;
707/999.001 |
Current CPC
Class: |
C12Q 1/68 20130101; C12Q
1/6809 20130101; G16B 40/00 20190201; C12N 15/10 20130101; C12Q
1/6855 20130101; G16B 40/10 20190201; Y10S 707/99936 20130101; G16B
30/00 20190201 |
Class at
Publication: |
707/1 |
International
Class: |
G06F 007/00 |
Goverment Interests
[0001] This invention was made with United States Government
support under award number 70NANB5H1036 awarded by the National
Institute of Standards and Technology. The United States Government
has certain rights in the invention.
Claims
What is claimed is:
1. A method for identifying, classifying, or quantifying one or
more nucleic acids in a sample comprising a plurality of nucleic
acids having different nucleotide sequences, said method
comprising: (a) probing said sample with one or more recognition
means, each recognition means recognizing a different target
nucleotide subsequence or a different set of target nucleotide
subsequences; (b) generating one or more signals from said sample
probed by said recognition means, each generated signal arising
from a nucleic acid in said sample and comprising a representation
of (i) the length between occurrences of target subsequences in
said nucleic acid, and (ii) the identities of said target
subsequences in said nucleic acid or the identities of said sets of
target subsequences among which are included the target
subsequences in said nucleic acid; and (c) searching a nucleotide
sequence database to determine sequences that match or the absence
of any sequences that match said one or more generated signals,
said database comprising a plurality of known nucleotide sequences
of nucleic acids that may be present in the sample, a sequence from
said database matching a generated signal when the sequence from
said database has both (i) the same length between occurrences of
target subsequences as is represented by the generated signal, and
(ii) the same target subsequences as are represented by the
generated signal, or target subsequences that are members of the
same sets of target subsequences represented by the generated
signal, whereby said one or more nucleic acids in said sample are
identified, classified, or quantified.
2. The method of claim 1 wherein each recognition means recognizes
one target subsequence, and wherein a sequence from said database
matches a generated signal when the sequence from said database has
both the same length between occurrences of target subsequences as
is represented by the generated signal and the same target
subsequences as represented by the generated signal.
3. The method of claim 1 wherein each recognition means recognizes
a set of target subsequences, and wherein a sequence from said
database matches a generated signal when the sequence from said
database has both the same length between occurrences of target
subsequences as is represented by the generated signal, and the
target subsequences are members of the sets of target subsequences
represented by the generated signal.
4. The method of claim 1 further comprising dividing said sample of
nucleic acids into a plurality of portions and performing the steps
of claim 1 individually on a plurality of said portions, wherein a
different one or more recognition means are used with each
portion.
5. The method of claim 1 wherein the quantitative abundance of
nucleic acids containing said nucleotide sequences in the sample is
determined from the quantitative level of the one or more signals
determined to match said sequences.
6. The method of claim 1 wherein said plurality of nucleic acids
are DNA.
7. The method of claim 6 wherein the DNA is cDNA.
8. The method of claim 7 wherein the cDNA is prepared from a plant,
a single celled animal, a multicellular animal, a bacterium, a
virus, a fungus, or a yeast.
9. The method of claim 8 wherein said database comprises
substantially all the known expressed sequences of said plant,
single celled animal, multicellular animal, bacterium, virus,
fungus, or yeast.
10. The method of claim 7 wherein the cDNA is of total cellular RNA
or total cellular poly(A) RNA.
11. The method of claim 6 wherein the recognition means are one or
more restriction endonucleases whose recognition sites are said
target subsequences, and wherein the step of probing comprises
digesting said sample with said one or more restriction
endonucleases into fragments and ligating double stranded adapter
DNA molecules to said fragments to produce ligated fragments, each
said adapter DNA molecule comprising (i) a shorter stand having no
5' terminal phosphates and consisting of a first and second
portion, said first portion at the 5' end of the shorter strand and
being complementary to the overhang produced by one of said
restriction endonucleases, and (ii) a longer strand having a 3' end
subsequence complementary to said second portion of the shorter
strand; and wherein the step of generating further comprises
melting the shorter strand from the ligated fragments, contacting
the ligated fragments with a DNA polymerase, extending the ligated
fragments by synthesis with the DNA polymerase to produce
blunt-ended double stranded DNA fragments, and amplifying the
blunt-ended fragments by a method comprising contacting the
blunt-ended fragments with the DNA polymerase and primer
oligodeoxynucleotides, said primer oligodeoxynucleotides comprising
the longer adapter strand, and said contacting being at a
temperature not greater than the melting temperature of the primer
oligodeoxynucleotide from a strand of the blunt-ended fragments
complementary to the primer oligodeoxynucleotide and not less than
the melting temperature of the shorter strand of the adapter
nucleic acid from the blunt-ended fragments.
12. The method of claim 6 wherein the recognition means are one or
more restriction endonucleases whose recognition sites are said
target subsequences, and wherein the step of probing further
comprises digesting the sample with said one or more restriction
endonucleases.
13. The method of claim 12 further comprising: (a) identifying a
fragment of a nucleic acid in the sample which generates said one
or more signals; and (b) recovering said fragment.
14. The method of claim 13 wherein the signals generated by said
recovered fragment do not match a sequence in said nucleotide
sequence database.
15. The method of claim 13 which further comprises using at least a
hybridizable portion of said fragment as a hybridization probe to
bind to a nucleic acid that can generate said fragment upon
digestion by said one or more restriction endonucleases.
16. The method of claim 12 wherein the step of generating further
comprises after said digesting: removing from the sample both
nucleic acids which have not been digested and nucleic acid
fragments resulting from digestion at only a single terminus of the
fragments.
17. The method of claim 16 wherein prior to digesting, the nucleic
acids in the sample are each bound at one terminus to a biotin
molecule, and said removing is carried out by a method which
comprises contacting the nucleic acids in the sample with
streptavidin or avidin affixed to a solid support.
18. The method of claim 16 wherein prior to digesting, the nucleic
acids in the sample are each bound at one terminus to a hapten
molecule, and said removing is carried out by a method which
comprises contacting the nucleic acids in the sample with an
anti-hapten antibody affixed to a solid support.
19. The method of claim 12 wherein said digesting with said one or
more restriction endonucleases leaves single-stranded nucleotide
overhangs on the digested ends.
20. The method of claim 19 wherein the step of probing further
comprises hybridizing double-stranded adapter nucleic acids with
the digested sample fragments, each said adapter nucleic acid
having an end complementary to said overhang generated by a
particular one of the one or more restriction endonucleases, and
ligating with a ligase a strand of said adapter nucleic acids to
the 5' end of a strand of the digested sample fragments to form
ligated nucleic acid fragments.
21. The method of claim 20 wherein said digesting with said one or
more restriction endonucleases and said ligating are carried out in
the same reaction medium.
22. The method of claim 21 wherein said digesting and said ligating
comprises incubating said reaction medium at a first temperature
and then at a second temperature, wherein said one or more
restriction endonucleases are more active at the first temperature
than the second temperature and said ligase is more active at the
second temperature than the first temperature.
23. The method of claim 22 wherein said incubating at said first
temperature and said incubating at said second temperature are
performed repetitively.
24. The method of claim 20 wherein the step of probing further
comprises prior to said digesting: removing terminal phosphates
from DNA in said sample by incubation with an alkaline
phosphatase.
25. The method of claim 24 wherein said alkaline phosphatase is
heat labile and is heat inactivated prior to said digesting.
26. The method of claim 20 wherein said generating step comprises
amplifying the ligated nucleic acid fragments.
27. The method of claim 26 wherein said amplifying is carried out
by use of a nucleic acid polymerase and primer nucleic acid
strands, said primer nucleic acid strands being capable of priming
nucleic acid synthesis by said polymerase.
28. The method of claim 27 wherein the primer nucleic acid strands
have a G+C content of between 40% and 60%.
29. The method of claim 27 wherein each said adapter nucleic acid
has a shorter strand and a longer strand, the longer strand being
ligated to the digested sample fragments, and said generating step
comprises prior to said amplifying step the melting of the shorter
strand from the ligated fragments, contacting the ligated fragments
with a DNA polymerase, extending the ligated fragments by synthesis
with the DNA polymerase to produce blunt-ended double stranded DNA
fragments, and wherein the primer nucleic acid strands comprise a
hybridizable portion of the sequence of said longer strands, each
different primer nucleic acid strand priming amplification only of
blunt ended double stranded DNA fragments that are produced after
digestion by a particular restriction endonuclease.
30. The method of claim 27 wherein each said adapter nucleic acid
has a shorter strand and a longer strand, the longer strand being
ligated to the digested sample fragments, and said generating step
comprises prior to said amplifying step the melting of the shorter
strand from the ligated fragments, contacting the ligated fragments
with a DNA polymerase, extending the ligated fragments by synthesis
with the DNA polymerase to Produce blunt-ended double stranded DNA
fragments, and wherein the primer nucleic acid strands comprise the
sequence of said longer strands, each different primer nucleic acid
strand priming amplification only of blunt ended double stranded
DNA fragments that are produced after digestion by a particular
restriction endonuclease.
31. The method of claim 30 wherein during said amplifying step the
primer nucleic acid strands are annealed to the ligated nucleic
acid fragments at a temperature that is less than the melting
temperature of the primer nucleic acid strands from strands
complementary to the primer nucleic acid strands but greater than
the melting temperature of the shorter adapter strands from said
blunt-ended fragments.
32. The method of claim 30 wherein the primer nucleic acid strands
comprise primers, each primer specific for a particular restriction
endonuclease, and further comprising at the 3' end of and
contiguous with the longer strand sequence, the portion of the
restriction endonuclease recognition site remaining on a nucleic
acid fragment terminus after digestion by the restriction
endonuclease.
33. The method of claim 32 wherein each said primer specific for a
particular restriction endonuclease further comprises at its 3' end
one or more nucleotides 3' to and contiguous with the remaining
portion of the restriction endonuclease recognition site, whereby
the ligated nucleic acid fragment amplified is that comprising said
remaining portion of said restriction endonuclease recognition site
contiguous to said one or more additional nucleotides.
34. The method of claim 33 wherein said specific primers are
detectably labeled, such that said primers comprising a particular
said one or more additional nucleotides can be distinguishably
detected from said primers comprising a different said one or more
additional nucleotides.
35. The method of claim 6 wherein the recognition means are
oligomers of nucleotides, nucleotide-mimics, or a combination of
nucleotides and nucleotide-mimics, which are specifically
hybridizable with the target subsequences.
36. The method of claim 35 wherein the step of generating comprises
amplifying with a nucleic acid polymerase and with primers
comprising said oligomers, whereby fragments of nucleic acids in
the sample between hybridized oligomers are amplified.
37. The method of claim 36 further comprising: (a) identifying a
fragment of a nucleic acid in the sample which generates said one
or more signals; and (b) recovering said fragment.
38. The method of claim 37 wherein the signals generated by said
recovered fragment do not match a sequence in said nucleotide
database.
39. The method of claim 37 which further comprises using at least a
hybridizable portion of said fragment as a hybridization probe to
bind to a nucleic acid that can generate said fragment upon
amplification with said nucleic acid polymerase and said one or
more primers.
40. The method of claim 1 wherein said signals further comprise a
representation of whether an additional target subsequence is
present on said nucleic acid in the sample between said occurrences
of target subsequences.
41. The method of claim 40 wherein said additional target
subsequence is recognized by a method comprising contacting nucleic
acids in the sample with oligomers of nucleotides,
nucleotide-mimics, or mixed nucleotides and nucleotide-mimics,
which are hybridizable with said additional target subsequence.
42. The method of claim 1 wherein the step of generating comprises
suppressing said signals when an additional target subsequence is
present on said nucleic acid in the sample between said occurrences
of target subsequences.
43. The method of claim 42 wherein the step of generating comprises
amplifying nucleic acids in the sample, and wherein said additional
target subsequence is recognized by a method comprising contacting
nucleic acids in the sample with (a) oligomers of nucleotides,
nucleotide-mimics, or mixed nucleotides and nucleotide-mimics,
which hybridize with said additional target subsequence and disrupt
the amplifying step; or (b) restriction endonucleases which have
said additional target subsequence as a recognition site and digest
the nucleic acids in the sample at the recognition site.
44. The method of claim 12 or 36 wherein the step of generating
further comprises separating nucleic acid fragments by length.
45. The method of claim 44 wherein the step of generating further
comprises detecting said separated nucleic acid fragments.
46. The method of claim 45 wherein the quantitative abundance of a
nucleic acid comprising a particular nucleotide sequence in the
sample is determined from the quantitative level of the one or more
signals generated by said nucleic acid that are determined to match
said particular nucleotide sequence.
47. The method of claim 45 wherein said detecting is carried out by
a method comprising staining said fragments with silver, labeling
said fragments with a DNA intercalating dye, or detecting light
emission from a fluorochrome label on said fragments.
48. The method of claim 45 wherein said representation of the
length between occurrences of target subsequences is the length of
fragments determined by said separating and detecting steps.
49. The method of claim 45 wherein said separating is carried out
by use of liquid chromatography or mass spectrometry.
50. The method of claim 45 wherein said separating is carried out
by use of electrophoresis.
51. The method of claim 50 wherein said electrophoresis is carried
out in a slab gel or capillary configuration using a denaturing or
non-denaturing medium.
52. The method of claim 1 wherein a predetermined one or more
nucleotide sequences in said database are of interest, and wherein
the target subsequences are such that said sequences of interest
generate at least one signal that is not generated by other
nucleotide sequences in said database.
53. The method of claim 52 wherein the nucleotide sequences of
interest are a majority of the sequences in said database.
54. The method of claim 1 wherein the target subsequences have a
probability of occurrence in the nucleotide sequences in said
database of from approximately 0.01 to approximately 0.30.
55. The method of claim 1 wherein the target subsequences are such
that nucleotide sequences in said database contain on average a
sufficient number of occurrences of target subsequences in order to
on average generate a signal that is not generated by any other
nucleotide sequence in said database.
56. The method of claim 55 wherein the number of pairs of target
subsequences present on average in a nucleotide sequence in said
database is no less than 3, and wherein the average number of
signals generated from nucleotide sequences in said database is
such that the average difference between lengths represented by the
generated signals is greater than or equal to 1 nucleotide.
57. The method of claim 55 wherein the target subsequences have a
probability of occurrence, p, approximately given by the solution
of 7 R ( R + 1 ) p 2 2 = Aand 8 L Np 2 = Bwherein N=the number of
different nucleotide sequences in said database; L=the average
length of said different nucleotide sequences in said database;
R=the number of recognition means; A=the number of pairs of target
subsequences present on average in said different nucleotide
sequences in said database; and B=the average difference between
lengths represented by the signals generated from the sequences in
said database.
58. The method of claim 57 wherein A is greater than or equal to
3.
59. The method of claim 57 wherein B is greater than or equal to
1.
60. The method of claim 1 wherein the target subsequences are
selected according to the further steps comprising: (a) determining
a pattern of signals that can be generated and the sequences
capable of generating each such signal by simulating the steps of
probing and generating applied to sequences in said database of
nucleotide sequences; (b) ascertaining the value of said determined
pattern according to an information measure; and (c) choosing the
target subsequences in order to generate a new pattern that
optimizes the information measure.
61. The method of claim 60 wherein said choosing step selects
target subsequences which comprise the recognition sites of the one
or more restriction endonucleases.
62. The method of claim 60 wherein said choosing step selects
target subsequences which comprise the recognition sites of the one
or more restriction endonucleases contiguous with one or more
additional nucleotides.
63. The method of claim 60 wherein a predetermined one or more of
the nucleotide sequences present in said database of nucleotide
sequences are of interest, and the information measure optimized is
the number of such said sequences of interest which generate at
least one signal that is not generated by any other nucleotide
sequence present in said database.
64. The method of claim 63 wherein said nucleotide sequences of
interest are a majority of the nucleotide sequences present in said
database.
65. The method of claim 60 wherein said choosing step is by
exhaustive search of all combinations of target subsequences of
length less than approximately 10.
66. The method of claim 60 wherein said step of choosing target
subsequences is by a method comprising simulated annealing.
67. The method of claim 1 wherein the step of searching further
comprises: (a) determining a pattern of signals that can be
generated and the sequences capable of generating each such signal
by simulating the steps of probing and generating applied to each
sequence in said database of nucleotide sequences; and (b) finding
the one or more nucleotide sequences in said database that are able
to generate said one or more generated signals by finding in said
pattern those signals that comprise a representation of (i) the
same lengths between occurrences of target subsequences as is
represented by the generated signal, and (ii) the same target
subsequences as are represented by the Generated signal, or target
subsequences that are members of the same sets of target
subsequences represented by the generated signal.
68. The method of claim 60 or 67 wherein the step of determining
further comprises: (a) searching for occurrences of said target
subsequences or sets of target subsequences in nucleotide sequences
in said database of nucleotide sequences; (b) finding the lengths
between occurrences of said target subsequences or sets of target
subsequences in the nucleotide sequences of said database; and (c)
forming the pattern of signals that can be generated from the
sequences of said database in which the target subsequences were
found to occur.
69. The method of claim 20 wherein said restriction endonucleases
generate 5' overhangs at the terminus of digested fragments and
wherein each double stranded adapter nucleic acid comprises: (a) a
shorter nucleic acid strand consisting of a first and second
contiguous portion, said first portion being a 5' end subsequence
complementary to the overhang produced by one of said restriction
endonucleases; and (b) a longer nucleic acid strand having a 3' end
subsequence complementary to said second portion of the shorter
strand.
70. The method of claim 69 wherein said shorter strand has a
melting temperature from a complementary strand of less than
approximately 68.degree. C., and has no terminal phosphate.
71. The method of claim 70 wherein said shorter strand is
approximately 12 nucleotides long.
72. The method of claim 69 wherein said longer strand has a melting
temperature from a complementary strand of greater than
approximately 68.degree. C., is not complementary to any nucleotide
sequence in said database, and has no terminal phosphate.
73. The method of claim 72 wherein said ligated nucleic acid
fragments do not contain a recognition site for any of said
restriction endonucleases.
74. The method of claim 72 wherein said one or more restriction
endonucleases are heat inactivated before said ligating.
75. The method of claim 72 wherein said longer strand is
approximately 24 nucleotides long and has a G+C content between 40%
and 60%.
76. The method of claim 20 wherein said restriction endonucleases
generate 3' overhangs at the terminus of the digested fragments,
and wherein each double stranded adapter nucleic acid comprises:
(a) a longer nucleic acid strand consisting of a first and second
contiguous portion, said first portion being a 3' end subsequence
complementary to the overhang produced by one of said restriction
endonucleases; and (b) a shorter nucleic acid strand complementary
to the 3' end of said second portion of the longer nucleic acid
stand.
77. The method of claim 76 wherein said shorter strand has a
melting temperature from said longer strand of less than
approximately 68.degree. C., and has no terminal phosphates.
78. The method of claim 77 wherein said shorter strand is 12 base
pairs long.
79. The method of claim 76 wherein said longer strand has a melting
temperature from a complementary strand of greater than
approximately 68.degree. C., is not complementary to any nucleotide
sequence in said database, has no terminal phosphate, and wherein
said ligated nucleic acid fragments do not contain a recognition
site for any of said restriction endonucleases.
80. The method of claim 79 wherein said longer strand is 24 base
pairs long and has a G+C content between 40% and 60%.
81. A method for identifying or classifying a nucleic acid
comprising: (a) probing said nucleic acid with a plurality of
recognition means, each recognition means recognizing a target
nucleotide subsequence or a set of target nucleotide subsequences,
in order to generate a set of signals, each signal representing
whether said target subsequence or one of said set of target
subsequences is present or absent in said nucleic acid; and (b)
searching a nucleotide sequence database, said database comprising
a plurality of known nucleotide sequences of nucleic acids that may
be present in the sample, for sequences matching said generated set
of signals, a sequence from said database matching a set of signals
when the sequence from said database (i) comprises the same target
subsequences as are represented as present, or comprises target
subsequences that are members of the sets of target subsequences
represented as present by the generated sets of signals, and (ii)
does not comprise the target subsequences represented as absent or
that are members of the sets of target subsequences represented as
absent by the generated sets of signals, whereby the nucleic acid
is identified or classified.
82. The method of claim 81 wherein the set of signals are
represented by a hash code which is a binary number.
83. The method of claim 81 wherein the step of probing generates
quantitative signals of the numbers of occurrences of said target
subsequences or of members of said set of target subsequences in
said nucleic acid.
84. The method of claim 83 wherein a sequence matches said
generated set of signals when the sequence from said database
comprises the same target subsequences with the same number of
occurrences in said sequence as in the quantitative signals and
does not comprise the target subsequences represented as absent or
target subsequences within the sets of target subsequences
represented as absent.
85. The method of claim 81 wherein said plurality of nucleic acids
are DNA.
86. The method of claim 85 wherein the recognition means are
detectably labeled oligomers of nucleotides, nucleotide-mimics, or
combinations of nucleotides and nucleotide-mimics, and the step of
probing comprises hybridizing said nucleic acid with said
oligomers.
87. The method of claim 86 wherein said detectably labeled
oligomers are detected by a method comprising detecting light
emission from a fluorochrome label on said oligomers, or arranging
said labeled oligomers to cause light to scatter from a light pipe
and detecting said scattering.
88. The method of claim 86 wherein the recognition means are
oligomers of peptido-nucleic acids.
89. The method of claim 86 wherein the recognition means are DNA
oligomers, DNA oligomers comprising universal nucleotides, or sets
of partially degenerate DNA oligomers.
90. The method of claim 85 wherein the step of searching further
comprises: (a) determining a pattern of sets of signals of the
presence or absence of said target subsequences or said sets of
target subsequences that can be generated and the sequences capable
of generating each set of signals in said pattern by simulating the
step of probing as applied to each sequence in said database of
nucleotide sequences; and (b) finding one or more nucleotide
sequences that are capable of generating said generated set of
signals by finding in said pattern those sets that match said
generated set, where a set of signals from said pattern matches a
generated set of signals when the set from said pattern (i)
represents as present the same target subsequences as are
represented as present or target subsequences that are members of
the sets of target subsequences represented as present by the
generated sets of signals and (ii) represents as absent the target
subsequences represented as absent or that are members of the sets
of target subsequences represented as absent by the generated sets
of signals.
91. The method of claim 85 wherein the target subsequences are
selected according to the further steps comprising: (a) determining
(i) a pattern of sets of signals representing the presence or
absence of said target subsequences or of said sets of target
subsequences that can be generated, and (ii) the sequences capable
of generating each set of signals in said pattern by simulating the
step of probing as applied to each sequence in said database of
nucleotide sequences; (b) ascertaining the value of said pattern
generated according to an information measure; and (c) choosing the
target subsequences in order to generate a new pattern that
optimizes the information measure.
92. The method of claim 91 wherein the information measure is the
number of sets of signals in the pattern which are capable of being
generated by one or more sequences in said database.
93. The method of claim 91 wherein the information measure is the
number of sets of signals in the pattern which are capable of being
generated by only one sequence in said database.
94. The method of claim 91 wherein said choosing step is by a
method comprising exhaustive search of all combination of target
subsequences of length less than approximately 10.
95. The method of claim 91 wherein said choosing step is by a
method comprising simulated annealing.
96. The method of claim 90 or 91 wherein the step of determining by
simulating further comprises: (a) searching for the presence or
absence of said target subsequences or sets of target subsequences
in each nucleotide sequence in said database of nucleotide
sequences; and (b) forming the pattern of sets of signals that can
be generated from said sequences in said database.
97. The method of claim 96 where the step of searching is carried
out by a string search.
98. The method of claim 96 wherein the step of searching comprises
counting the number of occurrences of said target subsequences in
each nucleotide sequence.
99. The method of claim 81 wherein the target subsequences have a
probability of occurrence in a nucleotide sequence in said database
of nucleotide sequences of from 0.01 to 0.6.
100. The method of claim 99 wherein the target subsequences are
such that the presence of one target subsequence in a nucleotide
sequence in said database of nucleotide sequences is substantially
independent of the presence of any other target subsequence in the
nucleotide sequence.
101. The method of claim 99 wherein fewer than approximately 50
target subsequences are selected.
102. A programmable apparatus for analyzing signals comprising: (a)
an inputting device for inputting one or more actual signals
generated by probing a sample comprising a plurality of nucleic
acids with recognition means, each recognition means recognizing a
target nucleotide subsequence or a set of target nucleotide
subsequences, said signals comprising a representation of (i) the
length between occurrences of said target subsequences in a nucleic
acid of said sample, and (ii) the identities of said target
subsequences in said nucleic acid, or the identities of said sets
of target subsequences among which is included the target
subsequences in said nucleic acid; (b) a searching device
operatively coupled to said accepting device for searching a
sequence in a nucleotide sequence database for occurrences of said
target subsequences or target subsequences that are members of said
sets of target subsequences, and for the length between such
occurrences, said database comprising a plurality of known
nucleotide sequences that may be present in said sample; (c) a
comparing device operatively coupled to said accepting device and
to said searching device for finding a match between said one or
more actual signals and a sequence in said database, said one or
more actual signals matching a sequence from said database when the
sequence from said database has both (i) the same length between
occurrences of target subsequences as is represented by said one or
more actual signals, and (ii) the same target subsequences as are
represented by said one or more actual signals, or target
subsequences that are members of the sets of target subsequences
represented by said one or more actual signals; and (d) a control
device operatively coupled to said comparing device for causing
said comparing to be done for sequences in the database and for
outputting those database sequences that match said one or more
actual signals.
103. The programmable apparatus of claim 102 wherein said searching
device searches for said target subsequences or a set of target
nucleotide subsequences in said database sequences by performing a
string comparison of the nucleotides in said subsequences with
those in said database sequence.
104. The programmable apparatus of claim 102 wherein said control
device further comprises causing said searching device to search
all sequences in said database in order to determine a pattern of
signals that can be generated by probing said sample with said
recognition means, and wherein said control device further causes
said comparing device to find any matches between said one or more
actual signals and said pattern of signals, said one or more actual
signals matching a signal in said pattern of signals when the
signal from said pattern represents (i) the same length between
occurrences of target subsequences as is represented by said one or
more actual signals, and (ii) the same target subsequences as are
represented by said one or more actual signals, or target
subsequences that are members of the sets of target subsequences
represented by said one or more actual signals.
105. The programmable apparatus of claim 102 wherein said sample of
nucleic acids comprises cDNA of RNA of a cell or tissue type, and
said database comprises DNA sequences that are likely to be
expressed by said cell or tissue type.
106. A computer readable memory that can be used to direct a
programmable apparatus to function for analyzing signals according
to steps comprising: (a) inputting one or more actual signals
generated by probing a sample comprising a plurality of nucleic
acids with recognition means, each recognition means recognizing a
target nucleotide subsequence or a set of target nucleotide
subsequences, said signals comprising a representation of (i) the
length between occurrences of said target subsequences in a nucleic
acid of said sample, and (ii) the identities of said target
subsequences in said nucleic acid, or the identities of said sets
of target subsequences among which is included the target
subsequences in said nucleic acid; (b) searching a sequence in a
nucleotide sequence database for occurrences of said target
subsequences or target subsequences that are members of said sets
of target subsequences, and for the length between such
occurrences, said database comprising a plurality of known
nucleotide sequences that may be present in said sample; (c)
matching said one or more actual signals and a sequence in said
database when the sequence in said database has both (i) the same
length between occurrences of target subsequences as is represented
by said one or more actual signals and (ii) the same target
subsequences as are represented by said one or more actual signals,
or target subsequences that are members of the sets of target
subsequences as are represented by said one or more actual signals;
and (d) repetitively performing said searching and matching steps
for the majority of sequences in the database and outputting those
database sequences that match said one or more actual signals.
107. A programmable apparatus for selecting target subsequences
comprising: (a) an initial selection device for selecting initial
target subsequences or initial sets of target subsequences; (b) a
first control device; (c) a search device operatively coupled to
said initial selection device and to said first control device (i)
for searching sequences in a nucleotide sequence database for
occurrences of said initial target subsequences or occurrences of
target subsequences that are members of said initial sets of target
subsequences and for the length between such occurrences, and (ii)
for determining an initial pattern of signals that can be generated
from said selected initial target subsequences or said initial sets
of target subsequences, said database comprising a plurality of
known nucleotide sequences, said signals comprising a
representation of (i) the length between said occurrences in a
sequence in said database, and (ii) the identities of said initial
target subsequences that occur in said sequence in said database,
or the identities of target subsequences that are members of the
initial sets of target subsequences that occur in said sequence in
said database; and (d) an ascertaining device operatively coupled
to said searching device and to said first control device for
ascertaining the value of said determined initial pattern according
to an information measure; and wherein said first control device
causes further target subsequences to be selected and causes the
search device to determine a further pattern of signals and the
ascertaining device to ascertain a further value of said
information measure and accepts the further target subsequences
when said further pattern optimizes said further value of said
information measure.
108. The programmable apparatus of claim 107 wherein a
predetermined one or more of the sequences in said database are of
interest, and wherein said ascertaining device ascertains the value
of an information measure by counting the number of such sequences
of interest which generate in said determined pattern at least one
signal that is not generated by any other sequence in said
database.
109. The programmable apparatus of claim 108 wherein said one or
more of the sequences of interest comprise substantially all the
sequences in said database.
110. The programmable apparatus of claim 107 wherein said first
control device optimizes the value of said information measure
according to a method of exhaustive search, wherein said first
control device selects further target subsequences of length less
than approximately 10 and accepts the further target subsequences
if said further value of said information measure is greater than
the previous value.
111. The programmable apparatus of claim 107 wherein said first
control device optimizes the value of said information measure
according to a method comprising simulated annealing, wherein said
first control device repeatedly selects further target subsequences
and accepts the further target subsequences if said further value
of said information measure is not decreased by greater than a
probabilistic factor dependent on a simulated-temperature, and
wherein said programmable apparatus further comprises a second
control device operatively coupled to said first control device for
decreasing said simulated-temperature as said first control device
selects further target subsequences.
112. The programmable apparatus of claim 111 wherein said
probabilistic factor is an exponential function of the negative of
the decrease in the information measure divided by said
simulated-temperature.
113. The programmable apparatus of claim 107 wherein said database
comprises a majority of known DNA sequences that are likely to be
expressed in one or more cell types.
114. A computer readable memory that can be used to direct a
programmable apparatus to function for selecting target
subsequences according to steps comprising: (a) selecting initial
target subsequences or initial sets of target subsequences; (b)
searching a sequence in a nucleotide sequence database for
occurrences of said initial target subsequences or occurrences of
target subsequences that are members of said initial sets of target
subsequences and for the length between such occurrences, said
database comprising a plurality of known nucleotide sequences that
may be present in said sample; (c) determining an initial pattern
of signals that can be generated from said selected initial target
subsequences or said initial sets of target subsequences, said
signals comprising a representation of (i) the length between said
occurrences in a sequence in said database, and (ii) the identities
of said initial target subsequences that occur in said sequence in
said database, or the identities of target subsequences that are
members of the initial sets of target subsequences that occur in
said sequence in said database; and (d) ascertaining the value of
said determined initial pattern according to an information
measure; and (e) repetitively performing said selecting, searching,
determining, and ascertaining steps to determine a further pattern
of signals and a further value of said information measure, and
accepting the further target subsequences when said further pattern
optimizes said further value of said information measure.
115. A programmable apparatus for displaying data comprising: (a) a
selecting device for selecting target subsequences or sets of
target subsequences, such that recognition means for recognizing
said target subsequences or said sets of target subsequences can be
used to generate signals by probing a sample comprising a plurality
of nucleic acids, said signals comprising a representation of (i)
the length between occurrences of said target subsequences in a
nucleic acid of said sample and (ii) the identities of said target
subsequences in said nucleic acid or the identities of said sets of
target subsequences among which are included the target
subsequences in said nucleic acid; (b) an inputting device for
inputting one or more actual signals generated by probing said
sample with said recognition means; (c) an analyzing device for
analyzing signals operatively coupled to said selecting and
inputting devices that determines which sequences in a nucleotide
sequence database can generate said actual signals when subject to
said recognition means, said database comprising a plurality of
known nucleotide sequences that may be present in said sample; (d)
an input/output device operatively coupled to said selecting,
inputting, and analyzing devices that inputs user requests and
controls the selecting device to select target subsequences or sets
of target subsequences, controls the inputting device to accept
actual signals, controls the analyzing device to find the sequences
in said database that can generate said actual signals, and
displays output comprising said actual signals and said sequences
in said database that can generate said actual signals.
116. The programmable apparatus of 115 wherein said sample is a
cDNA sample prepared from a tissue specimen, and the apparatus
further comprises a storage device operatively coupled to the
input/output device for storing indications of the origin of said
tissue specimen and information concerning said tissue specimen,
and wherein said indications can be displayed upon user input.
117. The programmable apparatus of 116 wherein the indications and
information concerning said tissue specimen comprises histological
information comprising tissue images.
118. The programmable apparatus of claim 115 further comprising:
(a) one or more instrument devices for probing said sample with
said recognition means and for generating said actual signals; and
(b) a control device operatively coupled to said one or more
instrument devices and to said input/output device for controlling
the operation of said instrument devices, wherein said user can
input control commands for control of said instrument devices and
receive output concerning the status of said instrument
devices.
119. The programmable apparatus of 118 wherein the one or more
instrument devices are capable of automatic operation, whereby the
probing and generating can be performed without manual
Intervention.
120. The programmable apparatus of claim 115 wherein one or more of
said selecting, inputting, analyzing, and input/output devices are
physically collocated with each other.
121. The programmable apparatus of claim 115 wherein one or more of
said selecting, inputting, analyzing, and input/output devices are
physically spaced apart from each other and are connected by a
communication medium for exchanges of commands and information.
122. A computer readable memory that can be used to direct a
programmable apparatus to function for displaying data according to
steps comprising: (a) selecting target subsequences or sets of
target subsequences, such that recognition means for recognizing
said target subsequences or said sets of target subsequences can be
used to generate signals by probing a sample comprising a plurality
of nucleic acids, said signals comprising a representation of (i)
the length between occurrences of said target subsequences in a
nucleic acid of said sample and (ii) the identities of said target
subsequences in said nucleic acid or the identities of said sets of
target subsequences among which are included the target
subsequences in said nucleic acid; (b) inputting one or more actual
signals generated by probing said sample with said recognition
means; (c) analyzing said one or more actual signals to determine
which sequences in a nucleotide sequence database can generate said
actual signals when subject to said recognition means, said
database comprising a plurality of known nucleotide sequences that
may be present in said sample; and (d) inputting user requests to
control said selecting step to select target subsequences or sets
of target subsequences, said inputting step to input actual
signals, and said analyzing step to find the sequences in said
database that can generate said actual signals, and outputting in
response to further user requests information comprising said
actual signals and said sequences in said database that can
generate said actual signals.
123. A method for identifying, classifying, or quantifying DNA
molecules in a sample of DNA molecules having a plurality of
different nucleotide sequences, the method comprising the steps of:
(a) digesting said sample with one or more restriction
endonucleases, each said restriction endonuclease recognizing a
subsequence recognition site and digesting DNA at said recognition
site to produce fragments with 5' overhangs; (b) contacting said
fragments with shorter and longer oligodeoxynucleotides, each said
shorter oligodeoxynucleotide hybridizable with a said 5' overhang
and having no terminal phosphates, each said longer
oligodeoxynucleotide hybridizable with a said shorter
oligodeoxynucleotide; (c) ligating said longer
oligodeoxynucleotides to said 5' overhangs on said DNA fragments to
produce ligated DNA fragments; (d) extending said ligated DNA
fragments by synthesis with a DNA polymerase to produce blunt-ended
double stranded DNA fragments; (e) amplifying said blunt-ended
double stranded DNA fragments by a method comprising contacting
said DNA fragments with a DNA polymerase and primer
oligodeoxynucleotides, each said primer oligodeoxynucleotide having
a sequence comprising that of one of the longer
oligodeoxynucleotides; (f) determining the length of the amplified
DNA fragments; and (g) searching a DNA sequence database, said
database comprising a plurality of known DNA sequences that may be
present in the sample, for sequences matching one or more of said
fragments of determined length, a sequence from said database
matching a fragment of determined length when the sequence from
said database comprises recognition sites of said one or more
restriction endonucleases spaced apart by the determined length,
whereby DNA molecules in said sample are identified, classified, or
quantified.
124. The method of claim 123 wherein the sequence of each primer
oligodeoxynucleotide further comprises 3' to and contiguous with
the sequence of the longer oligodeoxynucleotide the portion of the
recognition site of said one or more restriction endonucleases
remaining on a DNA fragment terminus after digestion, said
remaining portion being 5' to and contiguous with one or more
additional nucleotides, and wherein a sequence from said database
matches a fragment of determined length when the sequence from said
database comprises subsequences that are the recognition sites of
said one or more restriction endonucleases contiguous with said one
or more additional nucleotides and when the subsequences are spaced
apart by the determined length.
125. The method of claim 123 wherein said determining step further
comprises detecting the amplified DNA fragments by a method
comprising staining said fragments with silver.
126. The method of claim 123 wherein said oligodeoxynucleotide
primers are detectably labeled, wherein the determining step
further comprises detection of said detectable labels, and wherein
a sequence from said database matches a fragment of determined
length when the sequence from said database comprises recognition
sites of the one or more restriction endonucleases, said
recognition sites being that are identified by the detectable
labels of said oligodeoxynucleotide primers, said recognition sites
being spaced apart by the determined length.
127. The method of claim 123 wherein said determining step further
comprises detecting the amplified DNA fragments by a method
comprising labeling said fragments with a DNA intercalating dye or
detecting light emission from a fluorochrome label on said
fragments.
128. The method of claim 123 further comprising, prior to said
determining step, the step of hybridizing the amplified DNA
fragments with a detectably labeled oligodeoxynucleotide
complementary to a subsequence, said subsequence differing from
said recognition sites of said one or more restriction
endonucleases, wherein the determining step further comprises
detecting said detectable label of said oligodeoxynucleotide, and
wherein a sequence from said database matches a fragment of
determined length when the sequence from said database further
comprises said subsequence between the recognition sites of said
one or more restriction endonucleases.
129. The method of claim 123 wherein the one or more restriction
endonucleases are pairs of restriction endonucleases, the pairs
being selected from the group consisting of Acc56I and HindIII,
Acc65I and NgoMI, BamHI and EcoRI BglII and HindIII, BglII and
NgoMI, BsiWI and BspHI, BspHI and BstYI, BspHI and NgoMI, BsrGI and
EcoRI, EagI and EcoRI, EagI and HindIII, EagI and NcoI, HindIII and
NgoMI, NgoMI and NheI, NgoMI and SpeI, BglII and BspHI, Bsp120I and
NcoI, BssHII and NgoMI, EcoRI and HindIII, and NgoMI and XbaI.
130. The method of claim 123 wherein the step of ligating is
performed with T4 DNA ligase.
131. The method of claim 123 wherein the steps of digesting,
contacting, and ligating are performed simultaneously in the same
reaction vessel.
132. The method of claim 123 wherein the steps of digesting,
contacting, ligating, extending, and amplifying are performed in
the same reaction vessel.
133. The method of claim 123 wherein the step of determining the
length is performed by electrophoresis.
134. The method of claim 123 wherein the step of searching said DNA
database further comprises: (a) determining a pattern of fragments
that can be generated and for each fragment in said pattern those
sequences in said DNA database that are capable of generating the
fragment by simulating the steps of digesting with said one or more
restriction endonucleases, contacting, ligating, extending,
amplifying, and determining applied to each sequence in said DNA
database; and (b) finding the sequences that are capable of
generating said one or more fragments of determined length by
finding in said pattern one or more fragments that have the same
length and recognition sites as said one or more fragments of
determined length.
135. The method of claim 123 wherein the steps of digesting and
ligating go substantially to completion.
136. The method of claim 123 wherein the DNA sample is cDNA of RNA
from a tissue or a cell type derived from a plant, a single celled
animal, a multicellular animal, a bacterium, a virus, a fungus, or
a yeast.
137. The method of claim 123 wherein the DNA sample is cDNA of RNA
from one or more cell types of a mammal.
138. The method of claim 137 wherein the mammal is a human.
139. The method of claim 137 wherein the mammal is a human having
or suspected of having a diseased condition.
140. The method of claim 139 wherein the diseased condition is a
malignancy.
141. The method of claim 123 wherein said DNA sample is cDNA
prepared from mRNA.
142. A method for identifying, classifying, or quantifying DNA
molecules in a sample of DNA molecules with a plurality of
nucleotide sequences, the method comprising the steps of: (a)
digesting said sample with one or more restriction endonucleases,
each said restriction endonuclease recognizing a subsequence
recognition site and digesting DNA to produce fragments with 3'
overhangs; (b) contacting said fragments with shorter and longer
oligodeoxynucleotides, each said longer oligodeoxynucleotide
consisting of a first and second contiguous portion, said first
portion being a 3' end subsequence complementary to the overhang
produced by one of said restriction endonucleases, each said
shorter oligodeoxynucleotide complementary to the 3' end of said
second portion of said longer oligodeoxynucleotide stand; (c)
ligating said longer oligodeoxynucleotides to said DNA fragments to
produce a ligated fragments; (d) extending said ligated DNA
fragments by synthesis with a DNA polymerase to form blunt-ended
double stranded DNA fragments; (e) amplifying said double stranded
DNA fragments by use of a DNA polymerase and primer
oligodeoxynucleotides to produce amplified DNA fragments, each said
primer oligodeoxynucleotide having a sequence comprising that of a
longer oligodeoxynucleotide; (f) determining the length of the
amplified DNA fragments; and (g) searching a DNA sequence database,
said database comprising a plurality of known DNA sequences that
may be present in the sample, for sequences matching one or more of
said fragments of determined length, a sequence from said database
matching a fragment of determined length when the sequence from
said database comprises recognition sites of said one or more
restriction endonucleases spaced apart by the determined length,
whereby DNA sequences in said sample are identified, classified, or
quantified.
143. A method of detecting one or more differentially expressed
genes in an in vitro cell exposed to an exogenous factor relative
to an in vitro cell not exposed to said exogenous factor
comprising: (a) performing the method of claim 1 wherein said
plurality of nucleic acids comprises cDNA of RNA of said in vitro
cell exposed to said exogenous factor; (b) performing the method of
claim 1 wherein said plurality of nucleic acids comprises cDNA of
RNA of said in vitro cell not exposed to said exogenous factor; and
(c) comparing the identified, classified, or quantified cDNA of
said in vitro cell exposed to said exogenous factor with the
identified, classified, or quantified cDNA of said in vitro cell
not exposed to said exogenous factor, whereby differentially
expressed genes are identified, classified, or quantified.
144. A method of detecting one or more differentially expressed
genes in a diseased tissue relative to a tissue not having said
disease comprising: (a) performing the method of claim 1 wherein
said plurality of nucleic acids comprises cDNA of RNA of said
diseased tissue, such that one or more cDNA molecules are
identified, classified, and/or quantified; (b) performing the
method of claim 1 wherein said plurality of nucleic acids comprises
cDNA of RNA of said tissue not having said disease, such that one
or more cDNA molecules are identified, classified, and/or
quantified; and (c) comparing said identified, classified, and/or
quantified cDNA molecules of said diseased tissue with said
identified, classified, and/or quantified cDNA molecules of said
tissue not having the disease, whereby differentially expressed
cDNA molecules are detected.
145. The method of claim 144 wherein the step of comparing further
comprises finding cDNA molecules which are reproducibly expressed
in said diseased tissue or in said tissue not having the disease
and further finding which of said reproducibly expressed cDNA
molecules have significant differences in expression between the
tissue having said disease and the tissue not having said
disease.
146. The method of claim 145 wherein said finding cDNA molecules
which are reproducibly expressed and said significant differences
in expression of said cDNA molecules in said diseased tissue and in
said tissue not having the disease are determined by a method
comprising applying statistical measures.
147. The method of claim 146 wherein said statistical measures
comprise finding reproducible expression if the standard deviation
of the level of quantified expression of a cDNA molecule in said
diseased tissue or said tissue not having the disease is less than
the average level of quantified expression of said cDNA molecule in
said diseased tissue or said tissue not having the disease,
respectively, and wherein a cDNA molecule has significant
differences in expression if the sum of the standard deviation of
the level of quantified expression of said cDNA molecule in said
diseased tissue plus the standard deviation of the level of
quantified expression of said cDNA molecule in said tissue not
having the disease is less than the absolute value of the
difference of the level of quantified expression of said cDNA
molecule in said diseased tissue minus the level of quantified
expression of said cDNA molecule in said tissue not having the
disease.
148. The method of claim 144 wherein the diseased tissue and the
tissue not having the disease are from one or more mammals.
149. The method of claim 144 wherein the disease is a
malignancy.
150. The method of claim 144 wherein the disease is a malignancy
selected from the group consisting of prostrate cancer, breast
cancer, colon cancer, lung cancer, skin cancer, lymphoma, and
leukemia.
151. The method of claim 144 wherein the disease is a malignancy
and the tissue not having the disease has a premalignant
character.
152. A method of staging or grading a disease in a human individual
comprising: (a) performing the method of claim 1 in which said
plurality of nucleic acids comprises cDNA of RNA prepared from a
tissue from said human individual, said tissue having or suspected
of having said disease, whereby one or more said cDNA molecules are
identified, classified, and/or quantified; and (b) comparing said
one or more identified, classified, and/or quantified cDNA
molecules in said tissue to the one or more identified, classified,
and/or quantified cDNA molecules expected at a particular stage or
grade of said disease.
153. A method for predicting a human patient's response to therapy
for a disease, comprising: (a) performing the method of claim 1 in
which said plurality of nucleic acids comprises cDNA of RNA
prepared from a tissue from said human patient, said tissue having
or suspected of having said disease, whereby one or more cDNA
molecules in said sample are identified, classified, and/or
quantified; and (b) ascertaining if the one or more cDNA molecules
thereby identified, classified, and/or quantified is correlates
with a poor or a favorable response to one or more therapies.
154. The method of claim 153 which further comprises selecting one
or more therapies for said patient for which said identified,
classified, and/or quantified cDNA molecules correlates with a
favorable response.
155. A method for evaluating the efficacy of a therapy in a mammal
having a disease, the method comprising: (a) performing the method
of claim 1 wherein said plurality of nucleic acids comprises cDNA
of RNA of said mammal prior to a therapy; (b) performing the method
of claim 1 wherein said plurality of nucleic acids comprises cDNA
of RNA of said mammal subsequent to said therapy; (c) comparing one
or more identified, classified, and/or quantified cDNA molecules of
said mammal prior to said therapy with one or more identified,
classified, and/or quantified cDNA molecules of said mammal
subsequent to therapy; and (d) determining whether the response to
therapy is favorable or unfavorable according to whether any
differences in the one or more identified, classified, and/or
quantified cDNA molecules after therapy are correlated with
regression or progression, respectively, of the disease.
156. The method of claim 155 wherein the mammal is a human.
157. A kit comprising: (a) one or more containers having one or
more restriction endonucleases; (b) one or more containers having
one or more shorter oligodeoxynucleotide strands; (c) one or more
containers having one or more longer oligodeoxynucleotide strands
hybridizable with said shorter strands, wherein either the longer
or the shorter oligodeoxynucleotide strands each comprise a
sequence complementary to an overhang produced by at least one of
said one or more restriction endonucleases; and (d) instructions
packaged in association with said one or more containers for use of
said restriction endonucleases, shorter strands, and longer strands
for identifying, classifying, or quantifying one or more DNA
molecules in a DNA sample, said instructions comprising: i. digest
said sample with said restriction endonucleases into fragments,
each fragment being terminated on each end by a recognition site of
said one or more restriction endonucleases; ii. contact said
shorter and longer strands and said digested fragments to form
double stranded DNA adapters annealed to said digested fragments,
iii. ligate said longer strand to said fragments; iv. generate one
or more signals by separating and detecting such of said fragments
that are digested on each end, each signal comprising a
representation of the length of the fragment and the identity of
the recognition sites on both termini of the fragments; and v.
search a nucleotide sequence database to determine sequences that
match or the absence of any sequences that match said one or more
generated signals, said database comprising a plurality of known
nucleotide sequences of nucleic acids that may be present in the
sample, a sequence from said database matching a generated signal
when the sequence from said database has both (i) the same length
between occurrences of said recognition sites of said one or more
restriction endonucleases as is represented by the generated signal
and (ii) the same recognition sites of said one of more restriction
endonucleases as is represented by the generated signal.
158. The kit of claim 157 wherein said one or more restriction
endonucleases generate 5' overhangs at the terminus of digested
fragments, wherein each said shorter oligodeoxynucleotide strand
consists of a first and second contiguous portion, said first
portion being a 5' end subsequence complementary to the overhang
produced by one of said restriction endonucleases, and wherein each
said longer oligodeoxynucleotide strand comprises a 3' end
subsequence complementary to said second portion of said shorter
oligodeoxynucleotide strand.
159. The kit of claim 157 wherein said one or more restriction
endonucleases generate 3' overhangs at the terminus of the digested
fragments, wherein each said longer oligodeoxynucleotide strand
consists of a first and second contiguous portion, said first
portion being a 3' end subsequence complementary to the overhang
produced by one of said restriction endonucleases, and wherein each
said shorter oligodeoxynucleotide strand is complementary to the 3'
end of said second portion of said longer oligodeoxynucleotide
stand.
160. The kit of claim 157 wherein said instructions further
comprise those signals expected from one or more DNA molecules of
interest when said sample is digested with a particular one or more
restriction endonucleases selected from among said one or more
restriction endonucleases in said kit.
161. The kit of claim 160 wherein said one or more DNA molecules of
interest are cDNA molecules differentially expressed in a disease
condition.
162. The kit of claim 157 wherein the restriction endonucleases are
selected from the group consisting of Acc65I, AflII, AgeI, ApaLI,
ApoI, AscI, AvrI, BamHI, BclI, BglII, BsiWI, Bsp120I, BspEI, BspHI,
BsrGI, BssHII, BstYI, EagI, EcoRI, HindIII, MluI, NcoI, NgoMI,
NheI, NotI, SpeI, and XbaI.
163. The kit of claim 157 which comprises one or more containers
having one or more double stranded adapter DNA molecules formed by
annealing said longer and said shorter oligonucleotide strands.
164. The kit of claim 157 further comprising a computer readable
memory according to claim 106.
165. The kit of claim 157 further comprising a computer readable
memory according to claim 114.
166. The kit of claim 157 further comprising a computer readable
memory according to claim 122.
167. The kit of claim 157 further comprising in a container a DNA
ligase.
168. The kit of claim 157 further comprising in a container a
phosphatase capable of removing terminal phosphates from a DNA
sequence.
169. The kit of claim 157 further comprising in one or more
containers: (a) one or more primers, each said primer consisting of
a single stranded oligodeoxynucleotide comprising the sequence of
one of said longer strands; and (b) a DNA polymerase.
170. The kit of claim 169 wherein each of said one or more primers
further comprises (a) a first subsequence that is the portion of
the recognition site of one of said one or more restriction
endonucleases remaining at the terminus of a fragment after
digestion, and (b) a second subsequence of one or two additional
nucleotides contiguous with and 3' to said first subsequence,
wherein said primer is detectably labeled such that primers with
differing said one or two additional nucleotides have different
labels that can be distinguishably detected.
171. The kit of claim 157 wherein said instructions further
comprise: detect such of said fragments digested on each end by a
method comprising staining said fragments with silver, labeling
said fragments with a DNA intercalating dye, or detecting light
emission from a fluorochrome label on said fragments.
172. The kit of claim 157 further comprising: (a) reagents for
performing a cDNA sample preparation step; (b) reagents for
performing a step of digestion by one or more restriction
endonucleases; (c) reagents for performing a ligation step; and (d)
reagents for performing a PCR amplification step.
Description
1. FIELD OF THE INVENTION
[0002] The field of this invention is DNA sequence classification,
identification or determination, and quantification; more
particularly it is the quantitative classification, comparison of
expression, or identification of preferably all DNA sequences or
genes in a sample without performing any sequencing.
2. BACKGROUND
[0003] Over the past ten years, as biological and genomic research
have revolutionized our understanding of the molecular basis of
life, it has become increasingly clear that the temporal and
spatial expression of genes is responsible for all life's
processes, processes occurring in both health and in disease.
Science has progressed from an understanding of how single genetic
defects cause the traditionally recognized hereditary disorders,
such as the thalassemias, to a realization of the importance of the
interaction of multiple genetic defects along with environmental
factors in the etiology of the majority of more complex disorders,
such as cancer. In the case of cancer, current scientific evidence
demonstrates the key causative roles of altered expression of and
multiple defects in several pivotal genes. Other complex diseases
have similar etiology. Thus the more complete and reliable a
correlation that can be established between gene expression and
health or disease states, the better diseases can be recognized,
diagnosed and treated.
[0004] This important correlation is established by the
quantitative determination and classification of DNA expression in
tissue samples, and such a method which is rapid and economical
would be of considerable value. Genomic DNA ("gDNA") sequences are
those naturally occurring DNA sequences constituting the genome of
a cell. The state of gene, or gDNA, expression at any time is
represented by the composition of total cellular messenger RNA
("mRNA"), which is synthesized by the regulated transcription of
gDNA. Complementary DNA ("cDNA") sequences are synthesized by
reverse transcription from mRNA. cDNA from total cellular mRNA also
represents, albeit approximately, gDNA expression in a cell at a
given time. Consequently, rapid and economical detection of all the
DNA sequences in particular cDNA or gDNA samples is desired,
particularly so if such detection was rapid, precise, and
quantitative.
[0005] Heretofore, gene specific DNA analysis techniques have not
been directed to the determination or classification of
substantially all genes in a DNA sample representing total cellular
mRNA and have required some degree of sequencing. Generally,
existing cDNA, and also gDNA, analysis techniques have been
directed to the determination and analysis of one or two known or
unknown genetic sequences at one time. These techniques have used
probes synthesized to specifically recognize by hybridization only
one particular DNA sequence or gene. (See, e.g., Watson et al.,
1992, Recombinant DNA, chap 7, W. H. Freeman, New York.) Further,
adaptation of these methods to the problem of recognizing all
sequences in a sample would be cumbersome and uneconomical.
[0006] One existing method for finding and sequencing unknown genes
starts from an arrayed cDNA library. From a particular tissue or
specimen, mRNA is isolated and cloned into an appropriate vector,
which is then plated in a manner so that the progeny of individual
vectors bearing the clone of one cDNA sequence can be separately
identified. A replica of such a plate is then probed, often with a
labeled DNA oligomer selected to hybridize with the cDNA
representing the gene of interest. Thereby, those colonies bearing
the cDNA of interest are found and isolated, the cDNA harvested and
subject to sequencing. Sequencing can then be done by the Sanger
dideoxy chain termination method (Sanger et al., 1977, "DNA
sequencing with chain terminating inhibitors", Proc. Natl. Acad.
Sci. USA 74(12):5463-5467) applied to inserts so isolated.
[0007] The DNA oligomer probes for the unknown gene used for colony
selection are synthesized to hybridize, preferably, only with the
cDNA for the gene of interest. One manner of achieving this
specificity is to start with the protein product of the gene of
interest. If a partial sequence of 5 to 10-mer peptide fragment
from an active region of this protein can be determined,
corresponding 15 to 30-mer degenerate oligonucleotides can be
synthesized which code for this peptide. This collection of
degenerate oligonucleotides will typically be sufficient to
uniquely identify the corresponding gene. Similarly, any
information leasing to 15 to 30 long nucleotide subsequences can be
used to create a single gene probe.
[0008] Another existing method, which searches for a known gene in
a cDNA or gDNA prepared from a tissue sample, also uses single gene
or single sequence probes which are complementary to unique
subsequences of the already known gene sequences. For example, the
expression of a particular oncogene in sample can be determined by
probing tissue derived cDNA with a probe derived from a subsequence
of the oncogene's expressed sequence tag. Similarly the presence of
a rare or difficult to culture pathogen, such as the TB bacillus or
the HIV, can be determined by probing gDNA with a hybridization
probe specific to a gene of the pathogen. The heterozygous presence
of a mutant allele in a phenotypically normal individual, or its
homozygous presence in a fetus, can be determined by probing with
an allele specific probe complementary only to the mutant allele
(See, e.g., Guo et al., 1994, Nucleic Acid Research,
22:5456-65).
[0009] All existing methods using single gene probes, of which the
preceding examples are typical, if applied to determine all genes
expressed in a given tissue sample, would require many thousands to
tens of thousands of individual probes. It is estimated a single
human cell typically expresses approximately to 15,000 to 15,000
genes simultaneously and that the most complex tissue, e.g. the
brain, can express up to half the human genome (Liang et al., 1992,
"Differential Display of Eukaryotic Messenger RNA by Means of the
Polymerase Chain Reaction, Science, 257:967-971). Such an
application requiring such a number of probes is clearly too
cumbersome to be economic or, even, practical.
[0010] Another class of existing methods, known as sequencing by
hybridization ("SBH"), in contrast, use combinatorial probes which
are not gene specific (Drmanac et al., 1993, Science, 260:1649-52;
U.S. Pat. No. 5,202,231, Apr. 13, 1993, to Drmanac et al). An
exemplary implementation of SBH to determine an unknown gene
requires that a single cDNA clone be probed with all DNA oligomers
of a given length, say, for example, all 6-mers. Such a set of all
oligomers of a given length synthesized without any selection is
called a combinatorial probe library. From knowledge of all
hybridization results for a combinatorial library, say all the 4096
6-mer probe results, a partial DNA sequence for the cDNA clone can
be reconstructed by algorithmic manipulations. Complete sequences
are not determinable because, at least, repeated subsequences
cannot be fully determined. SBH adapted to the classification of
known genes is called oligomer sequence signatures ("OSS") (Lennon
et al., 1991, Trends In Genetics, 7(10):314-317). This technique
classifies a single clone based on the pattern of probe hits
against an entire combinatorial library, or a significant
sub-library. It requires that the tissue sample library be arrayed
into clones, each clone comprising only one pure sequence from the
library. It cannot be applied to mixtures.
[0011] These exemplary existing methods are all directed to finding
one sequence in an array of clones each expressing a single
sequence from a tissue sample. They are not directed to rapid,
economical, quantitative, and precise characterization of all the
DNA sequences in a mixture of sequences, such as a particular total
cellular cDNA or gDNA sample. Their adaptation to such a task would
be prohibitive. Determination by sequencing the DNA of a clone,
much less an entire sample of thousands of sequences, is not rapid
or inexpensive enough for economical and useful diagnostics.
Existing probe-based techniques of gene determination or
classification, whether the genes are known or unknown, require
many thousands of probes, each specific to one possible gene to be
observed, or at least thousands or even tens of thousands of probes
in a combinatorial library. Further, all of these methods require
the sample be arrayed into clones each expressing a single gene of
the sample.
[0012] In contrast to the prior exemplary existing gene
determination and classification techniques, another existing
technique, known as differential display, attempts to fingerprint a
mixture of expressed genes, as is found in a pooled cDNA library.
This fingerprint, however, seeks merely to establish whether two
samples are the same or different. No attempt is made to determine
the quantitative, or even qualitative, expression of particular,
determined genes (Liang et al., 1995, Current Opinions in
Immunology 7:274-280; Liang et al., 1992, Science 257:967-71; Welsh
et al., Nucleic Acid Res., 1992, 20:4965-70; McClelland et al.,
1993, Exs, 67:103-15; Lisitsyn, 1993, Science, 259:946-50).
Differential display uses the polymerase chain reaction ("PCR") to
amplify DNA subsequences of various lengths, which are defined by
being between the hybridization sites of arbitrarily selected
primers. Ideally, the pattern of lengths observed is characteristic
of the tissue from which the library was prepared. Typically, one
primer used in differential display is oligo(dT) and the other is
one or more arbitrary oligonucleotides designed to hybridize within
a few hundred base pairs of the poly-dA tail of a cDNA in the
library. Thereby, on electrophoretic separation, the amplified
fragments of lengths up to a few hundred base pairs should generate
bands characteristic and distinctive of the sample. Changes in
tissue gene expression may be observed as changes in one or more
bands.
[0013] Although characteristic banding patterns develop, no attempt
is made to link these patterns to the expression of particular
genes. The second arbitrary primer cannot be traced to a particular
gene. First, the PCR process is less than ideally specific. One to
a few base pair ("bp") mismatches ("bubbles") are permitted by the
lower stringency annealing step typically used and are tolerated
well enough so that a new chain can be initiated by the Tag
polymerase, often used in PCR reactions. Second, the location of a
single subsequence or its absence is insufficient information to
distinguish all expressed genes. Third, length information from the
arbitrary primer to the poly-dA tail is generally not found to be
characteristic of a sequence due to variations in the processing of
the 3' untranslated regions of genes, the variation in the
poly-adenylation process and variability in priming to the
repetitive sequence at a precise point. Thus, even the bands that
are produced often are smeared by the non-specific background
sequences present. Also known PCR biases to high G+C content and
short sequences further limit the specificity of this method. Thus
this technique is generally limited to "fingerprinting" samples for
a similarity or dissimilarity determination and is precluded from
use in quantitative determination of the differential expression of
identifiable genes.
[0014] Existing methods for gene or DNA sequence classification or
determination are in need of improvement in their ability to
perform rapid and economical as well as quantitative and specific
determination of the components of a cDNA mixture prepared from a
tissue sample. The preceding background review identifies the
deficiencies of several exemplary existing methods.
3. SUMMARY OF THE INVENTION
[0015] It is an object of this invention to provide methods for
rapid, economical, quantitative, and precise determination or
classification of DNA sequences, in particular genomic or
complementary DNA sequences, in either arrays of single sequence
clones or mixtures of sequences such as can be derived from tissue
samples, without actually sequencing the DNA. Thereby, the
deficiencies in the background arts just identified are solved.
This object is realized by generating a plurality of distinctive
and detectable signals from the DNA sequences in the sample being
analyzed. Preferably, all the signals taken together have
sufficient discrimination and resolution so that each particular
DNA sequence in a sample may be individually classified by the
particular signals it generates, and with reference to a database
of DNA sequences possible in the sample, individually determined.
The intensity of the signals indicative of a particular DNA
sequence depends quantitatively on the amount of that DNA present.
Alternatively, the signals together can classify a predominant
fraction of the DNA sequences into a plurality of sets of
approximately no more than two to four individual sequences.
[0016] It is a further object that the numerous signals be
generated from measurements of the results of as few a number of
recognition reactions as possible, preferably no more than
approximately 5-400 reactions, and most preferably no more than
approximately 20-50 reactions. Rapid and economical determinations
would not be achieved if each DNA sequence in a sample containing a
complex mixture required a separate reaction with a unique probe.
Preferably, each recognition reaction generates a large number of
or a distinctive pattern of distinguishable signals, which are
quantitatively proportional to the amount of the particular DNA
sequences present. Further, the signals are preferably detected and
measured with a minimum number of observations, which are
preferably capable of simultaneous performance.
[0017] The signals are preferably optical, generated by
fluorochrome labels and detected by automated optical detection
technologies. Using these methods, multiple individually labeled
moieties can be discriminated even though they are in the same
filter spot or gel band. This permits multiplexing reactions and
parallelizing signal detection. Alternatively, the invention is
easily adaptable to other labeling systems, for example, silver
staining of gels. In particular, any single molecule detection
system, whether optical or by some other technology such as
scanning or tunneling microscopy, would be highly advantageous for
use according to this invention as it would greatly improve
quantitative characteristics.
[0018] According to this invention, signals are generated by
detecting the presence (hereinafter called "hits") or absence of
short DNA subsequences (hereinafter called "target" subsequences)
within a nucleic acid sequence of the sample to be analyzed. The
presence or absence of a subsequence is detected by use of
recognition means, or probes, for the subsequence. The subsequences
are recognized by recognition means of several sorts, including but
not limited to restriction endonucleases ("REs"), DNA oligomers,
and PNA oligomers. REs recognize their specific subsequences by
cleavage thereof; DNA and PNA oligomers recognize their specific
subsequences by hybridization methods. The preferred embodiment
detects not only the presence of pairs of hits in a sample sequence
but also include a representation of the length in base pairs
between adjacent hits. This length representation can be corrected
to true physical length in base pairs upon removing experimental
biases and errors of the length separation and detection means. An
alternative embodiment detects only the pattern of hits in an array
of clones, each containing a single sequence ("single sequence
clones").
[0019] The generated signals are then analyzed together with DNA
sequence information stored in sequence databases in computer
implemented experimental analysis methods of this invention to
identify individual genes and their quantitative presence in the
sample.
[0020] The target subsequences are chosen by further computer
implemented experimental design methods of this invention such that
their presence or absence and their relative distances when present
yield a maximum amount of information for classifying or
determining the DNA sequences to be analyzed. Thereby it is
possible to have orders of magnitude fewer probes than there are
DNA sequences to be analyzed, and it is further possible to have
considerably fewer probes than would be present in combinatorial
libraries of the same length as the probes used in this invention.
For each embodiment, target subsequences have a preferred
probability of occurrence in a sequence, typically between 5% and
50%. In all embodiments, it is preferred that the presence of one
probe in a DNA sequence to be analyzed is independent of the
presence of any other probe.
[0021] Preferably, target subsequences are chosen based on
information in relevant DNA sequence databases that characterize
the sample. A minimum number of target subsequences may be chosen
to determine the expression of all genes in a tissue sample
("tissue mode"). Alternatively, a smaller number of target
subsequences may be chosen to quantitatively classify or determine
only one or a few sequences of genes of interest, for example
oncogenes, tumor suppressor genes, growth factors, cell cycle
genes, cytoskeletal genes, etc ("query mode").
[0022] A preferred embodiment of the invention, named quantitative
expression analysis ("QEA"), produces signals comprising target
subsequence presence and a representation of the length in base
pairs along a gene between adjacent target subsequences by
measuring the results of recognition reactions on cDNA (or gDNA)
mixtures. Of great importance, this method does not require the
cDNA be inserted into a vector to create individual clones in a
library. Creation of these libraries is time consuming, costly, and
introduces bias into the process, as it requires the cDNA in the
vector to be transformed into bacteria, the bacteria arrayed as
clonal colonies, and finally the growth of the individual
transformed colonies.
[0023] Three exemplary experimental methods are described herein
for performing QEA: a preferred method utilizing a novel
RE/ligase/amplification procedure; a PCR based method; and a method
utilizing a removal means, preferably biotin, for removal of
unwanted DNA fragments. The preferred method generates precise,
reproducible, noise free signatures for determining individual gene
expression from DNA in mixtures or libraries and is uniquely
adaptable to automation, since it does not require intermediate
extractions or buffer exchanges. A computer implemented gene
calling step uses the hit and length information measured in
conjunction with a database of DNA sequences to determine which
genes are present in the sample and the relative levels of
expression. Signal intensities are used to determine relative
amounts of sequences in the sample. Computer implemented design
methods optimize the choice of the target subsequences.
[0024] A second specific embodiment of the invention, termed colony
calling ("CC"), gathers only target subsequence presence
information for all target subsequences for arrayed, individual
single sequence clones in a library, with cDNA libraries being
preferred. The target subsequences are carefully chosen according
to computer implemented design methods of this invention to have a
maximum information content and to be minimum in number. Preferably
from 10-20 subsequences are sufficient to characterize the
expressed cDNA in a tissue. In order to increase the specificity
and reliability of hybridization to the typically short DNA
subsequences, preferable recognition means are PNAs. Degenerate
sets of longer DNA oligomers having a common, short, shared, target
sequence can also be used as a recognition means. A computer
implemented gene calling step uses the pattern of hits in
conjunction with a database of DNA sequences to determine which
genes are present in the sample and the relative levels of
expression.
[0025] The embodiments of this invention preferably generate
measurements that are precise, reproducible, and free of noise.
Measurement noise in QEA is typically created by generation or
amplification of unwanted DNA fragments, and special steps are
preferably taken to avoid any such unwanted fragments. Measurement
noise in colony calling is typically created by mis-hybridization
of probes, or recognition means, to colonies. High stringency
reaction conditions and DNA mimics with increased hybridization
specificity may be used to minimize this noise. DNA mimics are
polymers composed of subunits capable of specific,
Watson-Crick-like hybridization with DNA. Also useful to minimize
noise in colony calling are improved hybridization detection
methods. Instead of the conventional detection methods based on
probe labeling with fluorochromes, new methods are based on light
scattering by small 100-200 .mu.m particles that are aggregated
upon probe hybridization (Stimson et al., 1995, "Real-time
detection of DNA hybridization and melting on oligonucleotide
arrays by using optical wave guides", Proc. Natl. Acad. Sci. USA,
92:6379-6383). In this method, the hybridization surface forms one
surface of a light pipe or optical wave guide, and the scattering
induced by these aggregated particles causes light to leak from the
light pipe. In this manner hybridization is revealed as an
illuminated spot of leaking light on a dark background. This latter
method makes hybridization detection more rapid by eliminating the
need for a washing step between the hybridization and detection
steps. Further by using variously sized and shaped particles with
different light scattering properties, multiple probe
hybridizations can be detected from one colony.
[0026] Further, the embodiments of the invention can be adapted to
automation by eliminating non-automatable steps, such as
extractions or buffer exchanges. The embodiments of the invention
facilitate efficient analysis by permitting multiple recognition
means to be tested in one reaction and by utilizing multiple,
distinguishable labeling of the recognition means, so that signals
may be simultaneously detected and measured. Preferably, for the
QEA embodiments, this labeling is by multiple fluorochromes. For
the CC embodiments, detection is preferably done by the light
scattering methods with variously sized and shaped particles.
[0027] An increase in sensitivity as well as an increase in the
number of resolvable fluorescent labels can be achieved by the use
of fluorescent, energy transfer, dye-labeled primers. Other
detection methods, preferable when the genes being identified will
be physically isolated from the gel for later sequencing or use as
experimental probes, include the use of silver staining gels or of
radioactive labeling. Since these methods do not allow for multiple
samples to be run in a single lane, they are less preferable when
high throughput is needed.
[0028] Because this invention achieves rapid and economical
determination of quantitative gene expression in tissue or other
samples, it has considerable medical and research utility. In
medicine, as more and more diseases are recognized to have
important genetic components to their etiology and development, it
is becoming increasingly useful to be able to assay the genetic
makeup and expression of a tissue sample. For example, the presence
and expression of certain genes or their particular alleles are
prognostic or risk factors for disease (including disorders).
Several examples of such diseases are found among the
neurodegenerative diseases, such as Huntington's disease and
ataxia-telangiectasia. Several cancers, such as neuroblastoma, can
now be linked to specific genetic defects. Finally, gene expression
can also determine the presence and classification of those foreign
pathogens that are difficult or impossible to culture in vitro but
which nevertheless express their own unique genes.
[0029] Disease progression is reflected in changes in genetic
expression of an affected tissue. For example, expression of
particular tumor promoter genes and lack of expression of
particular tumor suppressor genes is now known to correlate with
the progression of certain tumors from normal tissue, to
hyperplasia, to cancer in situ, and to metastatic cancer. Return of
a cell population to a normal pattern of gene expression, such as
by using anti-sense technology, can correlate with tumor
regression. Therefore, knowledge of gene expression in a cancerous
tissue can assist in staging and classifying this disease.
[0030] Expression information can also be used to chose and guide
therapy. Accurate disease classification and staging or grading
using gene expression information can assist in choosing initial
therapies that are increasingly more precisely tailored to the
precise disease process occurring in the particular patient. Gene
expression information can then track disease progression or
regression, and such information can assist in monitoring the
success or changing the course of an initial therapy. A therapy is
favored that results in a regression towards normal of an abnormal
pattern of gene expression in an individual, while therapy which
has little effect on gene expression or its progression can need
modification. Such monitoring is now useful for cancers and will
become useful for an increasing number of other diseases, such as
diabetes and obesity. Finally, in the case of direct gene therapy,
expression analysis directly monitors the success of treatment.
[0031] In biological research, rapid and economical assay for gene
expression in tissue or other samples has numerous applications.
Such applications include, but are not limited to, for example, in
pathology examining tissue specific genetic response to disease, in
embryology determining developmental changes in gene expression, in
pharmacology assessing direct and indirect effects of drugs on gene
expression. In these applications, this invention can be applied,
e.g., to in vitro cell populations or cell lines, to in vivo animal
models of disease or other processes, to human samples, to purified
cell populations perhaps drawn from actual wild-type occurrences,
and to tissue samples containing mixed cell populations. The cell
or tissue sources can advantageously be a plant, a single celled
animal, a multicellular animal, a bacterium, a virus, a fungus, or
a yeast, etc. The animal can advantageously be laboratory animals
used in research, such as mice engineered or bread to have certain
genomes or disease conditions or tendencies. The in vitro cell
populations or cell lines can be exposed to various exogenous
factors to determine the effect of such factors on gene expression.
Further, since an unknown signal pattern is indicative of an as yet
unknown gene, this invention has important use for the discovery of
new genes. In medical research, by way of further example, use of
the methods of this invention allow correlating gene expression
with the presence and progress of a disease and thereby provide new
methods of diagnosis and new avenues of therapy which seek to
directly alter gene expression.
[0032] This invention includes various embodiments and aspects,
several of which are described below.
[0033] In a first embodiment, the invention provides a method for
identifying, classifying, or quantifying one or more nucleic acids
in a sample comprising a plurality of nucleic acids having
different nucleotide sequences, said method comprising probing said
sample with one or more recognition means, each recognition means
recognizing a different target nucleotide subsequence or a
different set of target nucleotide subsequences; generating one or
more signals from said sample probed by said recognition means,
each generated signal arising from a nucleic acid in said sample
and comprising a representation of (i) the length between
occurrences of target subsequences in said nucleic acid and (ii)
the identities of said target subsequences in said nucleic acid or
the identities of said sets of target subsequences among which is
included the target subsequences in said nucleic acid; and
searching a nucleotide sequence database to determine sequences
that match or the absence of any sequences that match said one or
more generated signals, said database comprising a plurality of
known nucleotide sequences of nucleic acids that may be present in
the sample, a sequence from said database matching a generated
signal when the sequence from said database has both (i) the same
length between occurrences of target subsequences as is represented
by the generated signal and (ii) the same target subsequences as is
represented by the generated signal, or target subsequences that
are members of the same sets of target subsequences represented by
the generated signal, whereby said one or more nucleic acids in
said sample are identified, classified, or quantified.
[0034] This invention further provides in the first embodiment
additional methods wherein each recognition means recognizes one
target subsequence, and wherein a sequence from said database
matches a generated signal when the sequence from said database has
both the same length between occurrences of target subsequences as
is represented by the generated signal and the same target
subsequences as represented by the generated signal, or optionally
wherein each recognition means recognizes a set of target
subsequences, and wherein a sequence from said database matches a
generated signal when the sequence from said database has both the
same length between occurrences of target subsequences as is
represented by the generated signal, and target subsequences that
are members of the sets of target subsequences represented by the
generated signal.
[0035] This invention further provides in the first embodiment
additional methods further comprising dividing said sample of
nucleic acids into a plurality of portions and performing the
methods of this object individually on a plurality of said
portions, wherein a different one or more recognition means are
used with each portion.
[0036] This invention further provides in the first embodiment
additional methods wherein the quantitative abundance of a nucleic
acid comprising a particular nucleotide sequence in the sample is
determined from the quantitative level of the one or more signals
generated by said nucleic acid that are determined to match said
particular nucleotide sequence.
[0037] This invention further provides in the first embodiment
additional methods wherein said plurality of nucleic acids are DNA,
and optionally wherein the DNA is cDNA, and optionally wherein the
cDNA is prepared from a plant, an single celled animal, a
multicellular animal, a bacterium, a virus, a fungus, or a yeast,
and optionally herein the cDNA is of total cellular RNA or total
cellular poly(A) RNA.
[0038] This invention further provides in the first embodiment
additional methods wherein said database comprises substantially
all the known expressed sequences of said plant, single celled
animal, multicellular animal, bacterium, or yeast.
[0039] This invention further provides in the first embodiment
additional methods wherein the recognition means are one or more
restriction endonucleases whose recognition sites are said target
subsequences, and wherein the step of robing comprises digesting
said sample with said one or more restriction endonucleases into
fragments and ligating double stranded adapter DNA molecules to
said fragments to produce ligated fragments, each said adapter DNA
molecule comprising (i) a shorter stand having no 5' terminal
phosphates and consisting of a first and second portion, said first
portion at the 5' end of the shorter strand being complementary to
the overhang produced by one of said restriction endonucleases and
(ii) a longer strand having a 3' end subsequence complementary to
said second portion of the shorter strand; and wherein the step of
generating further comprises melting the shorter strand from the
ligated fragments, contacting the sample with a DNA polymerase,
extending the ligated fragments by synthesis with the DNA
polymerase to produce blunt-ended double stranded DNA fragments,
and amplifying the blunt-ended fragments by a method comprising
contacting said blunt-ended fragments with a DNA polymerase and
primer oligodeoxynucleotides, said primer oligodeoxynucleotides
comprising the longer adapter strand, and said contacting being at
a temperature not greater than the melting temperature of the
primer oligodeoxynucleotide from a strand of the blunt-ended
fragments complementary to the primer oligodeoxynucleotide and not
less than the melting temperature of the shorter strand of the
adapter nucleic acid from the blunt-ended fragments.
[0040] This invention further provides in the first embodiment
additional methods wherein the recognition means are one or more
restriction endonucleases whose recognition sites are said target
subsequences, and wherein the step of probing further comprises
digesting the sample with said one or more restriction
endonucleases.
[0041] This invention further provides in the first embodiment
additional methods further comprising identifying a fragment of a
nucleic acid in the sample which generates said one or more
signals; and recovering said fragment, and optionally wherein the
signals generated by said recovered fragment do not match a
sequence in said nucleotide sequence database, and optionally
further comprising using at least a hybridizable portion of said
fragment as a hybridization probe to bind to a nucleic acid that
can generate said fragment upon digestion by said one or more
restriction endonucleases.
[0042] This invention further provides in the first embodiment
additional methods wherein the step of generating further comprises
after said digesting removing from the sample both nucleic acids
which have not been digested and nucleic acid fragments resulting
from digestion at only a single terminus of the fragments, and
optionally wherein prior to digesting, the nucleic acids in the
sample are each bound at one terminus to a biotin molecule or to a
hapten molecule, and said removing is carried out by a method which
comprises contacting the nucleic acids in the sample with
streptavidin or avidin or with an anti-hapten antibody,
respectively, affixed to a solid support.
[0043] This invention further provides in the first embodiment
additional methods wherein said digesting with said one or more
restriction endonucleases leaves single-stranded nucleotide
overhangs on the digested ends.
[0044] This invention further provides in the first embodiment
additional methods wherein the step of probing further comprises
hybridizing double-stranded adapter nucleic acids with the digested
sample fragments, each said adapter nucleic acid having an end
complementary to said overhang generated by a particular one of the
one or more restriction endonucleases, and ligating with a ligase a
strand of said adapter nucleic acids to the 5' end of a strand of
the digested sample fragments to form ligated nucleic acid
fragments.
[0045] This invention further provides in the first embodiment
additional methods wherein said digesting with said one or more
restriction endonucleases and said ligating are carried out in the
same reaction medium, and optionally wherein said digesting and
said ligating comprises incubating said reaction medium at a first
temperature and then at a second temperature, in which said one or
more restriction endonucleases are more active at the first
temperature than the second temperature and said ligase is more
active at the second temperature that the first temperature, or
wherein said incubating at said first temperature and said
incubating at said second temperature are performed
repetitively.
[0046] This invention further provides in the first embodiment
additional methods wherein the step of probing further comprises
prior to said digesting removing terminal phosphates from DNA in
said sample by incubation with an alkaline phosphatase, and
optionally wherein said alkaline phosphatase is heat labile and is
heat inactivated prior to said digesting.
[0047] This invention further provides in the first embodiment
additional methods wherein said generating step comprises
amplifying the ligated nucleic acid fragments, and optionally
wherein said amplifying is carried out by use of a nucleic acid
polymerase and primer nucleic acid strands, said primer nucleic
acid strands being capable of priming nucleic acid synthesis by
said polymerase, and optionally wherein the primer nucleic acid
strands have a G+C content of between 40% and 60%.
[0048] This invention further provides in the first embodiment
additional methods wherein each said adapter nucleic acid has a
shorter strand and a longer strand, the longer strand being ligated
to the digested sample fragments, and said generating step
comprises prior to said amplifying step the melting of the shorter
strand from the ligated fragments, contacting the ligated fragments
with a DNA polymerase, extending the ligated fragments by synthesis
with the DNA polymerase to produce blunt-ended double stranded DNA
fragments, and wherein the primer nucleic acid strands comprise a
hybridizable portion the sequence of said longer strands, or
optionally comprise the sequence of said longer strands, each
different primer nucleic acid strand priming amplification only of
blunt ended double stranded DNA fragments that are produced after
digestion by a particular restriction endonuclease.
[0049] This invention further provides in the first embodiment
additional methods wherein each primer nucleic acid strand is
specific for a particular restriction endonuclease, and further
comprises at the 3' end of and contiguous with the longer strand
sequence the portion of the restriction endonuclease recognition
site remaining on a nucleic acid fragment terminus after digestion
by the restriction endonuclease, or optionally wherein each said
primer specific for a particular restriction endonuclease further
comprises at its 3' end one or more nucleotides 3' to and
contiguous with the remaining portion of the restriction
endonuclease recognition site, whereby the ligated nucleic acid
fragment amplified is that comprising said remaining portion of
said restriction endonuclease recognition site contiguous to said
one or more additional nucleotides, and optionally such that said
primers comprising a particular said one or more additional
nucleotides can be distinguishably detected from said primers
comprising a different said one or more additional nucleotides.
[0050] This invention further provides in the first embodiment
additional methods wherein during said amplifying step the primer
nucleic acid strands are annealed to the ligated nucleic acid
fragments at a temperature that is less than the melting
temperature of the primer nucleic acid strands from strands
complementary to the primer nucleic acid strands but greater than
the melting temperature of the shorter adapter strands from the
blunt-ended fragments.
[0051] This invention further provides in the first embodiment
additional methods wherein the recognition means are oligomers of
nucleotides, nucleotide-mimics, or a combination of nucleotides and
nucleotide-mimics, which are specifically hybridizable with the
target subsequences, and optionally further provides additional
methods wherein the step of generating comprises amplifying with a
nucleic acid polymerase and with primers comprising said oligomers,
whereby fragments of nucleic acids in the sample between hybridized
oligomers are amplified.
[0052] This invention further provides in the first embodiment
additional methods wherein said signals further comprise a
representation of whether an additional target subsequence is
present on said nucleic acid in the sample between said occurrences
of target subsequences, and optionally wherein said additional
target subsequence is recognized by a method comprising contacting
nucleic acids in the sample with oligomers of nucleotides,
nucleotide-mimics, or mixed nucleotides and nucleotide-mimics,
which are hybridizable with said additional target subsequence.
[0053] This invention further provides in the first embodiment
additional methods wherein the step of generating comprises
suppressing said signals when an additional target subsequence is
present on said nucleic acid in the sample between said occurrences
of target subsequences, and optionally wherein, when the step of
generating comprises amplifying nucleic acids in the sample, said
additional target subsequence is recognized by a method comprising
contacting nucleic acids in the sample with (a) oligomers of
nucleotides, nucleotide-mimics, or mixed nucleotides and
nucleotide-mimics, which hybridize with said additional target
subsequence and disrupt the amplifying step; or (b) restriction
endonucleases which have said additional target subsequence as a
recognition site and digest the nucleic acids in the sample at the
recognition site.
[0054] This invention further provides in the first embodiment
additional methods wherein the step of generating further comprises
separating nucleic acid fragments by length, and optionally wherein
the step of generating further comprises detecting said separated
nucleic acid fragments, and optionally wherein said detecting is
carried out by a method comprising staining said fragments with
silver, labeling said fragments with a DNA intercalating dye, or
detecting light emission from a fluorochrome label on said
fragments.
[0055] This invention further provides in the first embodiment
additional methods wherein said representation of the length
between occurrences of target subsequences is the length of
fragments determined by said separating and detecting steps.
[0056] This invention further provides in the first embodiment
additional methods wherein said separating is carried out by use of
liquid chromatography, mass spectrometry, or electrophoresis, and
optionally wherein said electrophoresis is carried out in a slab
gel or capillary configuration using a denaturing or non-denaturing
medium.
[0057] This invention further provides in the first embodiment
additional methods wherein a predetermined one or more nucleotide
sequences in said database are of interest, and wherein the target
subsequences are such that said sequences of interest generate at
least one signal that is not generated by any other sequence likely
to be present in the sample, and optionally wherein the nucleotide
sequences of interest are a majority of sequences in said
database.
[0058] This invention further provides in the first embodiment
additional methods wherein the target subsequences have a
probability of occurrence in the nucleotide sequences in said
database of from approximately 0.01 to approximately 0.30.
[0059] This invention further provides in the first embodiment
additional methods wherein the target subsequences are such that
the majority of sequences in said database contain on average a
sufficient number of occurrences of target subsequences in order to
on average generate a signal that is not generated by any other
nucleotide sequence in said database, and optionally wherein the
number of pairs of target subsequences present on average in the
majority of sequences in said database is no less than 3, and
wherein the average number of signals generated from the sequences
in said database is such that the average difference between
lengths represented by the generated signals is greater than or
equal to 1 base pair.
[0060] This invention further provides in the first embodiment
additional methods wherein the target subsequences have a
probability of occurrence, p, approximately given by the solution
of 1 R ( R + 1 ) p 2 2 = A
[0061] and 2 L Np 2 = B
[0062] wherein N=the number of different nucleotide sequences in
said database; L=the average length of said different nucleotide
sequences in said database; R=the number of recognition means;
A=the number of pairs of target subsequences present on average in
said different nucleotide sequences in said database; and B=the
average difference between lengths represented by the signals
generated from the nucleic acids in the sample, and optionally
wherein A is greater than or equal to 3 and wherein B is greater
than or equal to 1.
[0063] This invention further provides in the first embodiment
additional methods wherein the target subsequences are selected
according to the further steps comprising determining a pattern of
signals that can be generated and the sequences capable of
generating each such signal by simulating the steps of probing and
generating applied to each sequences in said database of nucleotide
sequences; ascertaining the value of said determined pattern
according to an information measure; and choosing the target
subsequences in order to generate a new pattern that optimizes the
information measure, and optionally wherein said choosing step
selects target subsequences which comprise the recognition sites of
the one or more restriction endonucleases, and optionally wherein
said choosing step selects target subsequences which comprise the
recognition sites of the one or more restriction endonucleases
contiguous with one or more additional nucleotides.
[0064] This invention further provides in the first embodiment
additional methods wherein a predetermined one or more of the
nucleotide sequences present in said database of nucleotide
sequences are of interest, and the information measure optimized is
the number of such said sequences of interest which generate at
least one signal that is not generated by any other nucleotide
sequence present in said database, and optionally wherein said
nucleotide sequences of interest are a majority of the nucleotide
sequences present in said database.
[0065] This invention further provides in the first embodiment
additional methods wherein said choosing step is by exhaustive
search of all combinations of target subsequences of length less
than approximately 10, or wherein said step of choosing target
subsequences is by a method comprising simulated annealing.
[0066] This invention further provides in the first embodiment
additional methods wherein the step of searching further comprises
determining a pattern of signals that can be generated and the
sequences capable of generating each such signal by simulating the
steps of probing and generating applied to each sequence in said
database of nucleotide sequences; and finding the one or more
nucleotide sequences in said database that are able to generate
said one or more generated signals by finding in said pattern those
signals that comprise a representation of the (i) the same lengths
between occurrences of target subsequences as is represented by the
generated signal and (ii) the same target subsequences as is
represented by the generated signal, or target subsequences that
are members of the same sets of target subsequences represented by
the generated signal.
[0067] This invention further provides in the first embodiment
additional methods wherein the step of determining further
comprises searching for occurrences of said target subsequences or
sets of target subsequences in nucleotide sequences in said
database of nucleotide sequences; finding the lengths between
occurrences of said target subsequences or sets of target
subsequences in the nucleotide sequences of said database; and
forming the pattern of signals that can be generated from the
sequences of said database in which the target subsequences were
found to occur.
[0068] This invention further provides in the first embodiment
additional methods wherein said restriction endonucleases generate
5' overhangs at the terminus of digested fragments and wherein each
double stranded adapter nucleic acid comprises a shorter nucleic
acid strand consisting of a first and second contiguous portion,
said first portion being a 5' end subsequence complementary to the
overhang produced by one of said restriction endonucleases; and a
longer nucleic acid strand having a 3' end subsequence
complementary to said second portion of the shorter strand.
[0069] This invention further provides in the first embodiment
additional methods wherein said shorter strand has a melting
temperature from a complementary strand of less than approximately
68.degree. C., and has no terminal phosphate, and optionally
wherein said shorter strand is approximately 12 nucleotides
long.
[0070] This invention further provides in the first embodiment
additional methods wherein said longer strand has a melting
temperature from a complementary strand of greater than
approximately 68.degree. C., is not complementary to any nucleotide
sequence in said database, and has no terminal phosphate, and
optionally wherein said ligated nucleic acid fragments do not
contain a recognition site for any of said restriction
endonucleases, and optionally wherein said longer strand is
approximately 24 nucleotides long and has a G+C content between 40%
and 60%.
[0071] This invention further provides in the first embodiment
additional methods wherein said one or more restriction
endonucleases are heat inactivated before said ligating.
[0072] This invention further provides in the first embodiment
additional methods wherein said restriction endonucleases generate
3' overhangs at the terminus of the digested fragments and wherein
each double stranded adapter nucleic acid comprises a longer
nucleic acid strand consisting of a first and second contiguous
portion, said first portion being a 3' end subsequence
complementary to the overhang produced by one of said restriction
endonucleases; and a shorter nucleic acid strand complementary to
the 3' end of said second portion of the longer nucleic acid
stand.
[0073] This invention further provides in the first embodiment
additional methods wherein said shorter strand has a melting
temperature from said longer strand of less than approximately
68.degree. C., and has no terminal phosphates, and optionally
wherein said shorter strand is 12 base pairs long.
[0074] This invention further provides in the first embodiment
additional methods wherein said longer strand has a melting
temperature from a complementary strand of greater than
approximately 68.degree. C., is not complementary to any nucleotide
sequence in said database, has no terminal phosphate, and wherein
said ligated nucleic acid fragments do not contain a recognition
site for any of said restriction endonucleases, and optionally
wherein said longer strand is 24 base pairs long and has a G+C
content between 40% and 60%.
[0075] In a second embodiment, the invention provides a method for
identifying or classifying a nucleic acid comprising probing said
nucleic acid with a plurality of recognition means, each
recognition means recognizing a target nucleotide subsequence or a
set of target nucleotide subsequences, in order to generate a set
of signals, each signal representing whether said target
subsequence or one of said set of target subsequences is present or
absent in said nucleic acid; and searching a nucleotide sequence
database, said database comprising a plurality of known nucleotide
sequences of nucleic acids that may be present in the sample, for
sequences matching said generated set of signals, a sequence from
said database matching a set of signals when the sequence from said
database (i) comprises the same target subsequences as are
represented as present, or comprises target subsequences that are
members of the sets of target subsequences represented as present
by the generated sets of signals and (ii) does not comprise the
target subsequences represented as absent or that are members of
the sets of target subsequences represented as absent by the
generated sets of signals, whereby the nucleic acid is identified
or classified, and optionally wherein the set of signals are
represented by a hash code which is a binary number.
[0076] This invention further provides in the second embodiment
additional methods wherein the step of probing generates
quantitative signals of the numbers of occurrences of said target
subsequences or of members of said set of target subsequences in
said nucleic acid, and optionally wherein a sequence matches said
generated set of signals when the sequence from said database
comprises the same target subsequences with the same number of
occurrences in said sequence as in the quantitative signals and
does not comprise the target subsequences represented as absent or
target subsequences within the sets of target subsequences
represented as absent.
[0077] This invention further provides in the second embodiment
additional methods wherein said plurality of nucleic acids are
DNA.
[0078] This invention further provides in the second embodiment
additional methods wherein the recognition means are detectably
labeled oligomers of nucleotides, nucleotide-mimics, or
combinations of nucleotides and nucleotide-mimics, and the step of
probing comprises hybridizing said nucleic acid with said
oligomers, and optionally wherein said detectably labeled oligomers
are detected by a method comprising detecting light emission from a
fluorochrome label on said oligomers or arranging said labeled
oligomers to cause light to scatter from a light pipe and detecting
said scattering, and optionally wherein the recognition means are
oligomers of peptido-nucleic acids, and optionally wherein the
recognition means are DNA oligomers, DNA oligomers comprising
universal nucleotides, or sets of partially degenerate DNA
oligomers.
[0079] This invention further provides in the second embodiment
additional methods wherein the step of searching further comprises
determining a pattern of sets of signals of the presence or absence
of said target subsequences or said sets of target subsequences
that can be generated and the sequences capable of generating each
set of signals in said pattern by simulating the step of probing as
applied to each sequence in said database of nucleotide sequences;
and finding one or more nucleotide sequences that are capable of
generating said generated set of signals by finding in said pattern
those sets that match said generated set, where a set of signals
from said pattern matches a generated set of signals when the set
from said pattern (i) represents as present the same target
subsequences as are represented as present or target subsequences
that are members of the sets of target subsequences represented as
present by the generated sets of signals and (ii) represents as
absent the target subsequences represented as absent or that are
members of the sets of target subsequences represented as absent by
the generated sets of signals.
[0080] This invention further provides in the second embodiment
additional methods wherein the target subsequences are selected
according to the further steps comprising determining (i) a pattern
of sets of signals representing the presence or absence of said
target subsequences or of said sets of target subsequences that can
be generated, and (ii) the sequences capable of generating each set
of signals in said pattern by simulating the step of probing as
applied to each sequence in said database of nucleotide sequences;
ascertaining the value of said pattern generated according to an
information measure; and choosing the target subsequences in order
to generate a new pattern that optimizes the information
measure.
[0081] This invention further provides in the second embodiment
additional methods wherein the information measure is the number of
sets of signals in the pattern which are capable of being generated
by one or more sequences in said database, or optionally wherein
the information measure is the number of sets of signals in the
pattern which are capable of being generated by only one sequence
in said database.
[0082] This invention further provides in the second embodiment
additional methods wherein said choosing step is by a method
comprising exhaustive search of all combination of target
subsequences of length less than approximately 10, or optionally
wherein said choosing step is by a method comprising simulated
annealing.
[0083] This invention further provides in the second embodiment
additional methods wherein the step of determining by simulating
further comprises searching for the presence or absence of said
target subsequences or sets of target subsequences in each
nucleotide sequence in said database of nucleotide sequences; and
forming the pattern of sets of signals that can be generated from
said sequences in said database, and optionally where the step of
searching is carried out by a string search, and optionally wherein
the step of searching comprises counting the number of occurrences
of said target subsequences in each nucleotide sequence.
[0084] This invention further provides in the second embodiment
additional methods wherein the target subsequences have a
probability of occurrence in a nucleotide sequence in said database
of nucleotide sequences of from 0.01 to 0.6, or optionally wherein
the target subsequences are such that the presence of one target
subsequence in a nucleotide sequence in said database of nucleotide
sequences is substantially independent of the presence of any other
target subsequence in the nucleotide sequence, or optionally
wherein fewer than approximately 50 target subsequences are
selected.
[0085] In a third embodiment, the invention provides a programmable
apparatus for analyzing signals comprising an inputting device for
inputting one or more actual signals generated by probing a sample
comprising a plurality of nucleic acids with recognition means,
each recognition means recognizing a target nucleotide subsequence
or a set of target nucleotide subsequences, said signals comprising
a representation of (i) the length between occurrences of said
target subsequences in a nucleic acid of said sample, and (ii) the
identities of said target subsequences in said nucleic acid, or the
identities of said sets of target subsequences among which is
included the target subsequences in said nucleic acid; a searching
device operatively coupled to said accepting device for searching a
sequence in a nucleotide sequence database for occurrences of said
target subsequences or target subsequences that are members of said
sets of target subsequences, and for the length between such
occurrences, said database comprising a plurality of known
nucleotide sequences that may be present in said sample; a
comparing device operatively coupled to said accepting device and
to said searching device for finding a match between said one or
more actual signals and a sequence in said database, said one or
more actual signals matching a sequence from said database when the
sequence from said database has both (i) the same length between
occurrences of target subsequences as is represented by said one or
more actual signals and (ii) the same target subsequences as is
represented by said one or more actual signals or target
subsequences that are members of the same sets of target
subsequences represented by said one or more actual signals; and a
control device operatively coupled to said comparing device for
causing said comparing to be done for sequences in the database and
for outputting those database sequences that match said one or more
actual signals, and optionally wherein said searching device
searches for said target subsequences or a set of target nucleotide
subsequences in said database sequences by performing a string
comparison of the nucleotides in said subsequences with those in
said database sequence.
[0086] This invention further provides in the third embodiment that
said control device further comprises causing said searching device
to search substantially all sequences in said database in order to
determine a pattern of signals that can be generated by probing
said sample with said recognition means, and wherein said control
device further causes said comparing device to find any matches
between said one or more actual signals and said pattern of
signals, said one or more actual signals matching a signal in said
pattern of signals when the signal from said pattern represents (i)
the same length between occurrences of target subsequences as is
represented by said one or more actual signals and (ii) the same
target subsequences as is represented by said one or more actual
signals or target subsequences that are members of the same sets of
target subsequences represented by said one or more actual
signals.
[0087] This invention further provides in the third embodiment that
said sample of nucleic acids comprises cDNA from RNA of a cell or
tissue type, and said database comprises DNA sequences that are
likely to be expressed by d cell or tissue type.
[0088] This invention further provides in the third embodiment a
computer readable memory that can be used to direct a programmable
apparatus to function for analyzing signals according to steps
comprising inputting one or more actual signals generated by
probing a sample comprising a plurality of nucleic acids with
recognition means, each recognition means recognizing a target
nucleotide subsequence or a set of target nucleotide subsequences,
said signals comprising a representation of (i) the length between
occurrences of said target subsequences in a nucleic acid of said
sample, and (ii) the identities of said target subsequences in said
nucleic acid, or the identities of said sets of target subsequences
among which is included the target subsequences in said nucleic
acid; searching a sequence in a nucleotide sequence database for
occurrences of said target subsequences or target subsequences that
are members of said sets of target subsequences, and for the length
between such occurrences, said database comprising a plurality of
known nucleotide sequences that may be present in said sample;
matching said one or more actual signals and a sequence in said
database when the sequence in said database has both (i) the same
length between occurrences of target subsequences as is represented
by said one or more actual signals and (ii) the same target
subsequences as is represented by said one or more actual signals,
or target subsequences that are members of the same sets of target
subsequences as is represented by said one or more actual signals;
and repetitively performing said searching and matching steps for
the majority of sequences in the database and outputting those
database sequences that match said one or more actual signals, or
alternatively a computer readable memory for directing a
programmable apparatus to function in the manner of the third
object.
[0089] In a fourth embodiment, the invention provides a
programmable apparatus for selecting target subsequences comprising
an initial selection device for selecting initial target
subsequences or initial sets of target subsequences; a first
control device; a search device operatively coupled to said initial
selection device and to said first control device (i) for searching
sequences in a nucleotide sequence database for occurrences of said
initial target subsequences or occurrences of target subsequences
that are members of said initial sets of target subsequences and
for the length between such occurrences and (ii) for determining an
initial pattern of signals that can be generated from said selected
initial target subsequences or said initial sets of target
subsequences, said database comprising a plurality of known
nucleotide sequences, said signals comprising a representation of
(i) the length between said occurrences in a sequence in said
database, and (ii) the identities of said initial target
subsequences that occur in said sequence in said database, or the
identities of target subsequences that are members of the same
initial sets of target subsequences that occur in said sequence in
said database; and an ascertaining device operatively coupled to
said searching device and to said first control device for
ascertaining the value of said determined initial pattern according
to an information measure; and wherein said first control device
causes further target subsequences to be selected and causes the
search device to determine a further pattern of signals and the
ascertaining device to ascertain a further value of said
information measure and accepts the further target subsequences
when said further pattern optimizes said further value of said
information measure.
[0090] This invention further provides in the fourth object that a
predetermined one or more of the sequences in said database are of
interest, and wherein said ascertaining device ascertains the value
of an information measure by counting the number of such sequences
of interest which generate in said determined pattern at least one
signal that is not generated by any other sequence in said
database, and optionally that said one or more of the sequences of
interest comprise substantially all the sequences in said
database.
[0091] This invention further provides in the fourth embodiment
that said first control device optimizes the value of said
information measure according to a method of exhaustive search,
wherein said first control device selects further target
subsequences of length less than approximately 10 and accepts the
further target subsequences if said further value of said
information measure is greater than the previous value.
[0092] This invention further provides in the fourth embodiment
that said first control device optimizes the value of said
information measure according to a method comprising simulated
annealing, wherein said first control device repeatedly selects
further target subsequences and accepts the further target
subsequences if said further value of said information measure is
not decreased by greater than a probabilistic factor dependent on a
simulated-temperature, and wherein said programmable apparatus
further comprises a second control device operatively coupled to
said first control device for decreasing said simulated-temperature
as said first control device selects further target subsequences,
and optionally wherein said probabilistic factor is an exponential
function of the negative of the decrease in the information measure
divided by said simulated-temperature.
[0093] This invention further provides in the fourth embodiment
that the database comprises a majority of known DNA sequences that
are likely to be expressed by one or more cell types.
[0094] This invention further provides in the fourth embodiment a
computer readable memory that can be used to direct a programmable
apparatus to function for selecting target subsequences according
to steps comprising selecting initial target subsequences or
initial sets of target subsequences; searching a sequence in a
nucleotide sequence database for occurrences of said initial target
subsequences or occurrences of target subsequences that are members
of said initial sets of target subsequences and for the length
between such occurrences, said database comprising a plurality of
known nucleotide sequences that may be present in said sample;
determining an initial pattern of signals that can be generated
from said selected initial target subsequences or said initial sets
of target subsequences, said signals comprising a representation of
(i) the length between said occurrences in a sequence in said
database, and (ii) the identities of said initial target
subsequences that occur in said sequence in said database, or the
identities of target subsequences that are members of the initial
sets of target subsequences that occur in said sequence in said
database; ascertaining the value of said determined initial pattern
according to an information measure; and repetitively performing
said selecting, searching, determining, and ascertaining steps to
determine a further pattern of signals and a further value of said
information measure, and accepting the further target subsequences
when said further pattern optimizes said further value of said
information measure, or alternatively a computer readable memory
for directing a programmable apparatus to function in the manner of
the fourth object.
[0095] In a fifth embodiment, the invention provides a programmable
apparatus for displaying data comprising a selecting device for
selecting target subsequences or sets of target subsequences, such
that recognition means for recognizing said target subsequences or
said sets of target subsequences can be used to generate signals by
probing a sample comprising a plurality of nucleic acids, said
signals comprising a representation of (i) the length between
occurrences of said target subsequences in a nucleic acid of said
sample and (ii) the identities of said target subsequences in said
nucleic acid or the identities of said sets of target subsequences
among which are included the target subsequences in said nucleic
acid; an inputting device for inputting one or more actual signals
generated by probing said sample with said recognition means; an
analyzing device for analyzing signals operatively coupled to said
selecting and inputting devices that determines which sequences in
a nucleotide sequence database can generate said actual signals
when subject to said recognition means, said database comprising a
plurality of known nucleotide sequences that may be present in said
sample; an input/output device operatively coupled to said
selecting, inputting, and analyzing devices that inputs user
requests and controls the selecting device to select target
subsequences or sets of target subsequences, controls the inputting
device to accept actual signals, controls the analyzing device to
find the sequences in said database that can generate said actual
signals, and displays output comprising said actual signals and
said sequences in said database that can generate said actual
signals.
[0096] This invention further provides in the fifth embodiment that
said sample is a cDNA sample prepared from a tissue specimen, and
the apparatus further comprises a storage device operatively
coupled to the input/output device for storing indications of the
origin of said tissue specimen and information concerning said
tissue specimen, and wherein said indications can be displayed upon
user input, and optionally that the indications and information
concerning said tissue specimen comprises histological information
comprising tissue images.
[0097] This invention further provides in the fifth embodiment
additional apparatus further comprising one or more instrument
devices for probing said sample with said recognition means and for
generating said actual signals; and a control device operatively
coupled to said one or more instrument devices and to said
input/output device for controlling the operation of said
instrument devices, wherein said user can input control commands
for control of said instrument devices and receive output
concerning the status of said instrument devices, and optionally
wherein one or more of said selecting, inputting, analyzing, and
input/output devices are physically collocated with each other, or
are physically spaced apart from each other and are connected by a
communication medium for exchanges of commands and information.
[0098] This invention further provides in the fifth embodiment a
computer readable memory that can be used to direct a programmable
apparatus to function for displaying data according to steps
comprising selecting target subsequences or sets of target
subsequences, such that recognition means for recognizing said
target subsequences or said sets of target subsequences can be used
to generate signals by probing a sample comprising a plurality of
nucleic acids, said signals comprising a representation of (i) the
length between occurrences of said target subsequences in a nucleic
acid of said sample and (ii) the identities of said target
subsequences in said nucleic acid or the identities of said sets of
target subsequences among which are included the target
subsequences in said nucleic acid inputting one or more actual
signals generated by probing said sample with said recognition
means analyzing said one or more actual signals to determine which
sequences in a nucleotide sequence database can generate said
actual signals when subject to said recognition means, said
database comprising a plurality of known nucleotide sequences that
may be present in said sample; and inputting user requests to
control said selecting step to select target subsequences or sets
of target subsequences, said inputting step to input actual
signals, and said analyzing step to find the sequences in said
database that can generate said actual signals, and outputting in
response to further user requests information comprising said
actual signals and said sequences in said database that can
generate said actual signals, or alternatively a computer readable
memory for directing a programmable apparatus to function in the
manner of the fifth object.
[0099] In a sixth embodiment, the invention provides a method for
identifying, classifying, or quantifying DNA molecules in a sample
of DNA molecules having a plurality of different nucleotide
sequences, the method comprising the steps of digesting said sample
with one or more restriction endonucleases, each said restriction
endonuclease recognizing a subsequence recognition site and
digesting DNA at said recognition site to produce fragments with 5'
overhangs; contacting said fragments with shorter and longer
oligodeoxynucleotides, each said shorter oligodeoxynucleotide
hybridizable with a said 5' overhang and having no terminal
phosphates, each said longer oligodeoxynucleotide hybridizable with
a said shorter oligodeoxynucleotide; ligating said longer
oligodeoxynucleotides to said 5' overhangs on said DNA fragments to
produce ligated DNA fragments; extending said ligated DNA fragments
by synthesis with a DNA polymerase to produce blunt-ended double
stranded DNA fragments; amplifying said blunt-ended double stranded
DNA fragments by a method comprising contacting said DNA fragments
with a DNA polymerase and primer oligodeoxynucleotides, each said
primer oligodeoxynucleotide having a sequence comprising that of
one of the longer oligodeoxynucleotides; determining the length of
the amplified DNA fragments; and searching a DNA sequence database,
said database comprising a plurality of known DNA sequences that
may be present in the sample, for sequences matching one or more of
said fragments of determined length, a sequence from said database
matching a fragment of determined length when the sequence from
said database comprises recognition sites of said one or more
restriction endonucleases spaced apart by the determined length,
whereby DNA molecules in said sample are identified, classified, or
quantified.
[0100] This invention further provides in the sixth embodiment
additional methods wherein the sequence of each primer
oligodeoxynucleotide further comprises 3' to and contiguous with
the sequence of the longer oligodeoxynucleotide the portion of the
recognition site of said one or more restriction endonucleases
remaining on a DNA fragment terminus after digestion, said
remaining portion being 5' to and contiguous with one or more
additional nucleotides, and wherein a sequence from said database
matches a fragment of determined length when the sequence from said
database comprises subsequences that are the recognition sites of
said one or more restriction endonucleases contiguous with said one
or more additional nucleotides and when the subsequences are spaced
apart by the determined length.
[0101] This invention further provides in the sixth embodiment
additional methods wherein said determining step further comprises
detecting the amplified DNA fragments by a method comprising
staining said fragments with silver.
[0102] This invention further provides in the sixth embodiment
additional methods wherein said oligodeoxynucleotide primers are
detectably labeled, wherein the determining step further comprises
detection of said detectable labels, and wherein a sequence from
said database matches a fragment of determined length when the
sequence from said database comprises recognition sites of the one
or more restriction endonucleases, said recognition sites being
identified by the detectable labels of said oligodeoxynucleotide
primers, said recognition sites being spaced apart by the
determined length, and optionally wherein said determining step
further comprises detecting the amplified DNA fragments by a method
comprising labeling said fragments with a DNA intercalating dye or
detecting light emission from a fluorochrome label on said
fragments.
[0103] This invention further provides in the sixth embodiment
additional steps further comprising, prior to said determining
step, the step of hybridizing the amplified DNA fragments with a
detectably labeled oligodeoxynucleotide complementary to a
subsequence, said subsequence differing from said recognition sites
of said one or more restriction endonucleases, wherein the
determining step further comprises detecting said detectable label
of said oligodeoxynucleotide, and wherein a sequence from said
database matches a fragment of determined length when the sequence
from said database further comprises said subsequence between the
recognition sites of said one or more restriction
endonucleases.
[0104] This invention further provides in the sixth embodiment
additional methods wherein the one or more restriction
endonucleases are pairs of restriction endonucleases, the pairs
being selected from the group consisting of Acc56I and HindIII,
Acc65I and NgoMI, BamHI and EcoRI, BglII and HindIII, BglII and
NgoMI, BsiWI and BspHI, BspHI and BstYI, BspHI and NgoMI, BsrGI and
EcoRI, EagI and EcoRI, EagI and HindIII, EagI and NcoI, HindIII and
NgoMI, NgoMI and NheI, NgoMI and SpeI, BglII and BspHI, Bsp120I and
NcoI, BssHII and NgoMI, EcoRI and HindIII, and NgoMI and XbaI, or
wherein the step of ligating is performed with T4 DNA ligase.
[0105] This invention further provides in the sixth embodiment
additional methods wherein the steps of digesting, contacting, and
ligating are performed simultaneously in the same reaction vessel,
or optionally wherein the steps of digesting, contacting, ligating,
extending, and amplifying are performed in the same reaction
vessel.
[0106] This invention further provides in the sixth embodiment
additional methods wherein the step of determining the length is
performed by electrophoresis.
[0107] This invention further provides in the sixth embodiment
additional methods wherein the step of searching said DNA database
further comprises determining a pattern of fragments that can be
generated and for each fragment in said pattern those sequences in
said DNA database that are capable of generating the fragment by
simulating the steps of digesting with said one or more restriction
endonucleases, contacting, ligating, extending, amplifying, and
determining applied to each sequence in said DNA database; and
finding the sequences that are capable of generating said one or
more fragments of determined length by finding in said pattern one
or more fragments that have the same length and recognition sites
as said one or more fragments of determined length.
[0108] This invention further provides in the sixth embodiment
additional methods wherein the steps of digesting and ligating go
substantially to completion.
[0109] This invention further provides in the sixth embodiment
additional methods wherein the DNA sample is cDNA prepared from
mRNA, and optionally wherein the DNA is of RNA from a tissue or a
cell type derived from a plant, a single celled animal, a
multicellular animal, a bacterium, a virus, a fungus, a yeast, or a
mammal, and optionally wherein the mammal is a human, and
optionally wherein the mammal is a human having or suspected of
having a diseased condition, and optionally wherein the diseased
condition is a malignancy.
[0110] In a seventh embodiment, this invention provides additional
methods for identifying, classifying, or quantifying DNA molecules
in a sample of DNA molecules with a plurality of nucleotide
sequences, the method comprising the steps of digesting said sample
with one or more restriction endonucleases, each said restriction
endonuclease recognizing a subsequence recognition site and
digesting DNA to produce fragments with 3' overhangs; contacting
said fragments with shorter and longer oligodeoxynucleotides, each
said longer oligodeoxynucleotide consisting of a first and second
contiguous portion, said first portion being a 3' end subsequence
complementary to the overhang produced by one of said restriction
endonucleases, each said shorter oligodeoxynucleotide complementary
to the 3' end of said second portion of said longer
oligodeoxynucleotide stand; ligating said longer
oligodeoxynucleotide to said DNA fragments to produce a ligated
fragment; extending said ligated DNA fragments by synthesis with a
DNA polymerase to form blunt-ended double stranded DNA fragments;
amplifying said double stranded DNA fragments by use of a DNA
polymerase and primer oligodeoxynucleotides to produce amplified
DNA fragments, each said primer oligodeoxynucleotide having a
sequence comprising that of a longer oligodeoxynucleotides;
determining the length of the amplified DNA fragments; and
searching a DNA sequence database, said database comprising a
plurality of known DNA sequences that may be present in the sample,
for sequences matching one or more of said fragments of determined
length, a sequence from said database matching a fragment of
determined length when the sequence from said S database comprises
recognition sites of said one or more restriction endonucleases
spaced apart by the determined length, whereby DNA sequences in
said sample are identified, classified, or quantified.
[0111] In an eighth embodiment, this invention provides additional
methods of detecting one or more differentially expressed genes in
an in vitro cell exposed to an exogenous factor relative to an in
vitro cell not exposed to said exogenous factor comprising
performing the methods the first embodiment of this invention
wherein said plurality of nucleic acids comprises cDNA of RNA of
said in vitro cell exposed to said exogenous factor; performing the
methods of the first embodiment of this invention wherein said
plurality of nucleic acids comprises cDNA of RNA of said in vitro
cell not exposed to said exogenous factor; and comparing the
identified, classified, or quantified cDNA of said in vitro cell
exposed to said exogenous factor with the identified, classified,
or quantified cDNA of said in vitro cell not exposed to said
exogenous factor, whereby differentially expressed genes are
identified, classified, or quantified.
[0112] In a ninth embodiment, this invention provides additional
methods of detecting one or more differentially expressed genes in
a diseased tissue relative to a tissue not having said disease
comprising performing the methods of the first embodiment of this
invention wherein said plurality of nucleic acids comprises cDNA of
RNA of said diseased tissue such that one or more cDNA molecules
are identified, classified, and/or quantified; performing the
methods of the first embodiment of this invention wherein said
plurality of nucleic acids comprises cDNA of RNA of said tissue not
having said disease such that one or more cDNA molecules are
identified, classified, and/or quantified; and comparing said
identified, classified, and/or quantified cDNA molecules of said
diseased tissue with said identified, classified, and/or quantified
cDNA molecules of said tissue not having the disease, whereby
differentially expressed cDNA molecules are detected.
[0113] This invention further provides in the ninth embodiment
additional methods wherein the step of comparing further comprises
finding cDNA molecules which are reproducibly expressed in said
diseased tissue or in said tissue not having the disease and
further finding which of said reproducibly expressed cDNA molecules
have significant differences in expression between the tissue
having said disease and the tissue not having said disease, and
optionally wherein said finding cDNA molecules which are
reproducibly expressed and said significant differences in
expression of said cDNA molecules in said diseased tissue and in
said tissue not having the disease are determined by a method
comprising applying statistical measures, and optionally wherein
said statistical measures comprise determining reproducible
expression if the standard deviation of the level of quantified
expression of a cDNA molecule in said diseased tissue or said
tissue not having the disease is less than the average level of
quantified expression of said cDNA molecule in said diseased tissue
or said tissue not having the disease, respectively, and wherein a
cDNA molecule has significant differences in expression if the sum
of the standard deviation of the level of quantified expression of
said cDNA molecule in said diseased tissue plus the standard
deviation of the level of quantified expression of said cDNA
molecule in said tissue not having the disease is less than the
absolute value of the difference of the level of quantified
expression of said cDNA molecule in said diseased tissue minus the
level of quantified expression of said cDNA molecule in said tissue
not having the disease.
[0114] This invention further provides in the ninth embodiment
additional methods wherein the diseased tissue and the tissue not
having the disease are from one or more mammals, and optionally
wherein the disease is a malignancy, and optionally wherein the
disease is a malignancy selected from the group consisting of
prostrate cancer, breast cancer, colon cancer, lung cancer, skin
cancer, lymphoma, and leukemia.
[0115] This invention further provides in the ninth embodiment
additional methods wherein the disease is a malignancy and the
tissue not having the disease has a premalignant character.
[0116] In a tenth embodiment, this invention provides methods of
staging or grading a disease in a human individual comprising
performing the methods of the first embodiment of this invention in
which said plurality of nucleic acids comprises cDNA of RNA
prepared from a tissue from said human individual, said tissue
having or suspected of having said disease, whereby one or more
said cDNA molecules are identified, classified, and/or quantified;
and comparing said one or more identified, classified, and/or
quantified cDNA molecules in said tissue to the one or more
identified, classified, and/or quantified cDNA molecules expected
at a particular stage or grade of said disease.
[0117] In an eleventh embodiment, this invention provides
additional methods for predicting a human patient's response to
therapy for a disease, comprising performing the methods of the
first embodiment of this invention in which said plurality of
nucleic acids comprises cDNA of RNA prepared from a tissue from
said human patient, said tissue having or suspected of having said
disease, whereby one or more cDNA molecules in said sample are
identified, classified, and/or quantified; and ascertaining if the
one or more cDNA molecules thereby identified, classified, and/or
quantified correlates with a poor or a favorable response to one or
more therapies, and optionally which further comprises selecting
one or more therapies for said patient for which said identified,
classified, and/or quantified cDNA molecules correlates with a
favorable response.
[0118] In a twelfth embodiment, this invention provides additional
methods for evaluating the efficacy of a therapy in a mammal having
a disease, the method comprising performing the methods of the
first embodiment of this invention wherein said plurality of
nucleic acids comprises cDNA of RNA of said mammal prior to a
therapy; performing the method of the first embodiment of this
invention wherein said plurality of nucleic acids comprises cDNA of
RNA of said mammal subsequent to said therapy; comparing one or
more identified, classified, and/or quantified cDNA molecules in
said mammal prior to said therapy with one or more identified,
classified, and/or quantified cDNA molecules of said mammal
subsequent to therapy; and determining whether the response to
therapy is favorable or unfavorable according to whether any
differences in the one or more identified, classified, and/or
quantified cDNA molecules after therapy are correlated with
regression or progression, respectively, of the disease, and
optionally wherein the mammal is a human.
[0119] In a thirteenth embodiment, this invention provides a kit
comprising one or more containers having one or more restriction
endonucleases; one or more containers having one or more shorter
oligodeoxynucleotide strands; one or more containers having one or
more longer oligodeoxynucleotide strands hybridizable with said
shorter strands, wherein either the longer or the shorter
oligodeoxynucleotide strands each comprise a sequence complementary
to an overhang produced by at least one of said one or more
restriction endonucleases; and instructions packaged in association
with said one or more containers for use of said restriction
endonucleases, shorter strands, and longer strands for identifying,
classifying, or quantifying one or more DNA molecules in a DNA
sample, said instructions comprising (i) digest said sample with
said restriction endonucleases into fragments, each fragment being
terminated on each end by a recognition site of said one or more
restriction endonucleases; (ii) contact said shorter and longer
strands and said digested fragments to form double stranded DNA
adapters annealed to said digested fragments, (iii) ligate said
longer strand to said fragments; (iv) generate one or more signals
by separating and detecting such of said fragments that are
digested on each end, each signal comprising a representation of
the length of the fragment and the identity of the recognition
sites on both termini of the fragments; and (v) search a nucleotide
sequence database to determine sequences that match or the absence
of any sequences that match said one or more generated signals,
said database comprising a plurality of known nucleotide sequences
of nucleic acids that may be present in the sample, a sequence from
said database matching a generated signal when the sequence from
said database has both (i) the same length between occurrences of
said recognition sites of said one or more restriction
endonucleases as is represented by the generated signal and (ii)
the same recognition sites of said one of more restriction
endonucleases as is represented by the generated signal.
[0120] This invention further provides in the thirteenth embodiment
a kit wherein said one or more restriction erdonucleases generate
5' overhangs at the terminus of digested fragments, wherein each
said shorter oligodeoxynucleotide strand consists of a first and
second contiguous portion, said first portion being a 5' end
subsequence complementary to the overhang produced by one of said
restriction endonucleases, and wherein each said longer
oligodeoxynucleotide strand comprises a 3' end subsequence
complementary to said second portion of said shorter
oligodeoxynucleotide strand, or optionally wherein said one or more
restriction endonucleases generate 3' overhangs at the terminus of
the digested fragments, wherein each said longer
oligodeoxynucleotide strand consists of a first and second
contiguous portion, said first portion being a 3' end subsequence
complementary to the overhang produced by one of said restriction
endonucleases, and wherein each said shorter oligodeoxynucleotide
strand is complementary to the 3' end of said second portion of
said longer oligodeoxynucleotide stand.
[0121] This invention further provides in the thirteenth embodiment
a kit wherein said instructions further comprise those signals
expected from one or more DNA molecules of interest when said
sample is digested with a particular one or more restriction
endonucleases selected from among said one or more restriction
endonucleases in said kit, and optionally wherein said one or more
DNA molecules of interest are cDNA molecules differentially
expressed in a disease condition.
[0122] This invention further provides in the thirteenth embodiment
a kit wherein the restriction endonucleases are selected from the
group consisting of Acc65I, AflII, AgeI, ApaLI, ApoI, AscI, AvrI,
BamHI, BclI, BglII, BsiWI,. Bsp120I, BspEI, BspHI, BsrGI, BssHII,
BstYI, EagI, EcoRI, HindIII, MluI, NcoI, NgoMI, NheI, NotI, SpeI,
and XbaI.
[0123] This invention further provides in the thirteenth embodiment
a kit further comprising one or more containers having one or more
double stranded adapter DNA molecules formed by annealing said
longer and said shorter oligonucleotide strands.
[0124] This invention further provides in the thirteenth embodiment
a kit further comprising the computer readable memory of claim 106,
or optionally further comprising the computer readable memory of
claim 114, or optionally further comprising the computer readable
memory of claim 122.
[0125] This invention further provides in the thirteenth embodiment
a kit further comprising in a container a DNA ligase, or optionally
further comprising in a container a phosphatase capable of removing
terminal phosphates from a DNA sequence.
[0126] This invention further provides in the thirteenth embodiment
a kit further comprising one or more primers, each said primer
consisting of a single stranded oligodeoxynucleotide comprising the
sequence of one of said longer strands; and a DNA polymerase, and
optionally wherein each of said one or more primers further
comprises (a) a first subsequence that is the portion of the
recognition site of one of said one or more restriction
endonucleases remaining at the terminus of a fragment after
digestion, and (b) a second subsequence of one or two additional
nucleotides contiguous with and 3' to said first subsequence,
wherein said primer is detectably labeled such that primers with
differing said one or two additional nucleotides have different
labels that can be distinguishably detected.
[0127] This invention further provides in the thirteenth embodiment
a kit wherein said instructions further comprise: detect such of
said fragments digested on each end by a method comprising staining
said fragments with silver, labeling said fragments with a DNA
intercalating dye, or detecting light emission from a fluorochrome
label on said fragments.
[0128] This invention further provides in the thirteenth embodiment
a kit further comprising reagents for performing a cDNA sample
preparation step; reagents for performing a step of digestion by
one or more restriction endonucleases; reagents for performing a
ligation step; and reagents for performing a PCR amplification
step.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0129] These and other features, aspects, and advantages of the
present invention will become better understood by reference to the
accompanying drawings, following description, and appended claims,
where:
[0130] FIG. 1 shows exemplary results of the signals generated by
the QEA method of this invention;
[0131] FIGS. 2A, 2B, and 2C show DNA adapters for an RE/ligation
implementation of the QEA method of this invention, where the
restriction endonucleases generate 5' overhangs, open blocks
indicating strands of DNA;
[0132] FIGS. 3A and 3B show the DNA adapters for an RE/ligation
implementation of the QEA method of this invention, where the
restriction endonucleases generate 3' overhangs;
[0133] FIGS. 4A, 4B, and 4C show an exemplary biotin alternative
embodiment of the QEA method;
[0134] FIG. 5 shows the DNA primers for a PCR embodiment of the QEA
method;
[0135] FIGS. 6A and 6B show a method for DNA sequence database
selection according to this invention;
[0136] FIG. 7 shows an exemplary experimental description for the
QEA embodiment of this invention;
[0137] FIGS. 8A and 8B show an overview of a method for determining
a simulated database of experimental results for the QEA embodiment
of this invention;
[0138] FIG. 9 shows the detail of a method for simulating a QEA
reaction;
[0139] FIGS. 10A-F show exemplary results of the action of the
method of FIG. 9;
[0140] FIG. 11 shows the detail of a method for determining a
simulated database of experimental results for a QEA embodiment of
this invention;
[0141] FIGS. 12A, 12B, and 12C show an exemplary computer system
apparatus, and an alternative embodiment, implementing methods cf
this invention;
[0142] FIG. 13A shows exemplary detail of an experimental design
method for QEA and CC embodiments of this invention and FIG. 13B
shows exemplary detail of an experimental design method for a QEA
embodiment of this invention;
[0143] FIG. 14 shows an exemplary method for ordering the DNA
sequences found to be likely causes of a QEA signal in the order of
their likely presence in the sample;
[0144] FIG. 15 shows the detail of a method for determining a
simulated database of experimental results for a CC embodiment of
this invention;
[0145] FIGS. 16A, 16B, 16C, and 16D show exemplary reaction
temperature profiles for preferred manual and automated
implementations of a preferred RE embodiment of a QEA method.
5. DETAILED DESCRIPTION
[0146] According to the present invention, to uniquely identify an
expressed gene sequence, full or partial, and many components of
genomic DNA it is not necessary to determine actual, complete
nucleotide sequences of samples. Full sequences provide far more
information than is needed to merely classify or determine a gene
according to the invention. For example, in the human genome, it is
known that there are approximately 10.sup.5 expressed genes. Since
the average length of a coding sequence is approximately 2000
nucleotides, the total number of possible sequences is
approximately 4.sup.2000, or about 10.sup.1200. The actual number
of expressed human genes is an unimaginably small fraction
(10.sup.-1195) of the total number of possible DNA sequences. Even
sequencing a 50 bp fragment of a cDNA sequence generates about
10.sup.25 times more information than is needed for classification
of that sequence. Use of the present invention allows direct
classification of expressed gene sequences with far less
information than either a complete or a partial sequence
determination of a sample.
[0147] In computer science, codes which compactly identify a few
members from among a large set of possibilities are called hash
codes. An object of this invention is to construct hash codes for
expressed DNA sequences, or alternatively for any other existing
set of DNA sequences. In a fully populated code without any
unassigned code words, all human genes could be coded by an
approximately 17 bit binary number (2.sup.17=1.3.times.10.- sup.5).
A 20 bit code would be about 10% filled or 90% sparse
(2.sup.20=1.0.times.10.sup.6).
[0148] In this invention codes are constructed from signals which
represent the presence of short nucleic acid (preferably DNA)
subsequences (hereinafter called "target subsequences") in the
sample sequence and, preferably, in a QEA embodiment, include a
representation of the length along the sample sequence between
adjacent target subsequences. The presence of these subsequences is
recognized by subsequence recognition means, including, but not
limited to, REs, DNA binding proteins, and oligomers ("probes")
hybridizable to DNA of, for example, PNAs or DNAs. The subsequence
recognition means allow recognition of specific DNA subsequences by
the ability to specifically bind to or react with such
subsequences. The invention, and particularly its computer methods,
are adaptable to any subsequence recognition means available in the
art. Acceptable subsequence detection means preferably precisely
and reproducibly recognize target subsequences and generate a
recognition signal of adequate signal to noise ratio for all genes,
however rare, in a sample, and can also provide information on the
length between target subsequences.
[0149] The signals contain representations of target subsequence
occurrences and, preferably, a representation of the length between
target subsequence occurrences. In various embodiments of this
invention these representations may differ. In embodiments where
the target subsequences are exactly recognized, as where REs are
used, subsequence representation may simply be the actual identity
of the subsequences. In other embodiments where subsequence
recognition is less exact, as where short oligomers are used, this
representation may be "fuzzy". It may, for example, consist of all
subsequences which differ by one nucleotide from the target, or
some other set of possible subsequences, perhaps weighted by the
probability that each member of the set is the actual subsequence
in the sample sequence. Further, the length representation may
depend on the separation and detection means used to generate the
signals. In the case of electrophoretic separation, the length
observed electrophoretically may need to be corrected, perhaps up
to 5 to 10%, for mobility differences due to average base
composition differences or due to effects of any labeling moiety
used for detection. As these corrections may not be known until
target sequence recognition, the signal may contain the
electrophoretic length in bp and not the true physical length in
bp. For simplicity and without limitation, in most of the following
description unless otherwise noted the signals are presumed to
represent the information conveyed exactly, as if generated by
exact recognition means and error or bias free separation and
detection means. However, in particular embodiments, target
subsequences may be represented in a fuzzy fashion and length, if
present, with separation and detection bias present.
[0150] Target subsequences recognized are typically of contiguous
sequence. This is required for all known REs. However, oligomers
recognizing discontinuous subsequences can be used and can be
constructed by inserting degenerate nucleotides in any
discontinuous region. For example, a set of 16 oligomers
recognizing AGC--TAT, with a two nucleotide skip between the two
portions of the recognition subsequence, is could be constructed as
TCGNNATA, where N is any nucleotide. Alternately, such
discontiguous subsequences can be recognized by one oligomer of the
form TCGiiATA, where "i" is inosine, or any other "universal"
nucleotide, capable of hybridizing with any naturally occurring
base.
[0151] This invention is adaptable to analyzing any DNA sample for
which exists an accompanying database listing possible sequences in
the sample. More generally, the invention is adaptable to analyzing
the sequences of any biopolymer, built of a small number of
repeating units, whose naturally occurring representatives are far
fewer that the number of possible, physical polymers and in which
small subsequences can be recognized. Thus it is applicable to not
only naturally occurring DNA polymers but also to naturally
occurring RNA polymers, proteins, glycans, etc. Typically and
without limitation, however, the invention is applied to the
analysis of cDNA samples from any in vivo or in vitro sources. cDNA
can be synthesized either from total cellular RNA or from specific
sub-pools of RNA. These RNA sub-pools can be produced by RNA
pre-purification, for example, the separation of mRNA of the
endoplasmic reticulum from cytoplasmic mRNA, which thereby enriches
mRNA primarily encoding for cell surface or extracellular proteins
(Celis et al., 1994, Cell Biology, Academic Press, New York, N.Y.).
Such enriched mRNAs have increased diagnostic or therapeutic
utility due to their encoded protein's cell-surface or
extracellular roles, such as being a receptor. Such pre-purified
RNA pools can be used in all embodiments of this invention.
[0152] First strand cDNA synthesis can use any priming method known
in the art, for example, oligo(dT) primers, random hexamer primers,
phasing primers, mixtures thereof, etc. Phasing primers, containing
either an A,C, or G at the 3' end, can be used in separate cDNA
synthesis reactions to split the cDNA first strands into 3 pools,
each generated from poly(A) mRNA having a T, G, or C, respectively,
5' to the poly(A) tail. Fifteen mixtures can be synthesized by
using all 15 possible oligo(dT) primers containing a pair of non-T
nucleotides at the 3' end.
[0153] Two specific embodiments of the invention are respectively
termed "quantitative expression analysis" ("QEA") and "colony
calling" ("CC").
[0154] The specific embodiment, QEA, probes a sample with
recognition means, the recognition means generating signals, a
preferred signal being a triple comprising an indication of the
presence of a first target subsequence, an indication of the
presence of a second target subsequence, and a representation of
the length between the target subsequences in the sample nucleic
acids sequence. Each pair of target subsequences may occur more
than once in a sample nucleic acid, in which case the associated
lengths are between adjacent target subsequence occurrences.
[0155] The QEA embodiment is preferred for classifying and
determining sequences in cDNA mixtures, but is also adaptable to
samples with only one sequence. It is preferred for mixtures
because it affords the relative advantage over prior art methods
that cloning of sample nucleic acids is not required. Typically,
enough distinguishable signals are generated from pairs of target
subsequences to recognize a desired sequence in a sample mixture.
For example, first, any pair of target subsequences may hit more
than once in a single DNA molecule to be analyzed, thereby
generating several signals with differing lengths from one DNA
molecule. Second, even if the pair of target subsequences hits only
once in two different DNA molecules to be analyzed, the lengths
between the hits nay differ and thus distinguishable signals may be
generated.
[0156] The target subsequences used in QEA are preferably optimally
chosen by methods of this invention from DNA sequence databases
containing sequences likely to occur in the sample to be analyzed.
Efforts of the Human Genome Project in the United States, efforts
abroad, and efforts of private companies in the sequencing of the
human genome sequences, both expressed and genetic, are being
collected in several available databases (listed in .sctn.
5.1).
[0157] In a QEA "query mode" experiment, the focus is on
determining the expression of several genes, perhaps 1-100, of
interest and of known sequence. A minimal number of target
subsequences is chosen to generate signals, with the goal that each
of the several genes is discriminated by at least one unique
signal, which also discriminates it from all the other genes likely
to occur in the sample. In other words, the experiment is designed
so that each gene generates at least one signal unique to it (a
"good" gene, see infra). In a QEA "tissue mode" experiment, the
focus is on determining the expression of as many as possible,
preferably a majority, of the genes in a tissue, without the need
for any prior knowledge or interest in their expression. Target
subsequences are optimally chosen to discriminate the maximum
number of sample DNA sequences into classes comprising one or
preferably at most a few sequences. Signals are generated and
detected as determined by the threshold and sensitivity of a
particular experiment. Some important determinants of threshold and
sensitivity are the initial amount of mRNA and thus of cDNA, the
amount of molecular amplification performed during the experiment,
and the sensitivity of the detection means. Preferably, enough
signals are produced and detected so that the computer methods of
this invention can uniquely determine the expression of a majority,
or more preferably most, of the genes expressed in a tissue.
[0158] QEA signals are generated by methods utilizing recognition
means that include, but are not limited to REs in a preferred
RE/ligase method or in a method utilizing a removal means,
preferably contacting streptavidin linked to a solid phase with
biotin-labeled DNA, for removal of unwanted DNA fragments, and
nucleotide oligomer primers in a PCR method.
[0159] A preferred embodiment of the RE/ligase method is as
follows. The method employs recognition reactions with a pair (or
more) of REs which recognize target subsequences with high
specificity and cut the sequence at the recognition sites leaving
fragments with sticky ends characteristic of the particular RE. To
each sticky end, special primers are ligated which are
distinctively labeled with fluorochromes identifying the particular
RE making the cut, and thus the particular target subsequence. A
DNA polymerase is used to form blunt-ended DNA fragments. The
labeled fragments are then PCR amplified using the same special
primers a number of times preferably just sufficient to detect
signals from all sequences of interest while making relatively
small signals from the linearly amplifying singly cut fragments.
The amplified fragments are then separated by length using gel
electrophoresis, and the length and labeling of the fragments is
optically detected. Optionally, single stranded fragments can be
removed by a binding hydroxyapatite, or other single strand
specific, column or by digestion by a single strand specific
nuclease. Also, this invention is adaptable to other functionally
equivalent amplification and length separation means. In this
manner, the identity of the REs cutting a fragment, and thereby the
subsequences present, as well as the length between the cuts is
determined.
[0160] In a preferred PCR method for QEA, a suitable collection of
target subsequences is chosen by the computer implemented QEA
experimental design methods and PCR primers distinctively labeled
with fluorochromes are synthesized to hybridize with these
subsequences. The primers are designed as described in .sctn. 5.3
to reliably recognize short subsequences while achieving a high
specificity in PCR amplification. Using these primers, a minimum
number of PCR amplification steps amplifies those fragments between
the primed subsequences existing in DNA sequences in the sample.
The labeled, amplified fragments are separated by gel
electrophoresis and detected.
[0161] In an exemplary QEA method utilizing a removal means, which
has improved quantitative characteristics and is also adapted to
highly sensitive detection systems, cDNA is synthesized from a
tissue sample using at least one internally biotinylated primer.
The cDNA is then cyclized, cut with a pair of REs, and specifically
labeled primers are ligated to the cut ends, as discussed in .sctn.
5.2.2. The singly cut ends attached to the biotinylated synthesis
primers are removed with streptavidin or avidin beads leaving
highly pure labeled double cut cDNA fragments without any singly
cut and labeled background fragments. With a sufficiently sensitive
optical detection system, these pure doubly cut and labeled
fragments can be separated by length (e.g. by electrophoresis or
column chromatography) and directly detected without amplification.
If amplification is needed, absence of the DNA singly cut fragment
background improves signal to noise ratio permitting fewer
amplification steps and, thereby, decreased PCR amplification
bias.
[0162] Optional alternatives can provide increased discrimination
in QEA experiments. Two sequences producing two fragments of
identical end subsequences and length can be discriminated by
recognizing a third subsequence present in one of the fragments but
not in the other. In one alternative, a labeled probe recognizing
this third subsequence can be added before detection to generate
unique signals from the fragment containing that subsequence. In
another alternative, a probe can be added before amplification
which prevents amplification of the fragment with the third
subsequence and which thereby removes (suppresses) its signal. By
way of example, such a probe can be either an RE for recognizing
and cutting the fragment with the third subsequence or a PNA, or
modified DNA, probe which will hybridize with the third subsequence
and prevent its PCR amplification.
[0163] The signals generated from the recognition reactions of a
QEA experiment are analyzed by computer methods of this invention.
The analysis methods simulate a QEA experiment using a database
either of substantially all known DNA sequences or of substantially
all, or at least a majority of, the DNA sequences likely to be
present in a sample to be analyzed and a description of the
reactions to be performed. The simulation results in a digest
database which contains for all possible signals that can be
generated the sample sequences responsible. Thereby, finding the
sequences that can generate a signal involves a look-up in the
simulated digest database. Computer implemented design methods
optimize the choice of target subsequences in the QEA reactions in
order to maximize the information produced in an experiment. For
the tissue mode, the methods maximize the number of sequences
having unique signals by which their quantitative presence can be
unambiguously determined. For the query mode, the methods maximize
only the number of sequences of interest having unique signals,
ignoring other sequences that might be present in a sample.
[0164] A second specific embodiment, colony calling ("CC"),
generates subsequence hit data without length information. Since
this method requires only hybridizations, it is preferred for gene
identification in arrayed single-sequence clones constructed from a
tissue library. This embodiment constructs a binary code in which
each bit of the code represents the presence or absence of one
target subsequence. By probing four to eight target subsequences in
parallel, such as by using distinguishable fluorescent labeling of
the multiple probes, in view of the adequacy of a 20 bit code, the
presence or absence of any expressed human gene should be
determinable in just three to five separate probe steps. Such a
compact method with such economy in signal generation-is highly
useful. Alternatively, recent real time hybridization detection
methods (Stimson et al., 1995, Proc. Natl. Acad. Sci. USA,
92:6379-6383) based on optical wave guides can be used for
detection. These methods make hybridization detection more
efficient both by eliminating the washing step otherwise needed
between hybridization and detection and by speeding up the
detection step.
[0165] The hash code generated by the probe hybridization reactions
is interpreted by computer implemented methods of this invention.
The analysis methods simulate a CC experiment using a list of the
target subsequences and a database of the DNA sequences likely to
be present in a sample to be analyzed. The simulation results in a
hash code table which contains for each hash code all possible
sequences that can generate that code. Thereby, interpretation of a
detected hash code requires a look-up in the table to find the
possible sequences.
[0166] It is preferable that subsequences be carefully chosen in
order that a minimum set of targets be obtained, preferably no more
than approximately 20, that produce the maximum amount of
information. Computer implemented methods of this invention
determine optimum sets of target subsequences for a given database
of sequences likely to occur in the sample by optimizing the number
of non-empty hash codes in the simulated hash code table.
[0167] Maximum information is obtained when the target subsequences
occur completely randomly in the possible sample sequences, that
is, when their likelihood of occurrence is approximately 50% and
the presence of one subsequence is independent of the presence of
any other subsequence. Therefore, target subsequences chosen to
generate a signal should preferably occur in the DNA sequence
sample to be analyzed less than about 50% and at least more often
than 5-10%, preferably more often than 10-15%. The most preferable
occurrence probability is from 25-50%. Also the presence of one
target subsequence is preferably probabilistically independent of
the presence of any other subsequence.
[0168] Using data on expressed RNA from human DNA sequence
databases, this means that sub-sequences are preferably less than
about 5 to 8 bp long for cDNA classification. Typically, the
resulting preferable target subsequences are 4 to 6 bp long. Longer
sequences occur too infrequently to be preferred for use. However,
for classifying gDNA, longer subsequences, up to 20 to 40 bp, are
preferably used, because gDNA fragments are normally of much
greater length, from at least 5 kilobases ("kb") for plasmid
inserts to more the 100 kb for P1 inserts, and thus would typically
have more sequence variability, requiring longer target
subsequences.
[0169] The preferred hybridization probes for short target
subsequences are labeled peptido-nucleic acids (PNAs).
Alternatively sets of degenerate, longer DNA oligonucleotides are
used which include as a common subsequence the target subsequence.
These degenerate sets achieve improved hybridization specificity as
compared to 4 to 6-mers. Sets of probes, each probe distinctively
and distinguishably labeled with a fluorochrome, are hybridized in
conditions of high stringency to arrayed DNA sequence clones and
optically detected to detect the presence of target subsequences.
For example, in an embodiment wherein five fluorochromes are
simultaneously distinguished and 20 subsequences observations are
required for gene identification (a 20 bit code), any gene in a
colony can be identified in only four hybridization steps.
Alternately, efficient hybridization detection means based on
optical wave guide detection of DNA hybridization can be used. By
using differently sized and shaped particles associated with
different probes, the resultant differences in light scattering can
be used to detect hybridization of multiple probes simultaneously
with these wave guide methods.
[0170] Target subsequences can be chosen to discriminate not only
single genes but also, more coarsely, sets of genes. Fewer target
subsequences can be chosen so that a particular pattern of hits
will indicate the presence of a gene of a particular type. Types of
genes of interest might be oncogenes, tumor suppressor genes,
growth factors, cell cycle genes, or cytoskeletal genes, etc.
[0171] In embodiments of this invention where high stringency
hybridization are specified, such conditions generally comprise a
low salt concentration, equivalent to a concentration of SSC (173.5
g. NaCl, 88.2 g. Na Citrate, H.sub.2O to 1 1.) of less than
approximately 1 mM, and a temperature near or above the T.sub.m of
the hybridizing DNA. In contrast, conditions of low stringency
generally comprise a high salt concentration, equivalent to a
concentration of SSC of greater than approximately 150 mM, and a
temperature below the T.sub.m of the hybridizing DNA.
[0172] In embodiments of this invention where DNA oligomers are
specified for performing functions, including hybridization and
chain elongation priming, alternatively oligomers can be used that
comprise those of the following nucleotide mimics which perform
similar functions. Nucleotide mimics are subunits (other than
classical nucleotides) which can be polymerized to form molecules
capable of specific, Watson-Crick-like base pairing with DNA. The
oligomers can be DNA or RNA or chimeric mixtures or derivatives or
modified versions thereof. The oligomers can be modified at the
base moiety, sugar moiety, or phosphate backbone. The oligomers may
include other appending groups such as peptides,
hybridization-triggered cleavage agents (see, e.g., Krol et al.,
1988, BioTechniques 6:958-976), or intercalating agents (see, e.g.,
Zon, 1988, Pharm. Res. 5:539-549). The oligomers may be conjugated
to another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0173] The oligomers may also comprise at least one nucleotide
mimic that is a modified base moiety which is selected from the
group including but not limited to 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. The
oligomers may comprise at least one modified sugar moiety selected
from the group including but not limited to arabinose,
2-fluoroarabinose, xylulose, and hexose. The oligomers may comprise
at least one modified phosphate backbone selected from the group
consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0174] The oligomer may be an .alpha.-anomeric oligomer. An .alpha.
-anomeric oligomer forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15:6625-6641).
[0175] Oligomers of the invention may be synthesized by standard
methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate oligos may
be synthesized by the method of Stein et al. (1988, Nucl. Acids
Res. 16:3209), methylphosphonate oligos can be prepared by use of
controlled pore glass polymer supports (Sarin et al., 1988, Proc.
Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
[0176] In specific embodiments of this invention it is preferable
to use oligomers that can specifically hybridize to subsequences of
a DNA sequence too short to achieve reliably specific recognition,
such that a set of target subsequences is recognized. Further where
PCR is used, as Tag polymerase tolerates hybridization mismatches,
PCR specificity is generally less than hybridization specificity.
Where such oligomers recognizing short subsequences are preferable,
they may be constructed in manners including but not limited to the
following. To achieve reliable hybridization to shorter DNA
subsequences, degenerate sets of DNA oligomers may be used which
are constructed of a total length sufficient to achieve specific
hybridization with each member of the set containing a shorter
sequence complementary to the common subsequence to be recognized.
Alternatively, a longer DNA oligomer may be constructed with a
shorter sequence complementary to the subsequence to be recognized
and with additional universal nucleotides or nucleotide mimics,
which are capable of hybridizing to any naturally occurring
nucleotide. Nucleotide mimics are sub-units which can be
polymerized to form molecules capable of specific,
Watson-Crick-like base pairing with DNA. Alternatively, the
oligomers may be constructed from DNA mimics which have improved
hybridization energetics compared to naturally occurring
nucleotides.
[0177] A preferred mimic is a peptido-nucleic acid ("PNA") based on
a linked N-(2-aminoethyl)glycine backbone to which normal DNA bases
have been attached (Egholm et al., 1993, Nature, 365:566-67). This
PNA obeys specific Watson-Crick base pairing but with greater free
energy of binding and correspondingly higher melting temperatures.
Suitable oligomers may be constructed entirely from PNAs or from
mixed PNA and DNA oligomers.
[0178] In embodiments of this invention where DNA fragments are
separated by length, any length separation means known in the art
can be used. One alternative separation means employs a sieving
medium for separation by fragment length coupled with a force for
propelling the DNA fragments though the sieving medium. The sieving
medium can be a polymer or gel, such a polyacrylamide or agarose in
suitable concentrations to separate 10-1000 bp DNA fragments. In
this case the propelling force is a voltage applied across the
medium. The gel can be disposed in electrophoretic configurations
comprising thick or thin plates or capillaries. The gel can be
non-denaturing or denaturing. Alternately, the sieving medium can
be such as used for chromatographic separation, in which case a
pressure is the propelling force. Standard or high performance
liquid chromatographic ("HPLC") length separation means may be
used. An alternative separation means employs molecular
characteristics such as charge, mass, or charge to mass ratio. Mass
spectrographic means capable of separating 10-1000 bp fragments may
be used.
[0179] DNA fragment lengths determined by such a separation means
represent the physical length in base pairs between target
subsequences, after adjustment for biases or errors introduced by
the separation means and length changes due to experimental
variables (e.g., presence of a detectable label, ligation to an
adopter molecule). A represented length is the same as the physical
length between occurrences of target subsequences in a sequence
from said database when both said lengths are equal after applying
corrections for biases and errors in said separation means and
corrections based on experimental variables. For example,
represented lengths determined by electrophoresis can be adjusted
for mobility biases due to average base composition or mobility
changes due to an attached labeling moiety and/or adapter strand by
conventional software programs, such as Gene Scan Software from
Applied Biosystems, Inc. (Foster City, Calif.).
[0180] In embodiments of this invention where DNA fragments must be
labeled and detected, any compatible labeling and detection means
known in the art can be used. Advances in fluorochromes, in optics,
and in optical sensing now permit multiply labeled DNA fragments to
be distinguished even if they completely overlap in space, as in a
spot on a filter or a band in a gel. Results of several recognition
reactions or hybridizations can be multiplexed in the same gel lane
or filter spot. Fluorochromes are available for DNA labeling which
permit distinguishing 6-8 separate products simultaneously (Ju et
al., 1995, Proc. Natl. Acad Sci. USA, 92:4347-4351).
[0181] Exemplary fluorochromes adaptable to this invention and
methods of using such fluorochromes to label DNA are described in
.sctn. 6.10.
[0182] Single molecule detection by fluorescence is now becoming
possible (Eigen et al., 1994, Proc. Natl. Acad Sci. USA,
91:5740-5747), and can be adapted for use.
[0183] In embodiments of this invention where intercalating DNA
dyes are utilized to detect DNA, any such dye known in the art is
adaptable. In particular such dyes include but are not limited to
ethidium bromide, propidium iodide, Hoechst 33258, Hoechst 33342,
acridine orange, and ethidium bromide homodimers. Such dyes also
include POPO, BOBO, YOYO, and TOTO from Molecular Probes (Eugene,
Oreg.).
[0184] Finally alternative sensitive detection means available
include silver staining of polyacrylamide gels (Bassam et al.,
1991, Analytic Biochemistry, 196:80-83), and the use of
intercalating dyes. In this case the gel can be photographed and
the photograph scanned by scanner devices conventional in the
computer art to produce a computer record of the separated and
detected fragments. A further alternative is to blot an
electrophoretic separating gel onto a filter (e.g., nitrocellulose)
and then to apply any visualization means known in the art to
visualize adherent DNA. See, e.g., Kricka et al., 1995, Molecular
Probing, Blotting, and Sequencing, Academic Press, New York. In
particular, visualization means requiring secondary reactions with
one or more reagents or enzymes can be used, as can any means
employed in the CC embodiment.
[0185] A preferred separation and detection apparatus for use in
this invention is found in copending U.S. patent application Ser.
No. 08/438,231 filed May 9, 1995, which is hereby incorporated by
reference in its entirety. Other detection means adaptable to this
invention include the commercial electrophoresis machines from
Applied Biosystems Inc. (Foster City, Calif.), Pharmacia (ALF),
Hitachi, Licor. The Applied Biosystems machine is preferred among
these as it is the only machine capable of simultaneous 4 dye
resolution.
[0186] In the following subsections and the accompanying examples
sections the QEA and the CC embodiments are described in
detail.
5.1 QUANTITATIVE EXPRESSION ANALYSIS
[0187] This embodiment preferably generates one or more signals
unique to each cDNA sequence in a mixture of cDNAs, such as may be
derived from total cellular RNA or total cellular mRNA from a
tissue sample, and to quantitatively relate the strength of such a
signal or signals to the relative amount of that cDNA sequence in
the sample or library. Less preferably, the signals uniquely
determine only sets of a small number of sequences, typically 2-10
sequences. QEA signals comprise an indication of the presence of
pairs of target subsequences and the length between pairs of
adjacent subsequences in a DNA sample. Signals are generated in a
manner permitting straightforward automation with existing
laboratory robots. For simplicity of disclosure, and not by way of
limitation, the detailed description of this method is directed to
the analysis of samples comprising a plurality of cDNA sequences.
It is equally applicable to samples comprising a single sequence or
samples comprising sequences of other types of DNA or nucleic acids
generally.
[0188] While described in terms of cDNA hereinbelow, it will be
understood that the DNA sample can be cDNA and/or genomic DNA, and
preferably comprises a mixture of DNA sequences. In specific
embodiments, the DNA sample is an aliquot of cDNA of total cellular
RNA or total cellular mRNA, most preferably derived from human
tissue. The human tissue can be diseased or normal. In one
embodiment, the human tissue is malignant tissue, e.g., from
prostate cancer, breast cancer, colon cancer, lung cancer,
lymphatic or hematopoietic cancers, etc. In another embodiment, the
tissue may be derived from in vivo animal models of disease or
other biologic processes. In this cases the diseases modeled can
usefully include, as well as cancers, diabetes, obesity, the
rheumatoid or autoimmune diseases, etc. In yet another embodiment,
the samples can be derived from in vitro cultures and models. This
invention can also be advantageously applied to examine gene
expression in plants, yeasts, fungi, etc.
[0189] The cDNA, or the mRNA from which it is synthesized, must be
present at some threshold level in order to generate signals, this
level being determined to some degree by the conditions of a
particular QEA experiment. For example, such a threshold is that
preferably at least 1000, and more preferably at least 10,000, mRNA
molecules of the sequence to be detected be present in a sample. In
the case where one or only a few mRNAs of a type of interest are
present in each cell of a tissue from which it is desired to derive
the sample mRNA, at least a corresponding number of such cells
should be present in the initial tissue sample. In a specific
embodiment, the mRNA detected is present in a ratio to total sample
RNA of 1:10.sup.5 to 1:10.sup.6. With a lower ratio, more molecular
amplification can be performed during a QEA experiment.
[0190] The cDNA sequences occurring in a tissue derived pool
include short untranslated sequences and translated protein coding
sequences, which, in turn, may be a complete protein coding
sequence or some initial portion of a coding sequence, such as an
expressed sequence tag. A coding sequence may represent an as yet
unknown sequence or gene or an already known sequence or gene
entered into a DNA sequence database. Exemplary sequence databases
include those made available by the National Center for
Biotechnology Information ("NCBI") (Bethesda, Md.) (GenBank) and by
the European Bioinformatics Institute ("EMBL") (Hinxton Hall,
UK).
[0191] The QEA method is also applicable to samples of genomic DNA
in a manner similar to its application to cDNA. In gDNA samples,
information of interest includes occurrence and identity of
translocations, gene amplifications, loss of heterozygosity for an
allele, etc. This information is of interest in cancer diagnosis
and staging. In cancer patients, amplified sequences might reflect
an oncogene, while loss of heterozygosity might reflect a tumor
suppressor gene. Such sequences of interest can be used to select
target subsequences and to predict signals generated by a QEA
experiment. Even without prior knowledge of the sequences of
interest, detection and classification of QEA signal patterns is
useful for the comparison of normal and diseased states or for
observing the progression of a disease state. Gene expression
information concerning the progression of a disease state is useful
in order to elucidate the genetic mechanisms behind disease, to
find useful diagnostic markers, to guide the selection and observe
the results of therapies, etc. Signal differences identify the gene
or genes involved, whether already known or yet to be
sequenced.
[0192] Classification of QEA signal patterns, in an exemplary
embodiment, can involve statistical analysis to determine
significant differences between patterns of interest. This can
involve first grouping samples that are similar in one of more
characteristics, such characteristics including, for example,
epidemiological history, histopathological state, treatment
history, etc. Signal patterns from similar samples are then
compared, e.g., by finding the average and standard deviation of
each individual signals. Individual signal which are of limited
variability, for which the standard deviation is less than the
average, then represent genetic constants of samples of this
particular characteristic. Such limited variability signals from
one set of tissue samples can then be compared to limited
variability signals from another set of tissue samples. Signals
which differ in this comparison then represent significant
differences in the genetic expression between the tissue samples
and are of interest in reflecting the biological differences
between the samples, such as the differences caused by the
progression of a disease. For example, a significant difference in
expression is detected with the difference in the genetic
expression between two tissues exceed the sum of the standard
deviation of the expressions in the tissues. Other standard
statistical comparisons can also be used to establish level of
expression and the significance of differences in levels of
expressions.
[0193] Target subsequence choice is important in the practice of
this invention. The two primary considerations for selecting
subsequences are, first, redundancy, that is, that there be enough
subsequence pair hits per gene that a unique signal is likely to be
generated for each sample sequence, and second, resolution, that
is, that there not be so many primer pairs hitting with very
similar lengths in a sample that the signals cannot be
discriminated. For sufficient redundancy, it is preferable that
there be on average, approximately three pair hits per gene or DNA
sequence in the sample. It is highly preferable that there be at
least one pair hit per each gene In test of a database of
eukaryotic expressed sequences, it has been found that an average
value of three hits per gene appears to be generally a sufficient
guarantee of this minimum criterion.
[0194] Sufficient resolution depends on the separation and
detection means chosen. For a particular choice of separation and
detection means, a recognition reaction preferably should not
generate more fragments than can be separated and distinguishably
detected. In a preferred embodiment, gel electrophoresis is the
separation means used to separate DNA fragments by length. Existing
electrophoretic techniques allow an effective resolution of three
base pair ("bp") length differences in sequences of up to 1000 bp
length. Given knowledge of fragment base composition, effective
resolution down to 1 bp is possible by predicting and correcting
for the small differences in mobility due to differing base
composition. However and without limitation, an easily achievable
three bp resolution is assumed by way of example in the description
of the invention herein. It is preferable for increased detection
efficiency that the distinguishably labeled products from as many
recognition reactions as possible be combined for separation in one
gel lane. This combination is limited by the number of labels
distinguishable by the employed detection means. Any alternative
means for separation and detection of DNA fragments by length,
preferably with resolution of three bp or better, can be employed.
For example, such separation means can be thick or thin plate or
column electrophoresis, column chromatography or HPLC, or physical
means such as mass spectroscopy.
[0195] The redundancy and resolution criteria are probabilistically
expressed in Eqns. 1 and 2 in an approximation adequate to guide
subsequence choice. In these equations the number of genes in the
cDNA sequence mixture is N, the average gene length is L, the
number of target subsequence pairs is M (the number of pairs of
recognition means), and the probability of each target subsequence
hitting a typical gene is p. Since each target subsequences is
preferably selected to independently hit each pooled sequence, the
probability of an arbitrary subsequence pair hitting is then
p.sup.2. Eqn. 1 expresses the redundancy condition of three hits
per gene, assuming the probabilities of target subsequence hits are
independent. 3 Mp 2 = 3 ( 1 )
[0196] Eqn 2 expresses the resolution condition of having fragments
with lengths no closer on average than 3 base pairs. This equation
approximates the actual fragment length distribution with a uniform
distribution. 4 L Np 2 = 3 ( 2 )
[0197] Given expected values of N, the number of sequences in the
library or pool to analyze (library complexity), and L, the average
expressed sequence (or gene) length, Eqns 1 and 2 are solved for
the subsequence hit probability and number of subsequences
required. This solution depends on the particular redundancy and
resolution criteria dictated by the particular experimental method
chosen to implement QEA. Alternative values may be required for
other implementations of this embodiment.
[0198] For example, it is estimated that the entire human genome
contains approximately 10.sup.5 protein coding sequences with an
average length of 2000. The solution of Eqns 1 and 2 for these
parameters is p=0.082 and M=450. Thereby the gene expression of all
genes in all human tissues can be analyzed with 450 target
subsequence pairs, each subsequence having an independent
probability of occurrence of 8.2%. In an embodiment in which eight
fluorescently labeled subsequence pairs can be optically
distinguished and detected per electrophoresis lane, such as is
possible when using the separation and detection apparatus
described in copending U.S. patent application Ser. No. 08/438,231
filed May 9, 1995, 450 reactions can be analyzed in only 57 lanes.
Thereby only one electrophoresis plate is needed in order to
completely determine all human genome expression levels. Since the
best commercial machines known to the applicants can discriminate
only four fluorescent labels in one lane, a corresponding increase
in the number of lanes is required to perform a complete genome
analysis with such machines.
[0199] As a further example, it is estimated that a typically
complex human tissue expresses approximately 15,000 genes. The
solution for N=15000 and L=2000 is p=0.21 and M= 68. Thus
expression in a typical tissue can be analyzed with 68 target
subsequence pairs, each subsequence having an independent
probability of occurrence of 21%. Assuming 4 subsequence pairs can
be run per gel electrophoresis lane, the 68 reactions can be
analyzed in 17 lanes in order to determine the gene expression
frequencies in any human tissue. Thus it is clear that this method
leads to greatly simplified quantitative gene expression analysis
within the capabilities of existing electrophoretic systems.
[0200] These equations provide an adequate guide to picking
subsequence pairs. Typically, preferred probabilities of target
subsequence occurrence are from approximately 0.01 to 0.30.
Probabilities of occurrence of subsequences and RE recognition
sites can be determined from databases of DNA sample sequences.
Example 6.2 lists these probabilities for exemplified RE
recognition sites. Appropriate target subsequences can be selected
from these tables. Computer implemented QEA experimental design
methods can then optimize this initial selection.
[0201] Another use of QEA is to compare directly the expression of
only a few genes, typically 1 to 10, between two different tissues,
the query mode, instead of seeking to determine the expression of
all genes in a tissue, the tissue ode. In this query mode, a few
target subsequences are selected to identify the genes of interest
both among themselves and from all other sequences possibly
present. The computer design methods described hereinbelow can make
this selection. If 4 subsequence pairs are sufficient for
identification, then the fragments from the 4 recognition reactions
performed on each tissue are preferably separated and detected on
two separate lanes in the same gel. If 2 subsequence pairs are
sufficient for identification, the two tissues are preferably
analyzed in the same gel lane. Such comparison of signals from the
same gel improves quantitative results by eliminating measurement
variability due to . differences between separate electrophoretic
runs. For example, expression of a few target genes in diseased and
normal tissue samples can be rapidly and reliably analyzed.
[0202] The query mode of QEA is also useful even if the sequences
of the particular genes of interest are not yet known. For example,
fluorescent traces produced by subjecting separate samples to gel
electrophoretic separation means and then fluorescent detection
means are compared to identify feature differences. Such
differentially expressed features created in a particular
recognition reaction are then retrieved from the gel by methods
known in the art (e.g. electro-elution from the gel) and their
contained DNA fragments are analyzed by conventional techniques,
such as by sequencing. If partial, such sequences can then be used
as probes (e.g. in PCR or Southern blot hybridization) to recover
full-length sequences. In this manner, QEA techniques can guide the
discovery of new differentially expressed cDNA or of changes of the
state of gDNA. The sequences of the newly identified genes, once
determined, can then be used to guide QEA target subsequence choice
for further analysis of the differential expression of the new
genes.
[0203] Three specific embodiments of the QEA method are described
herein. These embodiments differ in how probing is performed by
recognition means to recognize the selected target subsequences.
There are also certain secondary consequential differences in how
the signals are generated from the recognition means. For the PCR
implementation of the QEA method, the target subsequences are
recognized by oligomers which hybridize to a DNA sequence to be
analyzed and act as PCR primers for the amplification of the
segments between adjacent primer pairs. Amplified fragments from a
sample are preferably separated by electrophoresis. Selection of
target subsequences, or primer binding sites, meeting the
probability of occurrence and independence criteria is preferably
made from a database containing sequences expected to be present in
the samples to be analyzed, for example human GenBank sequences,
and optimized by the experimental design methods. Subsequence
selection begins by compiling oligomer frequency tables containing
the frequencies of, preferably, all 4 to 8-mers by using a sequence
database. From these tables, target subsequences with the necessary
probabilities of occurrence are selected and checked for
independence, by, for example, checking that the conditional
probability for a hit by any selected pair of subsequences is the
product of the probabilities of the individual subsequence hit
probabilities. The initial choice can be optimized to determine
target subsequence sets producing unique fragments from the
greatest number of genes, that is so that each sequence uniquely
produces at least one signal. PCR primers are synthesized with a 3'
end complementary to the chosen subsequences and used in the PCR
embodiment. Example 6.1 illustrates the signals output by this
method in a specific example.
[0204] The other two specific embodiments described herein use REs
to recognize and cleave target subsequences in the sample DNA. In
one implementation, the desired doubly cut fragments are amplified
by an amplification means in order to dilute remaining, unwanted
singly cut fragments. Alternatively, the singly cut fragments are
removed by physical means (e.g. hydroxyapatite column separation)
or enzymatic means (e.g. single strand specific nucleases). In
another implementation, the unwanted singly cut ends are removed by
a removal means from the desired doubly cut fragments without an
amplification step, as described in .sctn. 5.2.2. For these
implementations, RE recognition sites define the possible target
subsequences and are selected in a manner similar to the above in
order to meet the previous probability or occurrence and
independence criteria. The probabilities of occurrence of various
RE recognition sites are determined from a database of potential
sample sequences, and those REs are chosen with recognition
sequences whose probabilities of occurrence meet the criterion of
Eqns 1 and 2 as closely as possible. If multiple REs satisfy the
selection criteria, a subset is selected by including only those
REs with independently occurring recognition sequences, determined,
for example in the previous manner using conditional probabilities.
An initial choice can be optionally optimized by the computer
implemented experimental design methods.
[0205] A number, R.sub.e, of REs are preferably selected so that
the number of RE pairs is approximately M, where the relation
between M and R.sub.e is given by Eqn 3. 5 M = R e ( R e + 1 ) 2 (
3 )
[0206] For example, a set a set of 20 acceptable REs results in 210
subsequence pairs.
[0207] There are numerous REs currently available whose recognition
sequences have a wide range of occurrence probabilities, from which
REs can be selected for the present invention. A sample of these
are presented in Example 6.2.
[0208] The PCR and the RE embodiments have different accuracy and
flexibility characteristics. The RE embodiments are generally more
accurate, with fewer false positive and negative identifications,
since the RE/ligase recognition reaction is generally more specific
than the hybridization of PCR primers to their short subsequence
targets, even under stringent hybridization conditions.
[0209] Restriction endonucleases ("RE") generally bind with
specificity only to their short four to eight bp recognition sites,
cleaving the DNA preferably with 4 bp complementary sequences. It
is preferable that REs used in this embodiment produce overhangs
characteristic of the particular RE. Thus REs, such as those known
as class IIS restriction enzymes, which produce overhangs of
unknown sequence are less preferable. Further, ligases, which are
used in an embodiment of the invention to ligate an adapter strand
to a digested terminus, are highly specific in their hybridization
requirements; even one bp mismatch near the ligation site will
prevent ligation (U.S. Pat. No. 5,366,877, Nov. 22, 1994, to Keith
et al.; U.S. Pat. No. 5,093,245, Mar. 3, 1992, to Keith et al.).
PCR and the preferred Taq polymerase used therein tolerates
hybridization mis-matches of elongation primers. Thus the PCR
embodiments may generate false positive signals which arise for
mis-matches in the hybridization of the oligomer probes with target
subsequences.
[0210] However, the PCR embodiments are more flexible since any
desired subsequences can be targets, while the RE embodiments are
limited to the recognition sequences of acceptable REs. However,
more than 150 to 200 REs are now commercially available recognizing
a wide variety of nucleotide sequences.
[0211] QEA experiments are also adaptable to distinguish sequences
into small sets, typically comprising 2 to 10 sequences, which
require fewer target subsequence pairs. Such coarser grain analysis
of gene expression or genomic composition requires fewer
recognition reactions and analysis time. Alternatively, smaller
numbers of target subsequence pairs can be optimally chosen to
distinguish individually a specific set of genes of interest from
all the other genes in the sample. These target subsequences can be
chosen either from REs that produce fragments from the desired
genes or, in the case of the PCR embodiment, from a more complete
set of subsequences optimized for this smaller set of DNA
sequences.
[0212] Detailed descriptions of exemplary implementations for
practicing the QEA recognition reactions and the computer
implemented experimental analysis and design methods are presented
in the following subsections followed by detailed experimental
protocols in Examples subsections. The implementations are
illustrative and not limiting, as this embodiment of the invention
may be practiced by any method generating the previously described
QEA signals.
5.2 RE EMBODIMENTS OF QEA
[0213] The restriction endonuclease ("RE") embodiments of the QEA
method use novel implementations of simultaneous RE and ligase
enzymatic reactions for generating labeled fragments of the genes
or sequences to be analyzed. These fragments are then separated by
length by a separation means and detected by a detection means to
yield QEA signals comprising the identity of the REs cutting each
fragment together with each fragment's length. The recognition
reactions can specifically and reproducibly generate QEA signals
with good signal to noise ratios and without any intermediate
extractions or buffer exchanges, which would hinder automatic
execution.
[0214] REs bind with specificity to short DNA target subsequences,
usually 4 to 8 bp long, that are termed recognition sites and are
characteristic of each RE. REs that are used cut the sequence at
(or near) these recognition sites preferably producing
characteristic ("sticky") ends with single-stranded overhangs,
which usually incorporate part of the recognition site.
[0215] Preferred REs have a 6 bp recognition site and generate a 4
bp 5' overhang. The RE embodiments are also adaptable to a 2 bp 5'
overhang, which is less preferred since 2 bp overhangs have a lower
ligase substrate activity than 4 bp overhangs. All RE embodiments
can be adapted to 3' overhangs of two and four bp. Further
preferred REs have the following additional properties. Their
recognition sites and overhang sequences are preferably such that
an adapter can be designed whose ligation does not recreate the
recognition site. They preferably have sufficient activity below
37.degree. C. and are heat inactivated at 65.degree. C. Heat
inactivation is preferable so that RE inactivation can be performed
prior to adding PCR reagents and conducting the PCR reaction in the
same vial. They preferably have low non-specific cutting and
nuclease activities and cut to completion. Of course, REs selected
for a particular experiment preferably have recognition sites
meeting the previously described occurrence and independence
criteria.
[0216] Preferred pair of REs for analyzing human and mouse cDNA are
listed on .sctn. 6.9.
[0217] Only doubly cut sequence fragments are of interest, and thus
in all RE QEA embodiments the desired doubly cut fragments are
distinguished from the unwanted singly cut fragments. Singly cut
fragments have a non-specific and non-reproducible length
distribution derived from the distribution of overall cDNA lengths,
which depends strongly on cDNA synthesis conditions. Only the
doubly cut fragments have a specific and reproducible length
distribution dependent only on the DNA sequence analyzed and
independent of cDNA synthesis conditions. To make this distinction,
the preferred RE embodiment of QEA exponentially amplifies doubly
cut fragments, so that their signals quickly overwhelm signals from
singly cut fragments, which are at most linearly amplified. PCR is
the preferred amplification means.
[0218] Alternative amplification means known in the art are
adaptable to this invention. If a removal means for singly cut ends
is not utilized in an embodiment, alternative amplification means
must preferentially amplify doubly cut ends over singly cut ends in
order that signals from singly cut ends be relatively suppressed.
On the other hand, if a removal means for singly cut ends is
utilized in an embodiment, then alternative amplification means
need have no amplification preference, as no singly cut ends are
present at the amplification step. Known alternative amplification
means are listed in Kricka et al., 1995, Molecular Probing,
Blotting, and Sequencing, chap. 1 and table IX, Academic Press, New
York. Of these alternative means, those employing the T7 RNA
polymerase are preferred.
[0219] The other two specific embodiments use a physical removal
means to directly remove singly cut fragments, preferably before
amplification. This can be accomplished, e.g., by labeling DNA
termini with a capture moiety prior to digestion. After digestion,
the singly cut fragments are removed by contacting the sample with
a binding partner of the capture moiety, affixed to a solid phase.
The preferred removal means is biotin-streptavidin. Other removal
means adaptable to this invention include various haptens; which
are removed by their corresponding antibodies. Exemplary haptens
include digoxigenin, DNP, and fluorescein (Holtke et al., 1992,
Sensitive chemiluminescent detection of digoxigenin labeled nucleic
acids: a fast and simple protocol for applications, Biotechniques,
12(1):104-113 and Olesen et al., 1993, Chemiluminescent DNA
sequencing with multiple labeling, Biotechniques, 15(3):480-485).
Alternately, single stranded fragments can be removed by single
stand specific column separation or single strand specific
nucleases.
[0220] RE embodiments of QEA use recognition moieties which are
specifically ligated to RE cut sticky ends so that in any one
recognition reaction ends cut by a particular RE receive a unique
moiety. Recognition moieties comprise oligomers capable of
specifically hybridizing to the RE generated sticky ends. In the
preferred RE embodiment, which uses PCR amplification, the
recognition moieties also provide primer means for the PCR.
[0221] The recognition moieties also provide for labeling and
recognition of RE cut ends. For example, using a pair of REs in one
recognition reaction generates doubly cut fragments some with the
recognition sequence of the first RE on both ends, some with the
recognition sequence of the second RE on both ends, and the
remainder with one recognition sequence of each RE on either end.
Using more REs generates doubly cut fragments with all pairwise
combinations of RE cut ends from adjacent RE recognition sites
along the sample sequences. All these cutting combinations need
preferably to be distinguished, since each provides unique
information on the presence of different. subsequences pairs
present in the original DNA sequence. Thus the recognition moieties
preferably have unique labels which label specifically each RE cut
made in a reaction. As many REs can be used in a single reaction as
labeled recognition moieties are available to uniquely label each
RE cut. If the detectable labeling in a particular system is, for
example, by fluorochromes, then fragments cut with one RE have a
single fluorescent signal from the one fluorochrome associated with
that RE, while fragments cut with two REs have mixed signals, one
from the fluorochrome associated with each RE. Thus all possible
pairs of fluorochrome labels are preferably distinguishable.
Alternatively, if certain target subsequence information is not
needed, the recognition moieties need not be distinctively labeled.
In embodiments using PCR amplification, corresponding primers would
not be labeled.
[0222] If silver staining is used to recognize fragments separated
on an electrophoresis gel, no recognition moiety need be labeled,
as fragments cut by the various RE combinations are not
distinguishable. In this case, when PCR amplification is used, only
primers are required.
[0223] The recognition reaction conditions are preferably selected,
as described in .sctn. 6.4, so that RE cutting and recognition
moiety ligation go to full completion: all recognition sites of all
REs in the reaction are cut and ligated to a recognition moiety. In
this manner, the fragments generated from a sequence analyzed lie
only between adjacent recognition sites of any RE in that reaction.
No fragments remain which include any RE recognition site, since
such a site is cut. Multiple REs can be used in one recognition
reaction. Too many REs in one reaction may cut the sequences too
frequently, generating a compressed length distribution with many
short fragments of lengths between 10 and a few hundred base pairs
long. Such a distribution may not be resolvable by the separation
means, for example gel electrophoresis, if the fragments are too
close in length, for example less than 3 bp apart on the average.
Too many REs also may generate fragments of the same length and end
subsequences from different sample sequences, thereby leading to
non-unique signals. Finally, where fragment labels are to be
distinguished, no more REs can be used than can have
distinguishably labeled sticky ends. These considerations limit the
number of REs optimally useable in one recognition reaction.
Preferably two REs are used, with one, three and four REs less
preferable. Preferable pairs of REs for the analysis of human cDNA
samples are listed in .sctn. 6.9.
[0224] An additional level of signal specificity is possible by
selecting or suppressing fragments having a third internal target
subsequence. Additional information on the presence or absence of
specific internal subsequences can be used along with the two end
subsequences and the length information to further distinguish
between otherwise identically classified fragments.
[0225] To select fragments with a third internal subsequence,
probes with distinguishable labels which bind to this target
subsequence are added to the fragments prior to detection, and
alternatively prior to separation and detection. On detection,
fragments with this third subsequence present will generate a
signal, preferably fluorescent, from the probe. Such a probe could
be a labeled PNA or DNA oligomer. Short DNA oligomers may need to
be extended with a universal nucleotide or degenerate sets of
natural nucleotides in order to provide for specific
hybridization.
[0226] Fragments with a third subsequence can be suppressed in
various manners in embodiments using PCR amplification. First, a
probe hybridizing with this third subsequence which prevents
polymerase elongation in PCR can be added prior to amplification.
Then sequences with this subsequence will be at most linearly
amplified and their signal thereby suppressed. Such a probe could
be a PNA or modified DNA oligomer (with the last nucleotide being a
ddNTP). Second, if the third subsequence is recognized by an RE,
this RE can be added to the RE-ligase reaction without any
corresponding specific primer. Fragments with the third subsequence
will be at most linearly amplified.
[0227] Both these alternatives can be extended to multiple internal
sequences by using multiple probes to recognize the sequences or to
disrupt exponential PCR amplification.
[0228] Construction of the recognition moieties, also herein called
adapters or linker-primers, is important and is described here in
advance of further details of the individual recognition reaction
steps. In the preferred embodiment, the adapters are partially
double stranded DNA ("dsDNA"). Alternatively, the adapters can be
constructed as oligomers of any nucleic acid, with corresponding
properties to the preferred DNA polymers. In an embodiment
employing an alternative amplification means, any polymer that can
serve with a template as a primer for that amplification means can
be used in that embodiment.
[0229] FIG. 2A illustrates the DNA molecules involved in the
ligation reaction as conventionally indicated with the 5' ends of
the top strands and the 3' ends of the bottom strands at left.
dsDNA 201 is a fragment of a sample cDNA sequence with an RE cut at
the left end generating, preferably, a four bp 5' overhang 202.
Adapter dsDNA 209 is a synthetic substrate provided by this
invention.
[0230] The precise characteristics of adapter 209 are selected in
order to ensure that RE digestion and adapter ligation preferably
go to completion, that generation of unwanted products and
amplification biases are minimized, and that unique labels are
attached to cut ends (if needed). Adapter 209 comprises strand 203,
called a primer, and a partially complementary strand 205, called a
linker. The primer is also known as the longer strand of the
adapter, and the linker is also known as the shorter strand of the
adapter.
[0231] The linker, or shorter strand, links the end of a cDNA cut
by an RE to the primer, or longer strand, by hybridization to the
sticky overhang of the cut end and to the primer in order that the
primer can be ligated to dsDNA 201. Therefore, linker 205 comprises
sequence 206 complementary to the sticky RE overhang 202 and
sequence 207 complementary to the 3' end of primer 203. Sequence
206 is preferably of the same length as the RE overhang. Sequence
207 is most preferably eight nucleotides long, less preferably from
4 to 12 nucleotides long, but can be of any length as long as the
linker reliably hybridizes with only one top primer in any one
recognition reaction and has an appropriate T.sub.m (preferably
less than approximately 68.degree. C.). Linker 205 also preferably
has no 5' terminal phosphate so that it will not ligate to the
bottom strand of dsDNA 201. Lack of terminal phosphate also
prevents the annealed adapters from ligating to each other, forming
dimers, and thereby competing with adapter ligation to RE cut
sample fragments. Adapter dimers would also be amplified in a
subsequent amplification step generating unwanted fragments.
Terminal phosphates can be removed using phosphatases known in the
art, followed by separation of the enzyme. An exemplary protocol
for an alkaline phosphatase reaction is found in .sctn. 6.4.1.
[0232] Further, the linker, or shorter strand, T.sub.m should
preferably be less than primer 203 self-annealing T.sub.m. This
ensures that subsequent PCR amplification conditions can be
controlled so that linkers present in the reaction mixture will not
hybridize and act as PCR primers, and, thereby, generate spurious
fragment lengths. The preferable T.sub.m is less than approximately
68.degree. C.
[0233] Primer, or longer strand, 203 further has a 3' end sequence
204 complementary to 3' end sequence 207 of bottom linker 205. In a
preferred aspect, in order that all RE cuts are properly ligated to
a unique top primer, in any single reaction, each primer should be
complementary to and hybridize with only one linker 205.
Consequently, all the linkers in any one reaction mixture
preferably have unique sequences 207 for hybridizing with unique
primers. In order that the ligation reaction go to completion,
primer 203 preferably should not recreate the recognition sequence
of any RE in the reaction mixture when it is ligated with cDNA end
202. Primer 203 has no 5' terminal phosphate in order to prevent
any self-ligations. To minimize amplification of undesired
sequences, termed amplification noise, in any subsequent PCR step
it is preferred that primer 203 not hybridize with any sequence
present in the original sample mixture. The T.sub.m of primer 203
is preferably high, in the range from 50.degree. to 80.degree. C.,
and more preferably above 68.degree. C. This ensures that the
subsequent PCR amplification can be controlled so that only primers
and not linkers initiate new chains. For example, this T.sub.m can
be achieved by use of a primer having a combination of a G+C
content preferably from 40-60%, most preferably from 55-60%, and a
primer length most preferably 24 nucleotides, and preferably from
18 to 30 nucleotides. Primer 203 is optionally labeled with
fluorochrome 208, although any DNA labeling system that preferably
allows multiple labels to be simultaneously distinguished is usable
in this invention.
[0234] Generally, the primer, or longer strand, are constructed so
that, preferably, they are highly specific, free of dimers and
hairpins, and form stable duplexes under the conditions specified,
in particular the desired T.sub.m. Software packages are available
for primer construction according to these principles, an example
being OLIGO.TM. Version 4.0 For Macintosh from National
Biosciences, Inc. (Plymouth, Minn.). In particular, a formula for
T.sub.m can be found in the OLIGO.TM. Reference Manual at Eqn. I,
page 2.
[0235] FIG. 2B illustrates two exemplary adapters and their
component primers and linkers constructed according to the above
description. Adapter 250 is specific for the RE BamHI, as it has a
3' end complementary to the 5' overhang generated by BamHI. Adapter
251 is similarly specific for the RE HindIII.
[0236] Example 6.9 contains a more comprehensive, non-limiting list
of adapters that can be used according to the invention. All
synthetic oligonucleotides of this invention are preferably as
short as possible for their functional roles in order to minimize
synthesis costs.
[0237] Alternatively, adapters can be constructed from hybrid
primers which are designed to facilitate the direct sequencing of a
fragment or the direct generation of RNA probes for in situ
hybridization with the tissue of origin of the DNA sample analyzed.
Hybrid primers for direct sequencing are constructed by ligating
onto the 5' end of existing primers the M13-21 primer, the M13
reverse primer, or equivalent sequences. Fragments generated with
such hybrid adapters can be removed from the separation means and
amplified and sequenced with conventional systems. Such sequence
information can be used both for a previously known sequence to
confirm the sequence determination and for a previously unknown
sequence to isolate the putative new gene. Hybrid primers for
direct generation of RNA hybridization probes are constructed by
ligating onto the 5' end of existing primers the phage T7 promoter.
Fragments generated with such hybrid adapters can be removed using
the separation means and transcribed into anti-sense RNA with
conventional systems. Such probes can be used for in situ
hybridization with the tissue of origin of the DNA sample to
determine in precisely what cell types a signal of interest is
expressed. Such hybrid adapters are illustrated in .sctn.
6.7.1.
[0238] A further alternative illustrated in FIG. 2C is to construct
an adapter by self hybridization of single stranded DNA in hairpin
loop configuration 212. The subsequences of loop 212 would have
similar properties to the corresponding subsequences of linker 205
and primer 203. Exemplary hairpin loop 211 sequences are C.sub.4 to
C.sub.10.
[0239] REs generating 3' overhangs are less preferred and require
the different adapter structure illustrated in FIG. 3A. dsDNA 301
is a fragment of a sample cDNA cut with a RE generating 3' sticky
overhang 302. Adapter 309 comprises primer, or longer strand, 304
and linker, or shorter strand, 305. Primer, or longer strand, 304
includes segment 306 complementary to and of the same length as 3'
overhang 302 and section 307 complementary to linker 305. It also
optionally has label 308 which distinctively labels primer 304. As
in the case of adapters for 5' overhangs, primer 304 has no 5'
terminal phosphate, in order to prevent self-ligations, and is such
that no recognition site for any RE in one recognition reaction is
created upon ligation of the primer with dsDNA 301. These condition
ensure that the RE digestion and ligation reactions go to
completion. Primer 304 should preferably not hybridize with any
sequence in the initial sample mixture. The T.sub.m of primer 304
is preferably high, in the range from 50.degree. to 80.degree. C.,
and more preferably above 68.degree. C. This ensures the subsequent
PCR amplification can be controlled so that only primers and not
linkers initiate new chains. For example, this T.sub.m can be
achieved by using a primer having a G+C content preferably from
40-60%, most preferably from 55-60%, and a primer length most
preferably of 24 nucleotide and less preferably of 18-30
nucleotides. Each primer 304 in a reaction can optionally have a
distinguishable label 308, which is preferably a fluorochrome.
[0240] Linker, or shorter strand, 305 is complementary to and
hybridizes with section 307 of primer 304 such that it is adjacent
to 3' overhang 302. Linker 305 is most preferably 8 nucleotides
long, less preferably from 4-16 nucleotides, and has no terminal
phosphates to prevent any self-ligation. This linker serves only to
promote ligation specificity and reaction speed. It does not
perform the function of linking primer 304 to the cut dsDNA, as it
did in the 5' case. Further, linker 305 T.sub.m should preferably
be less than primer 304 self-annealing T.sub.m. This insures that
subsequent PCR amplification conditions can be controlled so that
linkers present in the reaction mixture will not hybridize and act
as PCR primers, and, thereby, generate spurious fragment
lengths.
[0241] FIG. 3B illustrates an exemplary adapter with its primer and
linker for the case of the RE NlaIII. As in the 5' overhang case, a
3' adapter can also be constructed from a hairpin loop
configuration.
[0242] REs generating 5' and 3' overhangs are preferably not used
in the same recognition reaction. This is in order that a
complementary primer hybridization site can be presented on each of
the two strands of the product of the RE/ligase recognition
reaction.
[0243] Turning now to a detailed description of a preferred RE
embodiment of the QEA recognition reactions, the steps of this
preferred embodiment comprise, first, simultaneously cleaving a
mixed DNA sample with one or more REs and ligating recognition
moieties on the cut ends, second, amplifying the twice cut
fragments, if necessary, and third, separating the fragments by
length and detecting the lengths and labels, and the identities of
the REs cutting each fragment. If necessary, prior to the first
step, the cDNA sample is prepared by methods commonly known in the
art or as described in .sctn..sctn. 6.3 and 6.4.1. Following the
amplification step, optional steps to remove unwanted singly
stranded DNA fragments prior to detection can increase the signal
to noise ratio of the following detection. Two alternative RE
embodiments are described in following subsections. The number of
REs and associated adapters preferably are limited so that both a
compressed length distribution consisting of shorter fragments is
avoided and enough distinguishable labels are available for all the
REs used. Alternatively, REs can be used without associated
adapters in order that the amplified fragments not have the
associated recognition sequences. Absence of these sequences can be
used to additionally differentiate genes that happen to produce
fragments of identical length with particular REs.
[0244] In more detail, a cDNA preparation step may start with a
preexisting cDNA sample or with a tissue sample. When cDNA is
prepared from tissue samples, the exemplary methods and procedures
of Example 6.3 can be used. These consist of largely conventional
steps of RNA preparation from the tissue sample, preferably poly(A)
purified RNA is used but less preferably total cellular RNA can be
used, RNase extraction, DNase treatment; mRNA purification, and
first and second strand cDNA synthesis. Cloning into a vector is
not necessary.
[0245] The final preparation step of a DNA sample is removal of
terminal phosphates from all the cDNA. This is important to improve
the signal to noise ratio in the subsequent fragment length
separation and detection by eliminating amplification of unwanted,
singly cut fragments. Significant background signals arise from
exponential amplification of singly cut fragments whose blunt ends
have ligated to form a single dsDNA with two cut ends, an
apparently doubly cut fragment, which is exponentially amplified
like a normal doubly cut fragment. Since cDNA lengths vary
depending on synthesis condition, these unwanted, apparently doubly
cut fragments have a wide range of lengths and produce a diffuse
background on gel electrophoresis which obscures sharp bands from
the normally doubly cut fragments. This background can be
eliminated by preventing blunt end ligation of singly cut fragments
by initially removing all terminal phosphates from the cDNA sample,
without otherwise disrupting the integrity of the cDNA.
[0246] Terminal phosphate removal is preferably done with a
phosphatase. To prevent interference with the intended ligation of
adapters to doubly cut fragments, the phosphatase activity
preferably is removed prior to the RE digestion and adapter
ligation step. To avoid any phosphatase separation or extraction
step, the preferred phosphatase is a heat labile alkaline
phosphatase which is heat inactivated prior to the RE/ligase step.
A preferred phosphatase comes from cold living Barents Sea (arctic)
shrimp (U.S. Biochemical Corp.) ("shrimp alkaline phosphatase" or
"SAP"). Terminal phosphate removal need be done only once for each
population of cDNA being analyzed.
[0247] In other embodiments additional phosphatases my be used for
terminal phosphate removal, such as calf intestinal
phosphatase-alkaline from Boehringer Mannheim (Indianapolis, Ind.).
Those that are not heat inactivated require the addition of a step
to separate the phosphatase from the cDNA before the recognition
reactions, such as by phenol-chloroform extraction.
[0248] Preferably, the prepared cDNA is then separated into batches
of from 1 picogram ("pg") to 200 nanograms ("ng") of cDNA each, and
each batch is separately processed by the further steps of the
method. For a tissue mode experiment, to analyze gene expression,
preferably from a majority of expressed genes, from a single human
tissue requires determination of the presence of about 15,000
distinct cDNA sequences. By way of example, one sample is divided
into approximately 50 batches, each batch is then subject to the
RE/ligase recognition reaction and generates approximately 200-500
fragments, and more preferably 250 to 350 fragments of 10 to 1000
bp in length, the majority of fragments preferably having a
distinct length and being uniquely derived from one cDNA sequence.
A preferable example analysis would entail 50 batches generating
approximately 300 bands each.
[0249] For the query mode, fewer recognition reactions are employed
since only a subset of the expressed genes are of interest, perhaps
approximately from 1 to 100. The number of recognition reactions in
an experiment may then number approximately from 1 to 10 and an
appropriate number of cDNA batches is prepared.
[0250] Following cDNA preparation, the next step is simultaneous RE
cutting of and adapter ligation to the sample cDNA sequences. The
prepared sample is cut with one or more REs. The amount of RE
enzyme in the reaction is preferably approximately a 10 fold unit
excess. Substantially greater quantities are less preferred because
they can lead to star activity (non-specific cutting) while
substantially lower quantities are less preferred because they will
result in less rapid and only partial digestion, and hence
incomplete and inaccurate characterization of the subsequence
distribution.
[0251] In the same reaction, adapters and ligase enzyme are present
for simultaneous adapter ligation to the RE cut ends. The method is
adaptable to any ligase that is active in the temperature range 10
to 37.degree. C. T4 DNA ligase is the preferred ligase. In other
embodiments, cloned T4 DNA ligase or T4 RNA ligase can also be
used. In a further embodiment, thermostable ligases can be used,
such as Ampligase.TM. Thermostable DNA Ligase from Epicenpre
(Madison, Wis.), which has a low blunt end ligation activity. These
ligases in conjunction with the repetitive cycling of the basic
thermal profile for the RE-ligase reaction, described in the
following, permit more complete RE cutting and adapter
ligation.
[0252] Ligase activity can both generate unwanted products and
also, if an RE recognition site is regenerated, can cause an
endless cycle of further cutting and ligation. Terminal phosphate
removal during cDNA preparation prevents spurious ligation of the
blunt other ends of singly cut cDNA (and subsequent exponential
amplification of the results). Other unwanted products are fragment
concatamers formed when the sticky ends of cut cDNA fragments
hybridize and ligate. Such fragment concatamers are removed by
keeping the restriction enzymes active during ligation, thus
cutting unwanted concatamers once they form. Further, adapters,
once ligated, terminate further RE cutting, since adapters are
selected such that RE recognition sites are not recreated. A high
molar excess of adapters also is preferable since it limits
concatamer formation by driving the RE and ligase reactions toward
complete digestion and adapter ligation. Finally, unwanted adapter
self-ligation is prevented since primers and linker also lack
terminal phosphates (preferably due to synthesis without phosphates
or less preferably due to pretreatment thereof with
phosphatases).
[0253] The temperature profile of the RE/ligase reaction is
important for achieving complete cutting and ligation. The
preferred protocol has several stages. The first stage is at the
optimum RE temperature to achieve substantially complete cutting,
for example 37.degree. C. for 15 minutes. The second stage is a
ramp at -1.degree. C./min down to a temperature for substantially
compete annealing of adapters to the 4 bp sticky cut ends, for
example at 10.degree. C. During this ramp cutting and ligation
continue. The third stage is at the optimum temperature for adapter
annealing and ligation to the sticky ends. The fourth stage
achieves substantially complete ligation of cut products, and is,
for example, at 16.degree. C. for 30 minutes. The fifth stage is
again at the optimum RE to achieve complete cutting of all
recognition sites, for example at 37.degree. C. for 10 minutes. The
sixth stage is to heat inactivate the ligase and, preferably, also
the RE enzymes, and is, for example, 10 minutes at 65.degree. C.
The results are held at 4.degree. C.
[0254] A less preferred profile involves repetitive cycling of the
first five stages of the temperature protocol described above, that
is from an optimum RE temperature to optimum annealing and ligation
temperatures, and back to an optimum RE temperature. The additional
cycles further drive the RE/ligase reactions to completion. In this
embodiment, it is preferred to use thermostable ligase enzymes. The
majority of restriction enzymes are active at the conventional
16.degree. C. ligation temperature and hence prevent unwanted
ligation events without thermal cycling. However, temperature
profiles consisting of optimum ligation conditions interspersed
with optimum RE cutting conditions cause both enzymatic reactions
to proceed more rapidly than one constant temperature. An exemplary
profile comprises periodically cycling between a 37.degree. C.
optimum RE temperature to a 10.degree. C. optimum annealing and
ligation temperature at a ramp of -1.degree. C./min, then to a
16.degree. C. optimum ligation temperature, and then back to the
37.degree. C. optimum RE temperature. Following completion of
approximately 2 to 4 of these temperature cycles, the RE and ligase
enzymes are heat inactivated by a final stage at 65.degree. C. for
10 minutes. This avoids the need for separation or extractions
between steps. The results are held at 4.degree. C.
[0255] These thermal profiles are easily controlled and automated
by the use of commercially available computer controlled
thermocyclers, for example from MJ Research (Watertown, Mass.) or
Perkin Elmer (Norwalk, Conn.).
[0256] These reaction conditions are designed to achieve
substantially complete cutting of all RE recognition sites present
in the analyzed sequence mixture and complete ligation of reaction
terminating adapters on the cut ends, each adapter being unique in
one reaction for a particular RE cut end. The fragments generated
are limited by adjacent RE recognition sites and no fragment
includes internal undigested sites. Further, a minimum of unwanted
self-ligation products and concatamers is formed.
[0257] Following the RE/ligase step is amplification of the doubly
cut cDNA fragments. Although PCR protocols are described in the
exemplary embodiment, any amplification method that selects
fragments to be amplified based on end sequences is adaptable to
this invention (see above). With high enough sensitivity of
detection means, or even single molecule detection means, the
amplification step can be dispensed with entirely. This is
preferable as amplification inevitably distorts the quantitative
response of the method.
[0258] The PCR amplification protocol is designed to have maximum
specificity and reproducibility. First, the PCR amplification
produces fewer unwanted products if the amplification steps occur
at a temperature above the T, of the shorter linker so that it
cannot initiate unwanted DNA strands. The linker is preferably
melted by an initial incubation at 72.degree. C. without the Tag
polymerase enzyme or dNTP substrates present. A further incubation
at 72.degree. C. for 10 minutes with Tag polymerase and dNTPs is
performed in order to complete partial double strands to complete
double strands. Alternatively, linker melting and double strand
completion can be performed by a single incubation at 72.degree. C.
for 10 minutes with Tag polymerase. Subsequent PCR amplification
steps are carried out at temperatures sufficiently high to prevent
re-hybridization of the bottom linker.
[0259] Second, primer strand 203 of FIG. 2A (and 304 of FIG. 3A)
are typically used as PCR primers. They are preferably designed for
high amplification specificity and not to hybridize with any native
cDNA species to be analyzed.
[0260] They have high melting temperatures, preferably above
50.degree. C. and most preferably above 68.degree. C., to ensure
specific hybridization with a minimum of mismatches.
[0261] Third, the protocol's temperature profile is preferably
designed for specificity and reproducibility. The preferred profile
is 95.degree. C. for 30 seconds followed by 65.degree. C. for 1
minute. High annealing temperatures minimize primer
mis-hybridizations. Longer extension times reduce PCR bias in favor
of smaller fragments. Longer melting times reduces PCR
amplification bias in favor of high G+C content. Further, large
amplification volumes are preferred to reduce bias. Sufficient
amplification cycles are performed, typically between 15 and 30
cycles.
[0262] Any other techniques designed to raise specificity, yield,
or reproducibility of amplification are applicable to this method.
For example, one such technique is the use of 7-deaza-2'-dGTP in
the PCR reaction in place of dGTP. This has been shown to increase
PCR efficiency for G+C rich targets (Mutter et al., 1995, Nuc. Acid
Res., 23:1411-1418). For a further example, another such technique
is the addition of tetramethylammonium chloride to the reaction
mixture, which has the effect of raising the T.sub.m (Chevet et
al., 1995, Nucleic Acids Research, 23(16):3343-3344).
[0263] In one method of performing the PCR amplification, each
RE/ligase reaction sample is sub-divided into multiple aliquots,
and each aliquot is amplified with a different number of cycles.
Multiple amplifications with an increasing number of amplification
cycles, for example 10, 15, and 20 cycles, are preferable.
Amplifications with a lower number of cycles detect more prevalent
messages in a more quantitative manner. Amplification with a higher
number of cycles detect the presence of less prevalent genes but
less quantitatively. Multiple amplifications also serve as controls
for checking the reliability and quantitative response of the
process by comparing the size of the same signal in each
amplification.
[0264] Other methods of performing the PCR amplification are more
suited to automation. For example, the content of a reaction vial
can be configured as follows. First, 40 .mu.l of the PCR mix
without Mg ions is added followed by a wax bead that melts
approximately at 72.degree. C., such as Ampliwax beads
(Perkin-Elmer, Norwalk, Conn.). This bead is melted at 75.degree.
C. for 5 minutes and solidified at 25.degree. C. for 10 minutes.
Last 10 .mu.l of the RE/ligase mix with Mg ions is added. The
RE/ligase and PCR reactions are carried out by following the
temperature profile in FIG. 16D, which is a concatenation of the
RE/ligase and PCR profiles with an extra 10 minutes at 72.degree.
C. In this arrangement in the same vial, the RE/ligase reactions
can first be performed. The incubation at 72.degree. C. for 20
minutes permits the wax layer separating the mixtures to melt,
allows the RE/ligase mixture to mix with the PCR mix, and allows
completion of the partial double strands to complete double
strands. Then sufficient PCR cycles are performed, typically
between 15 and 30 cycles. This single tube implementation is well
adapted to automation. Other so called PCR "hot-start" procedures
can be used, such as those employing heat sensitive antibodies
(Invitrogen, CA) to initially block the activity of the
polymerase.
[0265] Following the amplification step, optional steps prior to
length separation and detection improve the method's signal to
noise ratio. First, single strands produced as a result of linear
amplification from singly cut fragments can e removed by the use of
single strand specific exonucleases. Mung Bean exonuclease (Exo) or
Exo I can be used, with Exo I referred because of its higher
specificity for single strands. Mung bean is less preferred and
even less preferred is S1 nuclease. Second, the amplified products
may be optionally concentrated by ethanol precipitation or column
separation.
[0266] Alternate PCR primers illustrated in FIG. 2D can be
advantageously used. In that figure, sample dsDNA 201 is
illustrated after the RE/ligase reaction and after incubation at
72.degree. C. for 10 minutes but just prior to the PCR
amplification steps. dsDNA 201 has been cleaved by an RE
recognizing subsequence 227 at position 221 producing overhang 202
and has been ligated to adapter primer strand 203. For definiteness
and without limitation, a particular relative position between RE
recognition subsequence 227 and overhang 202 is illustrated. Other
relative positions are known. The resulting DNA has been completed
to a blunt ended double strand by completing strand 220 by
incubation at 72.degree. C. for 10 minutes. Typically adapter
primer strand 203 is used as the PCR primer.
[0267] Alternatively strand 222, illustrated with its 5' end at the
left, can be advantageously used. Strand 222 comprises subsequence
223, with the same sequence as strand 203; subsequence 224, with
the same sequence as the RE overhang 202; subsequence 225, with a
sequence consisting of a remaining portion of RE recognition
subsequence 227, if any; and subsequence 226 of P nucleotides.
Length P is preferably from 1 to 6 and more preferably either 1 or
2. Subsequences 223 and 224 hybridize for PCR priming with
corresponding subsequences of dsDNA 201. Subsequence 225 hybridizes
with any remainder of recognition subsequence 227. Subsequence 226
hybridizes only with fragments 201 having complementary nucleotides
in corresponding positions 228. When P is 1, primer 223 selects for
PCR amplification 1 of the 4 possible dsDNAs 201 which may be
present; and when P is 2, 1 of the 16 is selected. If 4 (or 16)
primers 223 are synthesized, each with one of the possible (pairs
of) nucleotides, and if the RE/ligase reactions mix is separated in
4 (16) aliquots for use with one of these 4 (16) primers, the 4
(16) PCR reactions will select for amplification only one of the
possible dsDNAs 201. Thus these primers are similar to phasing
primers (European Patent Application No. 0 534 858 A1, published
Mar. 31, 1993).
[0268] The joint result of using primers 223 with subsequence 226
in multiple PCR reactions after one RE/ligase reaction is to extend
the effective target subsequence from the RE recognition
subsequence by concatenating onto the recognition subsequence a
subsequence which is complementary to subsequence 226. Thereby,
many additional target subsequences can be recognized while
retaining the specificity and exactness characteristic of the RE
embodiment. For example, REs recognizing 4 bp subsequences can be
used in such a combined reaction with an effective 5 or 6 bp target
subsequence, which need not be palindromic. REs recognizing 6 bp
sequences can be used in a combined reaction to recognize 7 or 8 bp
sequences. Such effective recognition sequences need to be
accounted for in the computer implemented design and analysis
methods subsequently described.
[0269] The next QEA step is the separation by length of the
amplified, labeled, cut cDNA fragments and observation of the
length distribution. Lengths of the sample of cut fragments will
typically span a range from a few tens of bp to perhaps 1000 bp.
For this range standard gel electrophoresis is capable of resolving
separate fragments which differ by three or more base pairs.
Knowledge of average fragment composition allows for correction of
composition induced small mobility differences and permits
resolution down to 1 bp. Any separation method with adequate length
resolution, preferably at least to three base pairs in a 1000 base
pair sequence, can also be used. The length distribution is
detected with means sensitive to the primer labels. In the case of
fluorochrome labels, since multiple fluorochrome labels can be
typically be resolved from a single band in a gel, the products of
one recognition reaction with several REs or other recognition
means or of several separate recognition reaction can be analyzed
in a single lane. The detection apparatus resolution for different
labels limits the number of RE products that can be simultaneously
detected.
[0270] Preferred protocols for the specific RE embodiments are
described in detail in .sctn. 6.4.
5.2.1. FIRST ALTERNATIVE RE EMBODIMENT
[0271] An alternative QEA protocol performs amplification prior to
the RE/ligase step. After the RE/ligase step, further amplification
is performed. Alternately, no further amplification is performed,
and in this case unwanted singly cut ends are removed as they are
not diluted by subsequent amplification.
[0272] Such removal is accomplished by first using primers that are
labeled with a capture moiety. A capture moiety is a substance
having a specific binding partner that can be affixed to a solid
substrate. For example, suitable capture moiety-binding partner
pairs include but are not limited to biotin-streptavidin,
biotin-avidin, a hapten (such as digoxigenin) and a corresponding
antibody, or other removal means known in the art. For example,
double stranded cDNA, perhaps prepared from a tissue sample
according to Example 6.3, is PCR amplified using a set of
biotin-labeled, arbitrary primers with no net sequence preference.
The result is partial cDNA sequences with biotin labels linked to
both ends. The amplified cDNA is cut with REs and ligated to
recognition moieties uniquely for each particular RE cut end. The
RE/ligase step is performed by procedures identical to those of the
prior section in order to drive the RE digestion and recognition
moiety ligation to completion and to prevent formation of
concatamers and other unwanted ligation products. The recognition
moieties can be the adapters previously described.
[0273] Next the unwanted singly cut fragments labeled with the
capture moiety are removed by contacting them with the binding
partner for the capture moiety affixed to a solid phase, followed
by removal of the solid phase. For example, where biotin is the
capture moiety, singly cut-fragments can be removed using
streptavidin or avidin magnetic beads, leaving only doubly cut
fragments that have RE-specific recognition moieties ligated to
each end. These products are then analyzed, also as in the previous
section, to determine the distribution of fragment lengths and RE
cutting combinations.
[0274] Other direct removal means may alternatively be used in this
invention. Such removal means include but are not limited to
digestion by single strand specific nucleases or passage though a
single strand specific chromatographic column, for example,
containing hydroxyapatite.
5.2.2. SECOND ALTERNATIVE RE EMBODIMENT
[0275] A second alternative embodiment in conjunction with
sufficiently sensitive detection means can eliminate altogether the
amplification step. In the preferred RE protocol, doubly cut
fragments ligated to adapters are exponentially amplified, while
unwanted, singly cut fragments are at best linearly amplified. Thus
amplification dilutes the unwanted fragments relative to the
fragments of interest. After ten cycles of amplification, for
example, signals from unwanted fragments are reduced to less than
approximately 0.1% of the signals from the doubly cut fragments.
Gene expression can then be quantitatively determined down to at
least this level. A greater number of amplification cycles results
in a greater relative dilution of signals from unwanted singly cut
fragments and, thereby, a greater sensitivity. But amplification
bias and non-linearities interfere with the quantitative response
of the method. For example, certain fragments will be
preferentially PCR amplified depending on such factors as length
and average base composition.
[0276] For improved quantitative response, it is preferred to
eliminate the bias accompanying the amplification steps. Then
output signal intensity is linearly responsive to the number of
input genes or sequences generating that signal. In the case of
common fluorescent detection means, a minimum of 6.times.10.sup.-18
moles of fluorochrome (approximately 10.sup.5 molecules) is
required for detection. Since one gram of cDNA contains about
10.sup.-6 moles of transcripts, it is possible to detect
transcripts to at least a 1% relative level from microgram
quantities of mRNA. With greater mRNA quantities, proportionately
rarer transcripts are detectable. Labeling and detection schemes of
increased sensitivity permit use of less mRNA. Such a scheme of
increased sensitivity is described in Ju et al., 1995, Fluorescent
energy transfer dye-labeled primers for DNA sequencing and
analysis, Proc. Natl. Acad. Sci. USA 92:4347-4351. Single molecule
detection means are about 10.sup.5 times more sensitive than
existing fluorescent means (Eigen et al., 1994, Proc. Natl. Acad.
Sci. USA, 91:5740-5747).
[0277] To eliminate amplification steps, a preferred protocol uses
a capture moiety separation means to directly remove singly cut
fragments from the desired doubly cut fragments. Only the doubly
cut fragments have a discrete length distribution dependent only on
the input gene sequences. The singly cut fragments have a broad
non-diagnostic distribution depending on cDNA synthesis conditions.
In this protocol, cDNA is synthesized using a primer labeled with a
capture moiety, is circularized, cut with REs, and ligated to
adapters. Singly cut ends are then removed by contact with a solid
phase to which a specific binding partner of the capture moiety is
affixed.
[0278] FIGS. 4A, 4B, and 4C illustrate a second alternative RE
protocol, which uses biotin as such a capture moiety for direct
removal of the singly cut 3' and 5' cDNA ends from the RE/ligase
mixture. cDNA first strands are synthesized according to the method
of Example 6.3 using, for example, an oligo(dT) primer with a
biotin molecule linked to one of the internal thymidine
nucleotides. For example, such a primer is T.sub.nT(biotin)T.sub.m,
with n approximately equal to m, and with n+m sufficiently large,
approximately 12 to 20, so that the primer will reliably hybridize
to the poly(A) tail of mRNA. Other biotin labeled primers may also
be used, such as random hexamers. Double stranded cDNA is then
synthesized, also according to Example 6.3, and any ends filled in
to form full dsDNA. Terminal phosphates are retained.
[0279] FIG. 4A illustrates such a cDNA 401 with ends 407 and 408,
poly(dA) sequence 402, poly(dT) primer 403 with biotin 404
attached. 405 is a recognition sequences for RE.sub.1; 406 is a
sequence for RE.sub.2. Fragment 409 is the cDNA sequence defined by
these adjacent RE recognition sequences. Fragments 423 and 424 are
singly cut fragments resulting from RE cleavages at sites 405 and
406.
[0280] Next, the cDNA is ligated into a circle. A ligation reaction
using, for example, T4 DNA ligase is performed under sufficiently
dilute conditions so that predominantly intramolecular ligations
occur circularizing the cDNA, with a only a minimum of
intermolecular, concatamer forming ligations. Reaction conditions
favoring circularization versus concatamer formation are described
in Maniatis, 1982, Molecular Cloning A Laboratory Manual, pp.
124-125, 286-288, Cold Spring Harbor, N.Y. Preferably, a DNA
concentration of less than approximately 1 .mu.g/ml has been found
adequate to favor circularization. Concatamers can be separated
from circularized single molecules by size separation using gel
electrophoresis, if necessary. FIG. 4B illustrates the circularized
cDNA. Blunt end ligation occurred between ends 407 and 408.
[0281] Then the circularized, biotin end labeled, cDNA is cut with
REs and ligated to adapters uniquely recognizing and perhaps
uniquely labeled for each particular RE cut. The RE/ligase step is
performed by procedures as described in the section hereinabove in
order to drive RE digestion and primer ligation to completion over
formation of concatamers and other unwanted ligation products.
Next, the unwanted singly cut ends are removed using streptavidin
or avidin magnetic beads, leaving only doubly cut fragments that
have RE-specific recognition sequences ligated to each end.
[0282] FIG. 4C illustrates these latter steps. Sequences 405 and
406 are cut by RE.sub.1 and RE.sub.2, respectively, and adapters
421 and 422 specific for cuts by RE.sub.1 and RE.sub.2,
respectively are ligated onto the sticky ends. Thereby, fragment
409 is freed from the circularized cDNA and adapters 421 and 422
are ligated to it. The remaining segment of the circularized cDNA
comprises singly cut ends 423 and 424 with ligated adapters 421 and
422. Both singly cut ends are joined to the primer sequence 403
with attached biotin 404. Removal is accomplished by contact with
streptavidin or avidin 420 which is fixed to substrate 425, perhaps
comprising magnetic beads. The doubly cut labeled fragment 409 can
now be simply separated from the singly cut ends affixed to the
substrate. Thereby, separation of the singly and doubly cut
fragments is achieved.
[0283] Signals from the uniquely labeled doubly cut ends can be
directly detected without any unwanted contamination from signals
from labeled singly cut ends. Importantly, since signals originate
only from cDNA sequences originally present in the sample, the
detected signals will quantitatively reflect cDNA sequence content
and thus gene expression levels. If the expression level is too low
for direct detection, the sample can be subjected to just the
minimum number of cycles of amplification, according to the methods
of Example 6.4, to detect the gene or sequence of interest. For
example, the number of cycles can be as small as four to eight
without any concern of background contamination or noise. Thus, in
this embodiment, amplification is not needed to suppress signals
from singly cut ends, and preferred more quantitative response
signal intensities result.
5.3 PCR EMBODIMENT OF QEA
[0284] An alternative implementation of the QEA method not using
REs is based on PCR, or alternative amplification means, to select
and amplify cDNA fragments between chosen target subsequences
recognized by amplification primers. See, generally, Innis et al.,
1989, PCR Protocols A Guide to Methods and Applications, Academic
Press, New York, and Innis et al., 1995, PCR Strategies, Academic
Press, New York.
[0285] Typically target subsequences between four and eight base
pairs long chosen by the methods previously described are preferred
because of their greater probability of occurrence, and hence
information content, as compared to longer subsequences. However,
DNA oligomers this short may not hybridize reliably and
reproducibly to their complementary subsequences to be effectively
used as PCR primers. Hybridization reliability depends strongly on
several variables, including primer composition and length,
stringency condition such as annealing temperature and salt
concentration, and cDNA mixture complexity. For the hash code to be
effective for gene calling, it is highly preferred that subsequence
recognition be as specific and reproducible as possible so that
well resolved bands representative only of the underlying sample
sequence are produced. Thus, instead of directly using single short
oligonucleotides complementary to the selected, target subsequences
as primers, it is preferable to use carefully designed primers.
[0286] The RE embodiments of QEA have been verified to produce
reproducible signal patterns over a 103 range on input DNA
concentrations. The PCR embodiment is less preferred because the
input DNA concentration, as well as the initial hybridization
temperature, must be closely to yield reproducible results.
[0287] The preferred primers are constructed according to the model
in FIG. 5. Primer 501 is constructed of three components, which,
listed 5' to 3', are 504, 503, and 502. Component 503, described
infra, is optional. Component 502 is a sequence which is
complementary to the subsequence which primer 501 is designed to
recognize. Component 502 is typically 4-8 bp long. Component 504 is
a 10-20 bp sequence chosen so the final primer does not hybridize
with any native sequence in the cDNA sample to be analyzed; that
is, primer 501 does not anneal with any sequence known to be
present in the sample to be analyzed. The sequence of component 504
is also chosen so that the final primer has a melting point above
50.degree. C., and preferably above 68.degree. C. The method for
controlling melting temperature selecting average primer
composition and primer length is described above.
[0288] Use of primer 501 in the PCR embodiment involves a first
annealing step, which allows the 3' end component 502 to anneal to
its target subsequence in the presence of end component 504, which
may not hybridize. Preferably, this annealing step is at a
temperature between 36 and 44.degree. C. that is empirically
determined to maximize reproducibility of the resulting signal
pattern. The DNA concentration is approximately 10 ng/50 ml and is
similarly determined to maximize reproducibility. Other PCR
conditions are standard and are described in .sctn. 6.5. Once
annealed, the 3' end serves as the primer elongation point for the
subsequent first elongation step. The first elongation step is
preferably at 72.degree. C. for 1 minute.
[0289] If stringency conditions are such that exact complementarity
is not required for hybridization, false positive signals can be
generated, that is signals resulting from inexact recognition of
the target subsequence. The generation of these false positive
bands can be accounted for in the experimental analysis methods in
order that DNA sample sequences can still be recognized, but,
perhaps, with some increased recognition ambiguity that may need
resolution. These bands are accounted for by allowing inexact
hybridization matches of the target subsequence, the degree of
inexactness depending on the stringency of the hybridization
conditions. In this case the signals generated contain only a fuzzy
representation of the actual subsequence in the sample, the degree
of fuzziness being a function of subsequence length and the
stringency condition, that is binding free energy, and the
temperature of the hybridization. Given the free energy and
temperature, the various possible actual subsequences can be
approximately determined by well known thermodynamic equilibrium
calculations.
[0290] Subsequent PCR cycles then use high temperature, high
stringency annealing steps. The high stringency annealing steps
ensure exact hybridization of the entire primer. No further false
positive bands are generated. Preferably, these PCR cycles
alternate between a 65.degree. C. annealing step and 95.degree. C.
melting step, each for 1 minute.
[0291] Optional component 503 can be used to improve the
specificity of the first low stringency annealing step and thereby
minimize false positive bands generated then. Component 503 can be
--(N).sub.j--, where N is any nucleotide and j is typically between
2 and 4, preferably 2. Use of all possible components 503 results
in a degenerate set of primers, 16 primers if j=2, which have a 3'
end subsequence effectively j bases longer than the target
subsequence. These longer complementary end sequences have improved
hybridization specificity. Alternately, component 503 can be
--(U).sub.3--, where N is a "universal" nucleotide and j is
typically between 2 and 4, preferably 3 or 4. A universal
nucleotide, such as inosine, is capable of forming base pairs with
any other naturally occurring nucleotide. In this alternative,
single primer 501 has a 3' end subsequence effectively j bases
longer than the target, and thus also has improved hybridization
specificity.
[0292] A less preferred primer design comprises sets of degenerate
oligonucleotides of sufficient length to achieve specific and
reproducible hybridization, where each member of a set includes a
shared subsequence complementary to one selected, target sequence.
For example, if a subsequence to be recognized is GATT, the set of
primers used may be all sequences of the form NNAATCNN, where N is
any nucleotide. Also sets of degenerate primers permit the
recognition of discontinuous subsequences. For example, GA--TT may
be recognized by all sequences of the form NAANNTCNN. Alternately,
a universal nucleotide can be used in place of the degenerate
nucleotides represented by `N`.
[0293] Each primer or primer set used in a single reaction is
preferably distinctively labeled for detection. In the preferred
embodiment using electrophoretic fragment separation, labeling is
by fluorochromes that can be simultaneously distinguished with
optical detection means.
[0294] An exemplary experimental protocol is summarized here, with
details presented in .sctn. 6.5. Total cellular mRNA or purified
sub-pools of cellular mRNA are used for cDNA synthesis. First
strand cDNA synthesis is performed according to .sctn. 6.3 using,
for example, an oligo(dT) primer or alternatively phasing primers.
Alternatively, cDNA samples can be prepared from any source or be
directly obtained.
[0295] Next, using a first strand cDNA sample, the primers of the
selected primer sets are used in a conventional PCR amplification
protocol. A high molar excess of primers is preferably used to
ensure only fragments between primer sites that are adjacent on a
target cDNA sequence or gene are amplified. With a high molar
excess of primers binding to all available primer binding sites, no
amplified fragment should include internally any primer recognition
site. As many primers can be used in one reaction as can be labeled
for concurrent separation and detection and which generate an
adequately resolved length distribution, as in the RE embodiments.
For example, if fluorochrome labeling is used, each pair of
fluorochromes preferably is distinguishable in one band and
separate pairs preferably are distinguishable in separate bands.
After amplification, the fragments are separated, re-suspended for
gel electrophoresis, electrophoretically separated, and optically
detected. Thereby the length distribution of fragments having
particular pairs of target subsequences at their ends is
ascertained.
[0296] Preferred protocols for the specific PCR embodiments are
described in detail in .sctn. 6.5.
5.4 QEA ANALYSIS AND DESIGN METHODS
[0297] This inventions provides two groups of methods for the
Quantitative Expression Analysis embodiment of this invention:
first, methods for QEA experimental design; and second, methods for
QEA experimental analysis. Although, logically, design precedes
analysis, the methods of experimental design depend on basic
methods described herein as part of experimental analysis.
Consequently, experimental analysis methods are described
first.
[0298] In the following, descriptions are often cast in terms of
the preferred QEA embodiment, in which REs are used to recognize
target subsequences. However, such description is not limiting, as
all the methods to be described are equally adaptable to all QEA
embodiments, including those in which target subsequences are
recognized by nucleic acid, or nucleic acid mimic, and probes which
recognize target subsequences by hybridization.
[0299] Further, the following descriptions are directed to the
currently preferred embodiments of these methods. However, it will
be readily apparent to those skilled in the computer and simulation
arts that many other embodiments of these methods are substantially
equivalent to those described and can be used to achieve
substantially the same results. This invention comprises such
alternative implementations as well as its currently preferred
implementation.
5.4.1 QEA EXPERIMENTAL ANALYSIS METHODS
[0300] The analysis methods comprise, first, selecting a database
of DNA sequences representative of the DNA sample to be analyzed,
second, using this database and a description of the experiment to
derive the pattern of simulated signals, contained in a database of
simulated signals, which will be produced by DNA fragments
generated in the experiment, and third, for any particular detected
signal, using the pattern or database of simulated signals to
predict the sequences in the original sample likely to cause this
signal. Further analysis methods present an easy to use user
interface and permit determination of the sequences actually
causing a signal in cases where the signal may arise from multiple
sequences, and perform statistical correlations to quickly
determine signals of interest in multiple samples.
[0301] The first analysis method is selecting a database of DNA
sequences representative of the sample to be analyzed. In the
preferred use of this invention, the DNA sequences to be analyzed
will be derived from a tissue sample, typically a human sample
examined for diagnostic or research purposes. In this use, database
selection begins with one or more publicly available databases
which comprehensively record all observed DNA sequences. Such
databases are GenBank from the National Center for Biotechnology
Information (Bethesda, Md.), the EMBL Data Library at the European
Bioinformatics Institute (Hinxton Hall, UK) and databases from the
National Center for Genome Research (Santa Fe, N.M.). However, as
any sample of a plurality of DNA sequences of any provenance can be
analyzed by the methods of this invention, any database containing
entries for the sequences likely to be present in such a sample to
be analyzed is usable in the further steps of the computer
methods.
[0302] FIG. 6A illustrates the preferred database selection method
starting from a comprehensive tissue derived database. Database
1001 is the comprehensive input database, having the exemplary
flat-file or relational structure 1010 shown in FIG. 6B, with one
row, or record, 1014 for each entered DNA sequence. Column, or
field, 1011 is the accession number field, which uniquely
identifies each sequence in database 1001. Most such databases
contain redundant entries, that is multiple sequence records are
present that are derived from one biological sequence. Column 1013
is the actual nucleotide sequence of the entry. The plurality of
columns, or fields, represented by 1012 contain other data
identifying this entry including, for example whether this is a
cDNA or gDNA sequence, if cDNA, whether this is a full length
coding sequence or a fragment, the species origin of the sequence
or its product, the name of the gene containing the sequence, if
known, etc. Although shown as one file, DNA sequence databases
often exits in divisions and selection from all relevant divisions
is contemplated by this invention. For example, GenBank has 15
different divisions, of which the EST division and the separate
database, dbEST, that contain expressed sequence tags ("EST") are
of particular interest, since they contain expressed sequences.
[0303] From the comprehensive database, all records are selected
which meet criteria for representing particular experiments on
particular tissue types. This is accomplished by conventional
techniques of sequentially scanning all records in the
comprehensive database, selecting those that match the criteria,
and storing the selected records in a selected database.
[0304] The following are exemplary selection methods. To analyze a
genomic DNA sample, database 1001 is scanned against criteria 1002
for human gDNA to create selected database 1003. To analyze
expressed genes (cDNA sequences), several selection alternatives
are available. First, a genomic sequence can be scanned in order to
predict which subsequences (exons) will be expressed. Thus selected
database 1005 is created by making selections according to
expression predictions 1004. Second, observed expressed sequences,
such as cDNA sequences, coding domain sequences ("CDS"), and ESTs,
can be selected 1006 to create selected database 1007 of expressed
sequences. Additionally, predicted and observed expressed sequences
can be combined into another, perhaps more comprehensive, selected
database of expressed sequences. Third, expressed sequences
determined by either of the prior methods may be further selected
by any available indication of interest 1008 in the database
records to create more targeted selected database 1009. Without
limitation, selected databases can be composed of sequences that
can be selected according to any available relevant field,
indication, or combination present in sequence databases.
[0305] The second analysis method uses the previously selected
database of sequences likely to be present in a sample and a
description of an intended experiment to derive a pattern of the
signals which will be produced by DNA fragments generated in the
experiment. This pattern can be stored in a computer implementation
in any convenient manner. In the following, without limitation, it
is described as being stored as a table of information. This table
may be stored as individual records or by using a database system,
such as any conventionally available relational database.
Alternatively, the pattern may simply be stored as the image of the
in-memory structures which represent the pattern.
[0306] A QEA experiment comprises several independent recognition
reactions applied to the DNA sample sequences, where in each of the
reactions labeled DNA fragments are produced from sample sequences,
the fragments lying between certain target subsequences in a sample
sequence. The target subsequences can be recognized and the
fragments generated by the preferred RE embodiments of the QEA
method or by the PCR embodiment of QEA. The following description
is focused on the RE embodiments.
[0307] FIG. 7 illustrates an exemplary description 1100 of a
preferred QEA embodiment. Field 1101 contains a description of the
tissue sample which is the source of the DNA sample. For example,
one experiment could analyze a normal prostrate sample; a second
otherwise identical experiment could analyze a prostrate sample
with premalignant changes; and a third experiment could analyze a
cancerous prostate sample. Differences in gene expression between
these samples then relate to the progress of the cancer disease
state. Such samples could be drawn from any other human cancer or
malignancy.
[0308] Major rows 1102, 1105, and 1109 describe the separate
individual recognition reactions to which the DNA from tissue
sample 1101 is subjected. Any number of reactions may be assembled
into an experiment, from as few as one to as many as there are
pairs of available recognition means to recognize subsequences.
FIG. 7 illustrates 15 reactions. For example, reaction 1 specified
by major row 1102 generates fragments between target subsequences
which are the recognition sites of restriction endonucleases 1 and
2 described in minor rows 1103 and 1104. Further, the RE1 cut end
is recognized by a labeling moiety labeled with LABEL1, and the RE2
end is recognized by LABEL2. Similarly, reaction 15, 1109, utilizes
restriction endonucleases 36 and 37 labeled with labels 3 and 4,
minor rows 1110 and 1111, respectively.
[0309] Major row 1105 describes a variant QEA reaction using three
REs and a separate probe. As described, many REs can be used in a
single recognition reaction as long as a useful fragment
distribution results. Too many REs results in a compressed length
distribution. Further, probes for target subsequences that are not
intended to be labeled fragment ends, but rather occur within a
fragment, can be used. For example, a labeled probe added after the
QEA PCR amplification step (if present in a given embodiment), a
post PCR probe, can recognize subsequences internal to a fragment
and thereby provide an additional signal which can be used to
discriminate between two sample sequences which produce fragments
of the same length and end sequence which otherwise have differing
internal sequences. For another example, a probe added before the
QEA PCR step and which cannot be extended by DNA polymerase will
prevent PCR amplification of those fragment containing the probe's
target subsequences. If PCR amplification is necessary to generate
detectable signals (in a given embodiment), such a probe will
prevent the detection of such a fragment. The absence of a fragment
may make a previously ambiguous detected band now unambiguous. Such
PCR disruption probes can be PNA oligomers or degenerate sets of
DNA oligomers, modified to prevent. polymerase extension (e.g. by
incorporation of a dideoxynucleotide at the 3' end).
[0310] Where alternative phasing PCR primers are used, their extra
recognition subsequences and labeling are described in rows
dependent to the RE/ligase reaction whose products they are used to
amplify.
[0311] Next FIG. 8A illustrates, in general, that from the database
selected to best represent the likely DNA sequences in the sample
analyzed, 1201, and the description of the QEA experiment, 1202,
the simulation methods, 1203, determine a pattern of simulated
signals stored in a simulated database, 1204, that represents the
results of the QEA experiment. The experimental simulation
generates the same fragment lengths and end subsequences from the
input database that will be generated in an actual experiment
performed on the same sample of DNA sequences.
[0312] Alternately, the simulated pattern or database may not be
needed, in which case the DNA database is searched sequence by
sequence, mock digestions are performed and compared against the
input signals. A simulated database is preferable if several
signals need to be searched or if the same QEA experiment is run
several times. Conversely, the simulated database can be dispensed
with when few signals from a few experiments need to searched. A
quantitative statement of when the simulated database is more
efficient depends upon an analysis of the costs of the various
operations and the size of DNA database, and can be performed as is
well known in the computer arts. Without limitation, in the
following the simulated database is described
[0313] FIG. 8B illustrates an exemplary structure for the simulated
database. Here, the simulated results of all the individual
recognition reactions defined for the experiment are gathered into
rectangular table 1210. The invention is equally adaptable to other
database structures containing equivalent information; such an
equivalent structure would be one, for example, where each reaction
was placed in a separate table. The rows of table 1210 are indexed
by the lengths of possible fragments. For example, row 1211
contains fragments of length 52. The columns of table 1210 are
indexed by the possible end subsequences and probe hits, if any, in
a particular experimental reaction. For example, columns 1212,
1213, and 1214 contain all fragments generated in reaction 1, R1,
which have both end subsequences recognized by RE1, one end
subsequence recognized by RE1 and the other by RE2, and both end
subsequences recognized by RE2, respectively. Other columns relate
to other reactions the experiment. Finally, the entries in table
1210 contain lists of the accession numbers of sequences in the
database that give rise to a fragment with particular length and
end subsequences. For example, entry 1215 indicates that only
accession number A01 generates a fragment of length 52 with both
end subsequences recognized by RE1 in R1. Similarly, entry 1216
indicates that accession numbers A01 and S003 generate a fragment
of length 151 with both end subsequences recognized by RE3 in
reaction 2.
[0314] In alternative embodiments, the contents of the table can be
supplemented with various information. In one aspect, this
information can aid in the interpretation of results produced by
the separation and detection means used. For example, if separation
is by electrophoresis, then the detected electrophoretic DNA length
can be corrected to obtain the true physical DNA length. Such
corrections are well known in the electrophoretic arts and depend
on such factors as average base composition and fluorochrome
labels. One commercially available package for making these
corrections is Gene Scan Software from Applied Biosystems, Inc.
(Foster City, Calif.). In this case, each table entry for a
fragment can contain additionally average base composition, perhaps
expressed as percent G+C content, and the experimental definition
can include primer average base composition and fluorochrome label
used. For a further example, if separation is by mass spectroscopy
or similar method, the additional information can be the molecular
weight of each fragment and perhaps a typically fragmentation
pattern. Use of other separation and detection means can suggest
the use of other appropriate supplemental data.
[0315] Where alternative phasing primers are used, supplemental
columns are used with RE pair in order to further identify the
effective target subsequence.
[0316] Before describing how this simulated database is generated,
it is useful first to describe how this database is used to predict
experimental results. Returning to FIG. 7, labels are used to
detect binding reaction events by subsequence recognition means to
the target DNA, to allow detection after separation of the
fragments by length. In an embodiment using fluorescent detection
means, these labels are fluorochromes covalently attached to the
primer strands of the adapters, as previously described, or to
hybridization probes, if any. Typically, all the fluorochrome
labels used in one reaction are simultaneously distinguishable so
that fragments with all possible combinations of target
subsequences can be fluorescently distinguished. For example,
fragments at entry 1217 in table 1210 (FIG. 8B) occur at length 175
and present simultaneous fluorescent signals LABEL1 and LABEL2 upon
stimulation, since these are the labels used with adapters which
recognize ends cuts by RE1 and RE2 respectively. For a further
example, in reaction 2, major row 1105 of experimental definition
1100 (FIG. 7), a fragment with ends cut by RE2 and RE3 and
hybridizing with probe P will present simultaneous signals LABEL2,
LABEL3, and LABEL4. Where effective target subsequences are
constructed with alternative phasing primers, this lookup is
appropriately modified.
[0317] Other labelings are within the scope of this invention. For
example, a certain group of target subsequences can be identically
labeled or not labeled at all, in which case the corresponding
group of fragments are not distinguishable. In this case, if RE1
and RE3 end subsequences were identically labeled in table 1210
(FIGS. 8B), a fragment of length 151 may be generated by sequence
T163, A01, or S003, or any combination of these sequences. In the
extreme, if silver (Ag) staining of an electrophoresis gel is used
in an embodiment to detect separated fragments, then all bands will
be identically labeled and only band lengths can be distinguished
within one electrophoresis lane.
[0318] Thus the simulated database together with the experimental
definition can be used to predict experimental results. If a signal
is detected in a recognition reaction, say Rn, whose end labelings
are LABEL1 and LABEL2 and whose representation of length is
corrected to physical length in base pairs of L, the length L row
of the simulated database is retrieved and it is scanned for Rn
entries with the detected subsequence labeling, by using the column
headings indicating observed subsequences and the experimental
definition indicating how each subsequence is labeled. If no match
is found, this fragment represents a new gene or sequence not
present in the selected database. If a match is found, then this
fragment, in addition to possibly being a new gene or sequence, can
also have been generated by those candidate sequences present in
the table entry(ies) found.
[0319] The simulated database lookup is described herein as using
the physical length of a detected fragment. In cases where the
separation and detection leans returns an approximation to the true
physical fragment length, lookup is augmented to account for such
as approximation. For example, electrophoresis, when used as the
separation means, returns the electrophoretic length, which
depending on average base composition and labeling moiety is
typically within 10% of the physical length. In this case database
lookup can search all relevant entries whose physical length is
within 10% of the reported electrophoretic length, perform
corrections to obtain electrophoretic length, and then check for a
match with the detected signal. Alternative lookup implementations
are apparent, one being to precompute the electrophoretic length
for all predicted fragments, construct an alternate table index
over the electrophoretic length, and then directly lookup the
electrophoretic length. Other separation and detection means can
require corresponding augmentations to lookup to correct for their
particular experimental biases and inaccuracies. It is understood
that where database lookup is referred to subsequently, either
simple physical lookup or augmented lookup is meant as
appropriate.
[0320] If matched candidate database sequences are found, then the
selected database can be consulted to determine other information
concerning these sequences, for example, gene name, tissue origin,
chromosomal location, etc. If an unpredicted fragment is found,
this fragment can be optionally retrieved from the length
separation means, cloned or sequenced, and used to search for
homologues in a DNA sequence database or to isolate or characterize
the previously unknown gene or sequence. In this manner this
invention can be used to rapidly discover and identify new
genes.
[0321] The computer methods of this invention are also adaptable to
other formats of an experimental definition. For example, the
labeling of the target subsequence recognition moieties can be
stored in a table separate from the table defining the experimental
reactions.
[0322] Now turning to the methods by which the simulated database
is generated, FIG. 9 illustrates a basic method, termed herein mock
fragmentation, which takes one sequence and the definition of one
reaction of an experiment and produces the predicted results of the
reaction on that sequence. Generation of the entire simulated
database requires repetitive execution of this basic method.
[0323] Turning first to a description of mock fragmentation, the
method commences at 1301 and at 1302 it inputs the sequence to be
fragmented and the definition of the fragmentation reaction, in the
following terms: the target end subsequences RE1 . . . REn, where n
is typically 2 or 3, and the subsequences to be recognized by post
PCR probes, P1 . . . Pn, where n is typically 0 or 1. Note that PCR
disruption probes act as unlabeled end subsequences and are so
treated for input to this method. The operation of the method is
illustrated by example in FIG. 10A-F for the case RE1, RE2 and
P1.
[0324] At step 1303, for each target end subsequence, the method
makes a "vector of ends", which has elements which are pairs of
nucleotide positions along the sequence, each pair being labeled by
the corresponding end subsequence. For embodiments where end
subsequences are recognized by hybridizing oligonucleotides, the
first member of each pair is the beginning of a target end
subsequence and the second member is the end of a target end
subsequence. For embodiments where target end subsequences are
recognized by restriction endonucleases, the first member of each
pair is the beginning of the overhang region that corresponds to
the RE recognition subsequence and the second member is the end of
that overhang region. It is preferred to use REs that generate 4 bp
overhangs. The actual target end subsequences are the RE
recognition sequences, which are preferably 4-8 bp long.
[0325] This vector is generated by a string operation which
compares the target end subsequence in a 5' to 3' direction against
the input sequence and seeks string matches, that is the
nucleotides match exactly. Where effective target subsequences are
formed by using alternative phasing primers, it is the effective
subsequences that are compared. This can be done by simply
comparing the end subsequence against the input sequence starting
at one end and proceeding along the sequence one base at time.
However, it is preferable to use a more efficient string matching
algorithm, such as the Knuth-Morris-Pratt or the Boyer-Moore
algorithms. These are described with sample code in Sedgewick,
1990, Algorithms in C, chap. 19, Addison-Wesley, Reading,
Mass..
[0326] In QEA embodiments where target subsequence are recognized
with accuracy, such as the RE embodiments, the comparison of target
subsequence against input sequence should be exact, that is the
bases should match in a one-to-one manner. In embodiments where
target subsequences are less accurately recognized, the string
match should be done in a less exact, or fuzzy, manner. For
example, in the PCR embodiments, a target subsequence of length T
can inaccurately recognize an input sequence, also of length T, by
matching only T-n bases exactly, where n is typically 1 or 2 and is
adjustable depending on experimental conditions. In this case the
string operation, which generates the vector of ends, should accept
partial T-n matches as well as exact matches. In this, the string
operations generate the false positive matches expected from the
experiments and permit these fragments to be identified. Ambiguity
in the simulated database, however, increases, since more fragments
leads to a greater chance of fragments of identical length and end
labels.
[0327] FIG. 10A illustrates end vectors 1401 and 1402, comprising
three and two ends, respectively, generated by RE1 and RE2, which
are for this example assumed to be REs with a 4 bp overhang. The
first overhang in vector 1401 occurs between nucleotide 10 and 14
in the input sequence.
[0328] Step 1304 of FIG. 9 merges all the end vectors for all the
end subsequences and sorts the elements on the position of the end.
Vector 1404 of FIG. 10B illustrates the result of this step for
example end vectors 1401 and 1402.
[0329] Step 1305 of FIG. 9 then creates the fragments generated by
the reaction by selecting the parts of the full input sequence that
are delimited by adjacent ends in the merged and sorted end vector.
Since the experimental conditions in conducting QEA should be
selected such that target end subsequence recognition is allowed to
go to completion, all possible ends are recognized. For the
restriction endonuclease embodiments, the cutting and ligase
reactions should be conducted such that all possible RE cuts are
made and to each cut end a labeled primer is ligated. These
conditions insure that no fragments contain internal unrecognized
target end subsequences and that only adjacent ends in the merged
and sorted vector define generated fragments.
[0330] Where additional information is needed for simulated
database entries to adapt to inaccuracies in particular separation
and detection means, such information can be collected at this
step. For example, in the case of electrophoretic separation,
fragment sequence can be determined and percent G+C content
computed and entered in the database along with the fragment
accession number.
[0331] For the PCR embodiments, the fragment length is the
difference between the end position of the second end subsequence
and the start position of the first end subsequence. For RE
embodiments, the fragment length is the difference between the
start position of the second end subsequence and the start position
of the first end subsequence plus twice the primer length (48 in
the preferred primer embodiment).
[0332] FIG. 10C illustrates the exemplary fragments generated, each
fragment being represented by a 4 member tuple comprising: the two
end subsequences, the length, and an indicator whether the probe
binds to this fragment. In FIG. 10C the position of this indicator
is indicated by a `*`. Fragment 1408 is defined by ends 1405 and
1406, and fragment 1409 by ends 1406 and 1407. There is no fragment
defined by ends 1405 and 1407 because the intermediate end
subsequence is recognized and either fully cut in an RE embodiment
or used as a fragment end priming position in a PCR embodiment. For
simplicity, the fragment lengths are illustrated for the RE
embodiment without the primer length addition.
[0333] Step 1306 of FIG. 9 checks if a hybridization probe is
involved in the experiment. If not, the method skips to step 1309.
If so, step 1307 determines the sequence of the fragment defined in
step 1305. FIG. 10D illustrates that the fragment sequences for
this example are the nucleotide sequences within the input sequence
that are between the indicated nucleotide positions. For example,
the first fragment sequence is the part of the input sequence
between positions 10 and 62. Step 1308 then checks each probe
subsequence against each fragment sequence to determine whether
there is any match (i.e., whether the probe has a sequence
complementary enough to the fragment sequence sufficient for it to
hybridize thereon). If a match is found, an indication is made in
the fragment 4 member tuple.
[0334] This match is done by string searching in a similar manner
to that described for generation of the end vectors.
[0335] Next at step 1309 of FIG. 9, all the fragment are sorted on
length and assembled into a vector of sorted fragments, which is
output from the mock fragmentation method at step 1310. This vector
contains the complete list of all fragments, with probe
information, defined by their end subsequences and lengths that the
input reaction will generate from the input sequence.
[0336] FIG. 10E illustrates the fragment vector of the example
sorted according to length. For illustrative purposes, probe P1 was
found to hybridize only to the third fragment 1412, where a `Y` is
marked. `N` is marked in all the other fragments, indicating no
probe binding.
[0337] The simulated database is generated by iteratively applying
the basic mock fragmentation method for each sequence in the
selected database and each reaction in the experimental definition.
FIG. 11 illustrates a simulated database generation method. The
method starts at 1501 and at 1502 inputs the selected
representative database and the experimental definition with, in
particular, the list of reactions and their related subsequences.
Step 1503 initializes the digest database table so that lists of
accession numbers may be inserted for all possible combinations of
fragment length and target end subsequences. Step 1504, a DO loop,
causes the iterative execution of steps 1505, 1506, and 1507 for
all sequences in the input selected database.
[0338] Step 1505 takes the next sequence in the database, as
selected by the enclosing DO loop, and the next reaction of the
experiment and performs the mock fragmentation method of FIG. 9, on
these inputs. Step 1506 adds the sorted fragment vector to the
simulated database by taking each fragment from the vector and
adding the sequence accession number to the list in the database
entry indexed by the fragment length and end subsequences and probe
(if any). FIG. 10F represents the simulated database entry list
additions that would result for the example mock fragmentation
reaction of FIGS. 10A-E. For example, accession number A01 is added
to the accession number list in the entry 1412 at length 151 and
with both end subsequences RE2.
[0339] Finally, step 1507 tests whether there is another reaction
in the input experiment that should be simulated against this
sequence. If so, step 1505 is repeated with this reaction. If not,
the DO loop is repeated to select another database sequence. If all
the database sequences have been selected, the step 1508 outputs
the simulated database and the method ends at 1509.
5.4.2. QEA EXPERIMENTAL DESIGN METHODS
[0340] The goal of the experimental design methods is to optimize
each experiment in order to obtain the maximum amount of
quantitative information. An experiment is defined by its component
recognition reactions, which are in turn defined by the target end
subsequences recognized, probes used, if any, and labels assigned.
If alternative phasing primers are used, effective target
subsequences are used. Any of several criteria can be used to
ascertain the amount of information obtained, and any of several
algorithms can be used to perform the reaction optimization.
[0341] A preferred criteria for ascertaining the amount of
information uses the concept of "good sequence." A good sequence
for an experiment is a sequence for which there is at least one
reaction in the experiment that produces a unique signal from that
sequence, that is, a fragment is produced from that good sequence,
by at least one recognition reaction, that has a unique combination
of length and labeling. For example, returning to FIG. 8B, the
sequence with accession number A01 is a good sequence because
reaction 1 produces signal 1215, with length 52 and with both
target end subsequences recognized by RE1, uniquely from sequence
A01. However, sequence S003 is not a good sequence because there
are no unique signals produced only from S003: reaction R2 produces
signal 1216 from both AO1 and S003 and signal 1219 from both Q012
and S003. Using the amount of good sequences as an information
measure, the greater the number of good sequences in an experiment
the better is the experimental design. Ideally, all possible
sequences in a sample would be good sequences.
[0342] Further, a quantitative measure of the expression of a good
sequence can simply be determined from the detected signal
intensity of the fragment uniquely produced from the good sequence.
Relative quantitative measures of the expression of different good
sequences can be obtained by comparing the relative intensities of
the signal uniquely produced from the good sequences. An absolute
quantitative measure of the expression of a good sequence can be
obtained by including a concentration standard in the original
sample. Such a standard for a particular experiment can consist of
several different good sequences known not to occur in the original
sample and which are introduced at known concentrations. For
example, exogenous good sequence 1 is added at a 1:10.sup.3
concentration in molar terms; exogenous good sequence 2 at a
1:10.sup.4 in molar terms; etc. Then comparison of the relative
intensity of the unique signal of a good sequence in the sample
with the intensities of the unique signal of the standards allows
determination of the molar concentrations of the sample sequence.
For example, if the good sequence has a unique signal intensity
half way between the unique signal intensities of good sequences 1
and 2, then it is present at a concentration half way between the
concentrations of good sequences 1 and 2.
[0343] Another preferred measure for ascertaining the amount of
information produced by an experiment is derived by limiting
attention to a particular set of sequences of interest, for example
a set of known oncogenes or a set of receptors known or expected to
be present in a particular tissue sample. An experiment is designed
according to this measure to maximize the number of sequences of
interest that are good sequences. Whether other sequences possibly
present in the sample are good sequences is not considered. These
other sequences are of interest only to the extent that the
sequences of interest produce uniquely labeled fragments without
any contribution from these other sequences.
[0344] This invention is adaptable to other measures for
ascertaining information from an experiment. For example, another
measure is to minimize on average the number of sequences
contributing to each detected signal. A further measure is, for
example, to minimize for each possible sequence the number of other
sequences that occur in common in the same signals. In that case
each sequence is linked by common occurrences in fragment labelings
to a minimum number of other sequences. This can simplify making
unambiguous signal peaks of interest (see infra).
[0345] Having chosen an information measure, for example the number
of good sequences, for an experiment, the optimization methods
choose target subsequences, and possibly probes, which optimize the
chosen measure. One possible optimization method is exhaustive
search, in which all subsequences in lengths less than
approximately 10 are tested in all combinations for that
combination which is optimum. This method requires considerable
computing power, and the upper bound is determined by the
computational facilities available and the average probability of
occurrence of subsequences of a given length. With adequate
resources, it is preferable to search all sequences down to a
probability of occurrence of about 0.005 to 0.01. Upper bounds may
range from 8 to 11 or 12.
[0346] A preferred optimization method is known as simulated
annealing. See Press et al., 1986, Numerical Recipes--The Art of
Scientific Computing, .sctn. 10.9, Cambridge University Press,
Cambridge, U.K. Simulated annealing attempts to find the minimum of
an "energy" function of the "state" of a system by generating small
changes in the state and accepting such changes according to a
probabilistic factor to create a "better" new state. While the
method progresses, a simulated "temperature", on which the
probabilistic factor depends and which limits acceptance of new
states of higher energy, is slowly lowered.
[0347] In the application to the methods of this invention, a
"state", denoted by S, is the experimental definition, that is the
target end subsequences and hybridization probes, if any, in each
recognition reaction of the experiment. The "energy", denoted E, is
taken to be 1.0 divided by the information measure, so that when
the energy is minimized, the information is maximized.
Alternatively, the energy can be any monotomically decreasing
function of the information measure. The computation of the energy
is denoted by applying the function E( ) to a state.
[0348] The preferred method of generating a new experiment, or
state, from an existing experiment, or state, is to make the
following changes, also called moves to the experimental
definition: (1) randomly change a target end subsequence in a
randomly chosen recognition reaction; (2) add a randomly chosen
target end subsequence to a randomly chosen reaction; (3) remove a
randomly chosen target end subsequence from a randomly chosen
reaction with three or more target subsequences; (4) add a new
reaction with two randomly chosen target end subsequences; and (5)
remove a randomly chosen reaction. If an RE embodiment of QEA is
being designed, all target end subsequences are limited to
available RE recognition sequences. If alternative phasing primers
are used to generate effective target subsequences, all
subsequences must be chosen from among such effective target
subsequences that can be generated from available REs. To generate
a new experimental definition, one of these moves is randomly
selected and carried out on the existing experimental definition.
Alternatively, the various moves can be unequally weighted. In
particular, if the number of reactions is to be fixed, moves (4)
and (5) are skipped. The invention is further adaptable to other
moves for generating new experiments. Preferable generation methods
will generate all possible experiments.
[0349] Several additional subsidiary choices are needed in order to
apply simulated annealing. The "Boltzman constant" is taken to be
1.0, so that the energy equals the temperature. The minimum of the
energy and temperature, denoted E.sub.0 and T.sub.0, respectively,
are defined by the maximum of the information measure. For example,
if the number of good sequences of interest is G and is used as the
information measure, then E.sub.0, which equals T.sub.0, equals
1/G. An initial temperature, denoted T.sub.1, is preferably chosen
to be 1. An initial experimental definition, or state, is chosen,
either randomly or guided by prior knowledge of previous .
experimental optimizations. Finally, two execution parameters are
chosen. These parameters define the "annealing schedule", that is
the manner in which the temperature is decreased during the
execution of the simulated annealing method. They are the number of
iterations in an epoch, denoted by N, which is preferably taken to
be 100 and the temperature decay factor, denoted by f, which is
preferably taken to be 0.95. Both N and f may be systematically
varied case-by-case to achieve a better optimization of the
experiment definition with a lower energy and a higher information
measure.
[0350] With choices for the information measure or energy function,
the moves for generating new experiments, an initial state or
experiment, and the execution parameters made as above, the general
application of simulated annealing to optimize an experimental
definition is illustrated in FIG. 13A. The information measure used
in this description is the number of good sequences of interest.
Any information measure, such as those previously described, may be
used alternately.
[0351] The method begins at step 1701. At step 1702 the temperature
is set to the initial temperature; the state to the initial state
or experimental definition; and the energy is set to the energy of
the initial state. At step 1703 the temperature and energy are
checked to determine whether either is less than or equal to the
minima for the information measure chosen, as the result of either
a fortuitous initial choice or subsequent computation steps. If the
energy is less than or equal to the minimum energy, no further
optimization is possible, and the final experimental definition and
its energy is output. If the temperature is less than or equal to
the minimum temperature, the optimization is stopped. Then the
inverse of the energy is the number of good sequences of interest
for this experimental definition.
[0352] Step 1706 is a DO loop which executes an epoch, or N
iterations, of the simulated annealing algorithm, Each iteration
consists of steps 1707 through 1711. Step 1707 generates a new
experimental definition, or state, S.sub.new, according to the
described generation moves. Step 1708 ascertains or determines the
information content, or energy, of S.sub.new. Step 1709 tests the
energy of the new state, and, if it is lower than the energy of the
current state, at step 1711, the new state and new energy are
accepted and replace the current state and current energy. If the
energy of the new state is higher than the energy of the current
state, step 1710 computes the following function. 6 EXP [ - ( E - E
new ) / T ]
[0353] This function defines the probabilistic factor controlling
acceptance. If this function is less than a random chosen number
uniformly distributed between 0 and 1, then the new state is
accepted at step 1711. If not, then the newly generated state is
discarded. These steps are equivalent to accepting a new state if
the energy is not increased by an amount greater than that
determined by function (4) in conjunction with the selection of a
random number. Or in other words, a new state is accepted if the
new information measure is not decreased by an amount greater than
indirectly determined by function (4).
[0354] Finally, after an epoch of the algorithm, at step 1712 the
temperature is reduced by the multiplicative factor f and the
method loops back to the test at step 1703.
[0355] Using this algorithm, starting from an initial experimental
definition which has certain information content, the algorithm
produces a final experimental definition with a higher information
content, or lower energy, by repetitively and randomly altering the
experimental definition in order to search for a definition with a
higher information content.
[0356] The computation of the energy of an experimental definition,
or state, in step 1708 is illustrated more detail in FIG. 13B. This
method starts at step 1720. Step 1721 inputs the current
experimental definition. Step 1722 determines a complete digest
database from this definition and a particular selected database by
the method of FIG. 11. Step 1723 scans the entire digest database
and counts the number of good sequences of interest. If the total
number of good sequences is the measure used, the total number of
good sequences can be counted. Alternatively, other information
measures may be applied to the digest database. Step 1724 computes
the energy as the inverse of the information measure.
Alternatively, another decreasing function of the information
content may be used as the energy. Step 1725 outputs the energy,
and the method ends at step 1726.
5.4.3. QEA AMBIGUITY RESOLUTION
[0357] In one utilization of this invention two related tissue
samples can be subject to the same experiment, perhaps consisting
of only one recognition reaction, and the outcomes compared. The
two tissue samples may be otherwise identical except for one being
normal and the other diseased, perhaps by infection or a
proliferative process, such as hyperplasia or cancer. One or more
signals may be detected in one sample and not in the other sample.
Such signals might represent genetic aspects of the pathological
process in one tissue. These signals are of particular
interest.
[0358] The candidate sequences that can produce a signal of
interest are determined, as previously described, by look-up in the
digest database. The signal may be produced by only one sequence,
in which case it is unambiguously identified. However, even if the
experiment has been optimized, the signal may be ambiguous in that
it may be produced by several candidate sequences from the selected
database. A signal of interest may be made unambiguous in several
manners which are described herein.
[0359] In a first manner of making unambiguous assume the signal of
interest is produced by several candidate sequences all of which
are good sequences for the particular experiment. Then which
sequences are present in the signal of interest can be ascertained
by determining the quantitative presence of the good sequences from
their unique signals. For example, referring to FIG. 8B, if the
signal 1217 of length 175 with the labeling 1213 is of interest,
the sequences actually present in the signal can be determined from
the quantitative determination of the presence of signals 1215 and
1218. Here, both the possible sequences contributing to this signal
are good sequences for this experiment.
[0360] The first manner of making unambiguous can be extended to
the case where one of the sequences possibly contributing to a
signal is not a good sequence. The quantitative presence of all the
possible good sequences can be determined from the quantitative
strength of their unique signals. The presence of the remaining
sequence which is not a good sequences can be determined by
subtracting from the quantitative presence of the signal of
interest the quantitative presences of all the good sequences.
[0361] Further extensions of the first manner can be made to cases
where more than one of the possible sequences is not a good
sequences if the sequences which are not good appear as
contributors to further signals involving good sequences in a
manner which allows their quantitative presences to be determined.
For example, suppose signal 1219 is of interest, where both
possible sequences are not good sequences. The quantitative
presence of sequence Q012 can be determined from signals 1220 and
1218 in the manner previously outlined. The quantitative presence
of sequence S003 can be determined from signals 1216 and 1215.
Thereby, the sequences contributing to signal 1219 can be
determined. More complex combinations can be similarly made
unambiguous.
[0362] An alternative extension of the first manner of making
unambiguous is by designing a further experiment in the possible
sequences contributing to a signal of interest are good sequences
even if they were not originally so. Since there are approximately
50 suitable REs that can be used in the RE embodiment of QEA
(Section 6.2),.there are approximately 600 RE reaction pairs that
can be performed, assuming that half of the theoretical maximum of
1,250 (50.times.50/2=1,250) are not useable. Since most RE pairs
produce on the average of 200 fragments and standard
electrophoretic techniques can resolve at least approximately 500
fragment lengths per lane, the RE QEA embodiment has the potential
of generating over 100,000 signals (500.times.200=100,000). The
number of possible signals is further increased by the use of
reactions with three or more REs and by the use of labeled probes.
Further, since the average complex human tissue, for example brain,
is estimated to express no more than approximately 25,000 genes,
there is a 4 fold excess of possible signals over the number of
possible sequences in a sample. Thus it is highly likely that for
any signal of interest, a further experiment can be designed and
optimized for which all possible candidates of the signal of
interest are good sequences. This design can be made by using the
prior optimization methods with an information measure the
sequences of interest in the signal of interest and starting with
an extensive initial experimental definition including many
additional reactions. In that manner, any signal of interest can be
made unambiguous.
[0363] A second manner of making unambiguous is by automatically
ranking the likelihood that the sequences possibly present in a
signal of interest are actually present using information from the
remainder of the experimental reactions. FIG. 14 illustrates a
preferred ranking method.
[0364] The method begins at step 1801 and at step 1802 inputs the
list of possible accession numbers in a signal of interest, the
experimental definition, and the actual experimental results.
DO-loop 1803 iterates once for each possible accession number. Step
1804 performs a simulated experiment by the method illustrated in
FIG. 11 in which, however, only the current accession number is
acted on. The output is a single sequence digest table, such as
illustrated in FIG. 10F.
[0365] Step 1805 determines a numerical score of ranking the
similarity of this digest table to the experimental results. One
possible scoring metric comprises scanning the digest table for all
fragment signals and adding 1 to the score if such a signal appears
also in the experimental results and subtracting 1 from the score
if such signal does not appear in the experimental results.
Alternate scoring metrics are possible. For example, the
subtraction of 1 may be omitted.
[0366] Step 1806 sorts the numerical scores of the likelihood that
each possible accession number is actually present in the sample.
Step 1807 outputs the sorted list and the method ends at step
1808.
[0367] By this method likelihood estimates of the presence of the
various possible sequences in a signal of interest can be
determined.
5.5. COLONY CALLING
[0368] The colony calling embodiment recognizes and classifies
single, individual genes or DNA sequences by determining the
presence or absence of target subsequences. No length information
is determined. This embodiment is directed to gene determination
and classification of arrayed samples or colonies, where each
sample or colony contains or expresses only one sequence or gene of
interest and is perhaps prepared from a tissue cDNA library. The
presence or absence of target subsequences in a colony is
determined by use of labeled hybridization recognition means, each
of which uniquely binds to one target subsequence. It is preferable
that this binding be highly specific and reproducible. Each sample
or colony, or an array of samples or colonies, is assayed for the
contained sequence by determining which of the set of probes
recognizes and thus hybridizes to target subsequences in the
sample(s) or colony(ies). Each sample is then characterized by a
hash code, each bit of which indicates which probes recognized
subsequences, or hits, in a particular sample. The sequence or gene
in a sample is determined from the hash code by computer
implemented methods.
[0369] The choice of the target subsequences is important. For
economical and rapid assay, the size of the set of recognition
means should be as small as possible, preferably less than 50
elements and more preferably from 15 to 25 elements. Further, it is
most preferable that all possible sequences or genes are recognized
and uniquely determined. It is preferable that 90 to 95% of all
possible sequences be recognized, with each sequence being
indistinguishable from, or ambiguous with, at most one or two other
sequences. Therefore, each target subsequence preferably occurs
frequently enough to minimize the number of different recognition
means needed. For example, it is not practical for this invention,
directed to rapid gene classification, if each probe recognized
only a few genes and therefore thousands of probes were needed.
However, each target subsequence preferably does not occur so
frequently that its presence conveys little information. For
example, a probe recognizing every gene conveys no information.
[0370] The optimal choice is for each target subsequence to have a
probability of occurrence in all the genes or sequences that can
appear in a sample or colony of approximately 50%; a preferable
choice is a probability of occurrence between 10 and 50%. Typically
for human cDNA libraries, target subsequences of length 4 to 6 meet
this condition, as longer sequences occur too infrequently to make
useful hash codes. Additionally, the presence of one target
subsequence is preferably independent of the presence of any other
target subsequence in the same sequence or gene. These two criteria
ensure that a hash code for a sample, consisting of indications of
which target subsequences are present, is maximally likely to
represent a unique gene or DNA sequence with minimum of wasted code
words not specifying any gene. Such a hash code is an efficient
representation of sequences or genes.
[0371] The maximal number of genes or sequences that can be
represented by a hash code is 2.sup.n, where n is the number of
target subsequences. A simple test to determine whether the target
subsequences occur frequently enough in the expected gene library
is made by comparing the actual probabilities of the two hash codes
that have all target subsequences either present or absent to the
ideal probabilities of these codes. If p is the probability that
any target subsequence occurs in a given sequence in the library,
then probability that none of the target subsequences occur in a
random gene is (1-p).sup.n. The closer the ratio
(1-p).sup.n/2.sup.-n is to 1 the more efficient is the code.
Similarly, the closer p.sup.n/2.sup.-n, the ratio of the
probabilities that all the target subsequences are present to the
ideal probability conveying maximum information, is to 1 the more
efficient is the code. We see the optimal p is close to
2.sup.-1.
[0372] The preferred method of selecting target subsequences
meeting the probability of occurrence and independence criteria is
to use a database containing sequences generally expected to be
present in the samples to be analyzed, for example human GenBank
sequences for human tissue derived samples. From a sequence
database, oligomer frequency tables are compiled containing the
frequencies of, preferably, all 4 to 8-mers. From these tables,
candidate subsequences with the desired probability of occurrence
are selected. Each candidate target subsequence is then checked for
independent occurrence, by, for example, checking that the
conditional probability for a hit by any selected pair of
candidates is approximately the product of the probabilities of the
individual candidate hit probabilities. Candidate target
subsequences meeting both occurrence and independence criteria are
possible target subsequences. A sufficient number, typically 20, of
any of these subsequences can be selected as target subsequences
for a hash code.
[0373] Preferably, but optionally, the initially set of target
subsequences can be optimized, using information on the actual
occurrences of the initially selected target subsequences in the
sequence database, resulting in a set of target subsequences
selected which recognizes a maximum number of genes with a minimum
number of sequences and with a minimum amount of recognition
ambiguity. Alternatively, this optimization can also be performed
on a sub-set of the database comprised of sequences or genes of
particular biological or medical interest, for example, the set of
all oncogenes or growth factors. In this manner, fewer target
subsequences can be chosen which distinguish more efficiently among
a set of sequences or genes of particular interest and distinguish
that set of genes from the sequences of the remainder of the
sample.
[0374] This combinatorial optimization problem is computationally
intensive to solve exactly. A number of approximate techniques can
be used to obtain efficient nearly optimal solutions. The preferred
but not limiting technique is to use simulated annealing (Press et
al., 1986, Numerical Recipes--The Art of Scientific Computing,
.sctn. 10.9, Cambridge University Press, Cambridge, U.K.). The
experimental design and optimization are described in detail in the
following section.
[0375] Example 6.6 illustrates the results of the simulated
annealing optimization method. Simulated annealing generally
produces a choice of subsequences that achieve the same resolution
while using approximately 20% fewer total sequences than a
selection guided only by the probability principles previously
described. This level of optimization is likely to improve with
larger and less redundant databases that represent longer
genes.
[0376] An alternative to using single target subsequences is to use
sets of target subsequences, recognized by sets of identically
labeled hybridization probes, to generate one presence or absence
indication for the hash code. In this alternative, sets of longer
target subsequences would be chosen such that the presence of any
target subsequence in the set is a presence indication. Absence
means no element of the set is present. If the sets are chosen so
that their probability of presence in a single sequence is near
50%, preferably from 10 to 50%, and the presence or absence of one
set is independent of the presence or absence of any other set,
such sets can be used to construct codes equally well as single
subsequences. A resulting code will be efficient and can be further
optimized by simulated annealing, as for single target subsequence
codes. Target sets of longer subsequences are preferable where
experimental recognition of shorter subsequences is less specific
and reproducible, as for example is true where short DNA oligomers
are used as hybridization probes for recognition. As a further
alternative, a code can consist of presence or absence indications
of mixed target sets of subsequences and single target
subsequences.
[0377] Probes for a target subsequence are preferably PNA
oligomers, or less preferably. DNA oligomers, which hybridize to
the subsequence of interest. Use of sets of degenerate DNA
oligomers to more specifically and reliably hybridize to short DNA
subsequences has been described in relation to the PCR
implementation of the QEA method. The use of PNAs is preferred in
the colony calling embodiment since PNA oligomers, due to their
more favorable hybridization energetics, more specifically and
reliably hybridize to shorter complementary DNA subsequences than
do DNA oligomers. Reliable hybridization occurs for PNA 6 to 8-mers
and longer.
[0378] Probing shorter subsequences preferably uses fully
degenerate sets of PNA oligomers, as is the case for DNA
oligomers.
[0379] PNAs are even more preferable when, in the alternative, the
hash code comprises presence or absence indication of target sets
of longer subsequences. In this case, many more DNA probes are
generally required than PNA probes. As PNA 6 to 8-mers reliably
hybridize, target sets can consist of subsequences of length 6 to
8. Since DNA oligomers of this length may not reliably hybridize,
each subsequence in the set must in turn be represented by a
further degenerate set of DNA oligomers, requiring thereby a set of
sets.
[0380] The experimental method of colony calling comprises three
principal steps: first, arraying cDNA libraries on filters or other
suitable substrates; second, PNA hybridization and detection,
alternatively DNA hybridization can be used; and third,
interpreting the resulting hash code to determine the sequence in
the sample.
[0381] The first step, which can be omitted if arrayed cDNA
libraries are already available, is constructing and arraying cDNA
libraries. Any methods known in the art may be used. For example,
cDNA libraries from normal or diseased tissues can be constructed
according to Example 6.3. Alternatively, the human cDNA libraries
constructed by M. B. Soares and colleagues are available as high
density arrays on filters and can be used for the practice of this
method. See Soares et al., 1994, Proc. Natl. Acad. Sci. USA,
91:9228-32. The ability to spot up to thousands of cDNA clones or
colonies on filters suitable for hybridization is an established
technology. This service is now provided by several companies,
including the preferred supplier Research Genetics (Huntsville,
Ala.). The protocol of Example 6.7 can be used to generate these
arrays from cDNA libraries.
[0382] The second step is probe (e.g. PNA) hybridization and
detection. Fluorescently labeled PNA oligomers are available from
PerSeptive Biosystems (Bedford, Mass.) or can be synthesized. PNAs
are designed to be complementary to the chosen target subsequences
and to have a maximum number of distinguishable labels for
simultaneous hybridization with multiple oligomers. PNA
hybridization is performed according to standard protocols
developed by the manufacturer and detailed in Example 6.7.
Detection of the PNA signals uses optical spectrographic means to
distinguish fluorochrome emissions similar to those used in DNA
analysis instruments, but appropriately modified to recognize spots
on filters as opposed to linearly arrayed bands.
[0383] The third step, interpretation of the hash code, is done by
the computer implemented method described in the following
section.
[0384] In an alternative embodiment, the intensity of the detected
hybridization signal indicates the number of times the probe binds
to the sample sequence. In this manner the number of recognized
target subsequences present in the sample can be determined. This
information can be used to more precisely classify of identify a
sample.
5.6. CC ANALYSIS AND DESIGN METHODS
[0385] The colony calling ("CC") computer implemented methods are
similar to the QEA computer methods. As for the QEA case, the
experimental analysis methods are described before the experimental
design methods.
5.6.1. CC EXPERIMENTAL ANALYSIS METHODS
[0386] The analysis methods make use of a mock experiment concept.
First, a database is selected to represent possible sequences in
the sample by the same methods as described for QEA analysis. These
are illustrated and described with reference to FIG. 6A. For CC, an
experimental definition is simply a list of N.sub.p target
subsequences, where N.sub.p is preferably between 16 and 20. Next,
a mock experiment generates one hash code for each sequence in the
selected database, each hash code being a string of N.sub.p binary
digits wherein the n'th digit is a 1 (0) if the n'th target
subsequence does (does not) hybridize with the sequence. The
results of all the mock experiments determine the pattern of hash
codes expected. This pattern is output in a code table of all
possible hash codes in which, for each hash code, there is a list
of all accession numbers of sequences with this code.
[0387] This method is illustrated in more detail in FIG. 15. The
method starts at step 1901 and at step 1902 it inputs a selected
database and on experimental definition consisting of N.sub.p
target subsequences. Step 1903 initializes a table which for each
of the 2.sup.Np hash codes can contain a list of possible accession
numbers which have this hash code. Step 1904 is a DO loop which
iterates through all sequences in the database. For a particular
sequence, step 1905 checks for each target subsequence whether that
subsequence hybridizes to the sequence. This is implemented by
string matching in a manner similar to step 1303 of FIG. 9. A
binary hash code is constructed from this hybridization
information, and step 1906 adds the accession number of the
sequence to the list of accession numbers associated with this hash
code in the code table. Step 1907 outputs the code table and the
method ends at step 1908.
[0388] Having built a pattern of simulated hash code in a code
table, analysis of an experiment requires only simple table
look-up. A colony is hybridized with each of the N.sub.p
recognition means for the target subsequences. The results of the
hybridization are used to construct a resulting hash code. This
code table for this hash code entry then contains a list of
sequence accession numbers that are possible candidates for the
sample sequence. If the list contains only one element, then the
sample has been uniquely identified. If the list contains more than
one element, the identification is ambiguous. If the list is empty,
the sample is not in the selected database and may possibly be a
previously unknown sequence.
[0389] Alternately, as for QEA experimental analysis, a code table
can be dispensed with if only a few hash codes need to be looked up
from only a few experiments. Then the DNA database is scanned
sequence by sequence for those sequences generating the hash code
of interest. If many hash codes from many experiments need to be
analyzed, a code table is more efficient. The quantitative decision
of when to build a code table depends on the costs of the various
operations and the size of DNA database, and can be performed as is
well known in the computer arts. Without limitation, this
description is built on the use of a code table.
[0390] For those embodiments where the recognition means can each
recognize a subset of target subsequences, code table construction
must be modified accordingly. Such embodiments, for example, can
involve DNA oligomer probes which due to their length can hybridize
with an intended target subsequences and those subsequences which
differ by 1 base pair from the intended target. In such
embodiments, step 1905 checks whether each member of such a set of
target subsequences is found in the sample sequence. If any member
is found in the sequence, then this information is used to
construct the hash code.
5.6.2. CC EXPERIMENTAL DESIGN METHODS
[0391] As for QEA, the goal of CC experimental design is to
maximize the amount of information from a CC hybridization
experiment. This is also performed by defining an information
measure and choosing an optimization method which maximizes this
measure.
[0392] The preferred information measure is the number of occupied
hash codes. This is equivalent to minimizing the number of
accession numbers which can result in a given hash code. In fact
for N.sub.p greater than about 17 to 18, that is for 2.sup.Np
greater than the number of expressed human genes (about 100,000),
maximizing the number of occupied hash codes can result in each
hash code representing a single sequence. Such a unique code
contains the maximum amount of information. The invention is
adaptable to other CC information measures. For example, if only a
subset of the possible sequences are of interest, an appropriate
measure would be the number of such sequences which are uniquely
represented by a hash code. As for QEA, these are sequences of
interest.
[0393] One optimization algorithm is exhaustive search. In
exhaustive search, all subsequences of length less than
approximately 10 are tried in all combinations in order to find the
optimum combination producing the best hash code according to the
chosen information measure. This method is inefficient. The
preferred algorithm for optimizing the information from an
experiment is simulated annealing. This is performed by the method
illustrated and described with respect to FIG. 13A. For CC, the
following preferred choices are made.
[0394] The energy is taken to be 1.0 divided by the information
content; alternatively, any monotonically decreasing function of
the information content can be used. The energy is determined by
performing the mock experiment of FIG. 15 using a particular
experimental definition and then applying the measure to the
resulting code table. For example, if the number of occupied hash
codes is the information measure, this number can be computed by
simply scanning the code table and counting the number of table
entries with non-empty accession number lists. The Boltzman
constant is again taken to be 1 so that the temperature equals the
energy. The initial temperature is preferably 1.0. The minimum
energy and temperature, E.sub.0 and T.sub.0, respectively, are
determined by the information measure. For example, with the prior
choices for energy function and information measure, E.sub.0, which
equals T.sub.0, is 1.0 divided by the number of sequences in the
selected database.
[0395] The method of generating a new experimental definition from
an existing definition is to pick randomly one target subsequence
and to perform one of the following moves: (1) randomly modifying
one or more nucleotides; (2) adding a random nucleotide; and (3)
removing a random nucleotide. A modification is discarded if it
results in two identical target subsequences. Further, it is
desirable to discard a modification if the resulting subsequence
has an extreme probability of binding to sequences in the database.
For example, if the modified subsequence binds with a probability
less than approximately 0.1 or more than approximately 0.5 to
sequences in the selected database, it should be discarded. To
generate a new experiment, one of these moves is randomly selected
and carried out on the existing experimental definition.
Alternatively, the various moves can be unequally weighted. The
invention is further adaptable to other methods of generating new
experiments. Preferably, generation methods used will randomly
generate all possible experiments. An initial experimental
definition can be picked by taking N.sub.p randomly chosen
subsequences or by using subsequences from prior optimization.
[0396] Finally, the two execution parameters defining the
"annealing schedule", that is the manner in which the temperature
is decreased during the execution of the simulated annealing
method, are defined and chosen as in the QEA case. The number of
iterations in an epoch, denoted by N, is preferably taken to be 100
and the temperature decay factor, denoted by f, is preferably taken
to be 0.95. Both N and f may be systematically varied case-by-case
to achieve a better experimental definition with lower energy and a
higher information measure.
[0397] With these choices the simulated annealing optimization
method of FIG. 13A can be performed to obtain an optimized set of
target subsequences. To determine an optimum N.sub.p, different
initial N.sub.p can be selected, the prior design optimization
performed, and the results compared. The Np with the maximum
information measure is optimum for the selected database.
5.6.3. CC QUANTITATIVE ALTERNATIVE
[0398] To make use of quantitative detection information the
pattern of simulated hash codes stored in the code table is
augmented with additional information. For each hash code in the
table and each sequence giving rise to that hash code, this
additional information comprises recording the number of times each
target subsequence is found in such a sequence. These numbers are
simply determined by scanning the entire sequence and counting the
number of occurrences of each target subsequence.
[0399] An exemplary method to perform hash code look up in this
augmented table is to first find the sequences giving rise to a
particular hash code as a binary number, and second to pick from
these the most likely sequence as that sequence having the most
similar pattern of subsequence counts to the detected quantitative
hybridization signal. An exemplary method to determine such
similarity is to linearly normalize the detected signal so that the
smallest hybridization signal is 1.0 and then to find the closest
sequence by using a Euclidean metric in an n-dimensional code
space.
[0400] For CC experimental design, each pattern of subsequence
counts may alternatively be considered as a distinct code entry for
evaluation of an information measure. This is instead of
considering each hash code alone a distinct entry.
5.7. APPARATUS FOR PERFORMING THE METHODS OF THE INVENTION
[0401] The apparatus of this invention includes means for
performing the recognition reactions of this invention in a
preferably automated fashion, for example by the protocols of
.sctn. 6.4.3, and means for performing the computer implemented
experimental analysis and design methods of this invention.
Although the subsequent discussion is directed to embodiments of
apparatus for the QEA embodiments of this invention, similar
apparatus is adaptable to the CC embodiments. Such adaption
includes using, in place of the corresponding components for the
QEA embodiments, automatic laboratory instruments appropriate for
making and hybridizing arrays of clones and for reading the results
of the hybridizations, and using programs implementing the computer
analysis and design methods for the CC embodiments described in
.sctn. 5.6.
[0402] FIG. 12A illustrates an exemplary apparatus for the QEA
embodiments of this invention, and with the described adaption,
also for the CC embodiments of this invention. Computer 1601 can
be, alternatively, a UNIX based work station type computer, an
MS-DOS or Windows based personal computer, a Macintosh personal
computer, or another equivalent computer. In a preferred
embodiment, computer 1601 is a PowerPC.TM. based Macintosh computer
with software systems capable of running both Macintosh and
MS-DOS/Windows programs.
[0403] FIG. 12B illustrates the general software structure in RAM
memory 1650 of computer 1601 in a preferred embodiment. At the
lowest software level is Macintosh operating system 1655. This
system contains features 1656 and 1657 for permitting execution of
UNIX programs and MS-DOS or Windows programs alongside Macintosh
programs in computer 1601. At the next higher software level are
the preferred languages in which the computer methods of this
invention are implemented. LabView 1658, from National Instruments
(Dallas, Tex.), is preferred for implementing control routines 1661
for the laboratory instruments, exemplified by 1651 and 1652, which
perform the recognition reactions and fragment separation and
detection. C or C++ languages 1659 are preferred for implementing
experimental routines 1662, which are described in .sctn..sctn. 5.4
and 5.6. Less preferred but useful for rapid prototyping are
various scripting languages known in the art. PowerBuilder 1660,
from Sybase (Denver, Colo.), is preferred for implementing the user
interfaces to the computer implemented routines and methods.
Finally, at the highest software level are the programs
implementing the described computer methods. These programs are
divided into instrument control routines 1661 and experimental
analysis and design routines 1662. Control routines 1661 interact
with laboratory instruments, exemplified by 1651 and 1652, which
physically perform the QEA and CC protocols. Experimental routines
1662 interact with storage devices, exemplified by devices 1654 and
1653, which store DNA sequence databases and experimental
results.
[0404] Returning to FIG. 12A, although only one processor is
illustrated, alternatively, the computer methods and instrument
control interface can be performed on a multiprocessor or on
several separate but linked processors, such that instrument
control methods 1661, computational experimental methods 1661, and
the graphical interface methods can be on different processors in
any combination or sub-combination.
[0405] Input/output devices include color display device 1620
controlled by a keyboard and standard mouse 1603 for output display
of instrument control information and experimental results and
input of user requests and commands. Input and output data are
preferably stored on disk devices such as 1604, 1605, 1624, and
1625 connected to computer 1601 through links 1606. The data can be
stored on any combination of disk devices as is convenient.
Thereby, links 1606 can be either local attachments, whereby all
the disks can be in the computer cabinet(s), LAN attachments,
whereby the data can be on other local server computers, or remote
links, whereby the data can be on distant servers.
[0406] Instruments 1630 and 1631 exemplify laboratory devices for
performing, in a partly or wholly automatic manner, the QEA
recognition reactions. These instruments can be, for example,
automatic thermal cyclers, laboratory robots, and controllable
separation and detection apparatus, such as is found in the
applicants' copending U.S. patent application Ser. No. 08/438,231
filed May 9, 1995. Links 1632 exemplify control and data links
between computer 1601 and controlled devices 1631 and 1632. They
can be special buses, standard LANs, or any suitable link known in
the art. These links can alternatively be computer readable medium
or even manual input exchanged between the instruments and computer
1601. Outline arrows 1634 and 1635 exemplify the physical flow of
samples through the apparatus for performing experiments 1607 and
1613. Sample flow can be either automatic, manual, or any
combination as appropriate. In alternative embodiments there may be
fewer or more laboratory devices, as dictated by the current state
of the laboratory automation art.
[0407] On this complete apparatus, a QEA experiment is designed,
performed, and analyzed, preferably in a manner as automatic as
possible. First, a QEA experiment is designed, according to the
methods specified in .sctn. 5.4.2 as implemented by experimental
routines 1662 on computer 1601. Input to the design routines are
databases of DNA sequences, which are typically representative
selected database 1605 obtained by selection from input
comprehensive sequence database 1604, as described in .sctn. 5.4.1.
Alternatively, comprehensive DNA databases 1604 can be used as
input. Database 1604 can be local to or remote from computer 1601.
Database selection performed by processor 1601 executing the
described methods generates one or more representative selected
databases 1605. Output from the experimental design methods are
tables, exemplified by 1609 and 1615, which, for a QEA RE
embodiment, specify the recognition reaction and the REs used for
each recognition reaction.
[0408] Second, the apparatus performs the designed experiment.
Exemplary experiment 1607 is defined by tissue sample 1608, which
may be normal or diseased, experimental definition 1609, and
physical recognition reactions 1610 as defined by 1609. Where
instrument 1630 is a laboratory robot for automating reaction,
computer 1601 commands and controls robot 1630 to perform reactions
1610 on cDNA samples prepared from tissue 1608. Where instrument
1631 is a separation and detection instrument, the results of these
reactions are then transferred, automatically or manually, to 1631
for separation and detection. Computer 1601 commands and controls
performance of the separation and receives detection information.
The detection information is input to computer 1601 over links 1632
and is stored on storage device 1624, along with the experimental
design tables and information on the tissue sample source for
processing. Since this experiment uses, for example, fluorescent
labels, detection results are stored as fluorescent traces
1611.
[0409] Experiment 1613 is processed similarly along sample pathway
1633, with robot 1630 performing recognition reactions 1616 on cDNA
from tissue 1608 as defined by definition 1615, and device 1631
performing fragment separation and detection. Fragment detection
data is input by computer 1601 and stored on storage device 1625.
In this case, for example, silver staining is used, and detection
data is image 1617 of the stained bands.
[0410] During experimental performance, instrument control routines
1661 provide the detailed control signals needed by instruments
1630 and 1631. These routines also allow operator monitoring and
control by displaying the progress of the experiment in process,
instrument status, instrument exceptions or malfunctions, and such
other data that can be of use to a laboratory operator.
[0411] Third, interactive experimental analysis is performed using
the database of simulated signals generated by analysis and design
routines 1662 as described in .sctn..sctn. 5.4.2 and 5.4.3.
Simulated database 1612 for experiment 1607 is generated by the
analysis methods executing on processor 1601 using as input the
appropriate selected database 1605 and experimental definition
1609, and is output in table 1612. Similarly table 1618 is the
corresponding simulated database of signals for experiment 1613,
and is generated from appropriate selected database 1605 and
experimental definition 1615. A signal is made unambiguous by
experimental routines 1662 that implement the methods described in
.sctn. 5.4.3.
[0412] Display device 1602 presents an exemplary user interface for
the data generated by the methods of this invention. This user
interface is programmed preferably by using the Powerbuilder
display front end. At 1620 are selection buttons which can be used
to select the particular experiment and the particular reaction of
the experiment whose results are to be displayed. Once the
experiment is selected, histological images of the tissue source of
the sample are presented for selection and display in window 1621.
These images are typically observed, digitized, and stored on
computer 1601 as part of sample preparation. The results of the
selected reaction of the selected experiment are displayed in
window 1622. Here, a fluorescent trace output of a particular
labeling is made available. Window 1622 is indexed by marks 1626
representing the possible locations of DNA fragments of successive
integer lengths. Window 1623 displays contents from simulated
database 1612. Using, for example, mouse 1603, a particular
fragment length index 1626 is selected. The processor then
retrieves from the simulated database the list of accession numbers
that could generate a peak of that length with the displayed end
labeling. This window can also contain further information about
these sequences, such as gene name, bibliographic data, etc. This
further information may be available in selected databases 1605 or
may require queries to the complete sequence database 1604 based on
the accession numbers. In this manner, a user can interactively
inquire into the possible sequences causing particular results and
can then scan to other reactions of the experiment by using buttons
1620 to seek other evidence of the presence of these sequences.
[0413] It is apparent that this interactive interface has further
alternative embodiments specialized for classes of users of
differing interests and goals. For a user interested in determining
tissue gene expression, in one alternative, a particular accession
number is selected from window 1623 with mouse 1603, and processor
1601 scans the simulated database for all other fragment lengths
and their recognition reactions that could be produced by this
accession number. In a further window, these lengths and reactions
are displayed, and the user allowed to select further reactions for
display in order to confirm or refute the presence of this
accession number in the tissue sample. If one of these other
fragments are generated uniquely by this sequence (a "good
sequence", see supra), that fragment can be highlighted as of
particular interest. By displaying the results of the generating
reaction of that unique fragment, a user can quickly and
unambiguously determine whether or not that particular accession
number is actually present in the sample.
[0414] In another interface alternative, the system displays two
experiments side by side, displaying two histological images 1621
and two experimental results 1622. This allows the user to
determine by inspection signals present in one sample and not
present in the other. If the two samples were diseased and normal
specimens of the same tissue, such signals would be of considerable
interest as perhaps reflecting differences due to the pathological
process. Having a signal of interest, preferably repeatable and
reproducible, a user can then determine the likely accession
numbers causing it by invoking the previously described interface
facilities. In a further elaboration of this embodiment, system
1601 can aid the determination of signals of interest by automating
the visual comparison by performing statistical analysis of signals
from samples of the same tissue in different states. First, signals
reproducibly present in tissue samples in the same state are
determined, and second, differences in these reproducible signals
across samples from the several states are compared. Display 1602
then shows which reproducible signals vary across the states,
thereby guiding the user in the selection of signals of
interest.
[0415] The apparatus of this invention has been described above in
an embodiment adapted to a single site implementation, where the
various devices are substantially local to computer 1601 of FIG.
12A, although the various links shown could also represent remote
attachments. An alternative, explicitly distributed embodiment of
this apparatus is illustrated in FIG. 12C. Shown here are
laboratory instruments 1670, DNA sequence database systems 1684,
and computer systems 1671 and 1673, all of which cooperate to
perform the methods of this invention as described above.
[0416] These systems are interconnected by communication medium
1674 and its local attachments 1675, 1676, and 1677 to the various
systems. This medium may be any dedicated or shared or local or
remote communication medium known in the art. For example, it can
be a "campus" LAN network extending perhaps a few kilometers, a
dedicated wide area communication system, or a shared network, such
as the Internet. The system local attachments are adapted to the
nature of medium 1674.
[0417] Laboratory instruments 1670 are commanded by computer system
1671 to perform the automatable steps of the recognition reactions,
separation of the reaction results, and detection and transmission
of resulting signals through link 1672. Link 1672 can be any local
or remote link known in the art that is adapted to instrument
control, and may even be routed through communication medium
1674.
[0418] DNA sequence database systems 1684 with various sequence
databases 1685 may be remote from the other systems, for example,
by being directly accessed at their sites of origin, such as
Genbank at Bethesda, MD. Alternatively, parts or all of these
databases nay be periodically downloaded for local access by
computer systems 1671 and 1672 onto such storage devices as discs
or CD-ROMs.
[0419] Computer system 1671, including computer 1681, storage 1682,
and display 1683, can perform various methods of this invention.
For example, it can perform solely the control routine for control
and monitoring of instrument system 1670, whereby experimental
design and analysis are performed elsewhere, as at computer system
1673. In this case, system 1671 it would typically be operated by
laboratory technicians. Alternatively, system 1671 can also perform
experimental designs, which meet the requirements of remote users
of sample analysis information. In another alternative, system 1671
can carry out all the computer implemented methods of this
invention, including final data display, in which case it would be
operated by the final users of the analysis information.
[0420] Computer system 1673, including computer 1678, storage 1679,
and display 1680, can perform a corresponding range of functions.
However, typically system 1673 is remotely located and would be
used by final users of the DNA sample information. Such users can
include clinicians seeking information to make a diagnosis, grade
or stage a disease, or guide therapy. Other users can include
pharmacologists seeking information useful for the design or
improvement of drugs. Finally, other users can include researchers
seeking information useful to basic studies in cell biology,
developmental biology, etc. It is also possible that a plurality of
computer systems 1673 can be linked to laboratory system 1670 and
control system 1671 in order to provide for the analysis needs of a
plurality of classes of users by designing and causing the
performance of appropriate experiments.
[0421] It will be readily apparent to those of skill in the
computer arts that alternative distributed implementations of the
apparatus of this invention, along with alternative functional
allocations of the computer implemented methods to the various
distributed systems, are equally possible.
[0422] All the computer implemented methods of this invention can
be recorded for storage and transport on any computer readable
memory devices known in the art. For example, these include, but
are not limited to, semiconductor memories--such as ROMs, PROMs,
EPROMs, EEPROMS, etc. of whatever technology or
configuration--magnetic memories--such as tapes, cards, disks, etc
of whatever density or size --optical memories--such as optical
read-only memories, CD-ROM, or optical wirteable memories--and any
other computer readable memory technologies.
[0423] Also, although this apparatus has been described primarily
with reference to QEA analysis of human tissue samples, the
laboratory instruments and associated control, design, and analysis
computer systems are not so limited.
[0424] They are also adaptable to performing the CC embodiment of
this invention and to the analysis of other samples, such as from
animal models or in vitro cultures.
[0425] The invention is further described in the following examples
which are in no way intended to limit the scope of the
invention.
6. EXAMPLES
6.1. SUBSEQUENCE HIT AND LENGTH INFORMATION
[0426] This example illustrates QEA signals generated by a PCR
embodiment. From the October 1994 GenBank database, 12,000 human
first continuous coding domain sequences ("CDS") were selected.
This selection resulted in pool of sequences with a bias toward
shorter genes, the average length of the selected CDSs being 1000
bp instead of the typical coding sequence length of 1800-2000 bp,
and with no guarantee that sequences were not be repeated in the
selection. From this set, tables containing the probability of
occurrence of all 4 to 6-mer sequences were constructed.
[0427] Then Eqns. 1 and 2 were solved for N=12,000 and L=1,000
resulting in p=0.17 and M=108. Five 6-mer target subsequences with
this probability of occurrence were chosen from the 6-mer tables
and grouped into four pairs: CAGATA--TCTCAC, CAGATA--GGTCTG,
CAGATA--GCTCAA CAGATA--CACACC. The pool of selected CDSs were then
scanned against these four pairs of target subsequences to
determine whether any pair hit and if so the length between the
hits.
[0428] The histogram of FIG. 1 presents the results of this scan.
Along axis 102 is the relative length between subsequence pair
hits. This would be the length observed in a gel separation of the
amplified fragments of a QEA PCR reaction using these target
subsequences. Along axis 101 is the number hits at any given
length. For example, spike 103 at a length of approximately 800
base pairs represents a fragment length having three hits. Multiple
hits at one length may occur either because several CDSs have one
target subsequence pair spaced this length, because one CDS has
several target subsequence pairs spaced this length, because of
redundancy in the selected CDSs, or because signals of this length
were generated by more than one pair of target subsequences. Spike
104 at a slightly longer length represents a relative length with
only one hit. This fragment is generated from a unique sequence and
provides a unique indication of its presence in a cDNA mixture,
that is, this is a good sequence.
6.2. RESTRICTION ENDONUCLEASES
[0429] Tables 1-4 list all palindromic 4-mer and 6-mer potential RE
recognition sequences. RE enzymes recognizing each site, where
known, are also listed, along with an exemplary commercial
supplier. Over 85% of possible sequences spanning a wide range of
occurrence probabilities have a known RE recognizing and cleaving
the sequence.
[0430] The frequency of these sequences was determined, as in
example 6.1, in 12,000 human first continuous coding domain
sequences selected from the October 1994 GenBank database. The
tables are sorted in order of increasing recognition occurrence
probability. The bar in the recognition sequence indicates the site
in the recognition sequence where the RE cuts.
[0431] The following vendor abbreviations are used: New England
Biolabs (Beverly, Mass.) ("NEB"), Stratagene (La Jolla, Calif.),
Boehringer Mannheim (Indianapolis, Ind.) ("BM"), and Gibco BRL
division of Life Technologies (Gaithersburg, Md.) ("BRL").
1TABLE 1 THE 4-MER RESTRICTION SITES Recognition CDS Sequence
Frequency RE Overhang Vendor C.vertline.GCG 0.36 SelI 2
C.vertline.TAG 0.44 MaeI 2 NEB T.vertline.TAA 0.45 MseI 2 NEB TATA
0.45 none GCG.vertline.C 0.50 HhaI 2 NEB ATAT 0.50 none
A.vertline.CGT 0.52 MaeII 2 BM T.vertline.CGA 0.53 TaqI 2 NEB
.vertline.AATT 0.53 Tsp5091 4 NEB C.vertline.CGG 0.61 MspI 2 NEB
G.vertline.TAC 0.64 Csp6I 2 NEB .vertline.GATC 0.67 Sau3AI 4 NEB
CATG.vertline. 0.68 NlaIII 4 NEB TG.vertline.CA 0.78 CviRI 0
AG.vertline.CT 0.78 AluI 0 NEB GG.vertline.CC 0.79 HaeIII 0 NEB
[0432]
2TABLE 2 THE FIRST 20 6-MER RESTRICTION SITES CDS Sequence
Frequency RE Overhang Vendor TCG.vertline.CGA 0.01 NruI 0 NEB
TAC.vertline.GTA 0.02 SnaBI 0 NEB C.vertline.GTACG 0.02 BsiWI 4 NEB
CGAT.vertline.CG 0.02 PvuI 2 NEB A.vertline.CGCGT 0.03 MluI 4 NEB
A.vertline.CTAGT 0.03 SpeI 4 NEB G.vertline.TCGAC 0.04 SalI 4 NEB
AA.vertline.CGTT 0.04 Psp1406I 2 NEB A.vertline.CCGGT 0.04 AgeI 4
NEB G.vertline.CTAGC 0.04 NheI 4 NEB TATATA 0.04 none
GTT.vertline.AAC 0.05 HpaI 0 NEB TAGCTA 0.05 none TAATTA 0.05 none
GTA.vertline.TAC 0.05 Bst1107I 0 NEB CTATAG 0.05 none CGCGCG 0.05
none C.vertline.CTAGG 0.06 AvrII 4 NEB TT.vertline.CGAA 0.06 SfaI 2
BM AT.vertline.CGAT 0.06 ClaI 2 NEB
[0433]
3TABLE 3 THE MIDDLE 20 6-MER RESTRICTION SITES CDS Sequence
Frequency RE Overhang Vendor C.vertline.TTAAG 0.06 AflII 4 NEB
T.vertline.CTAGA 0.06 Xbal 4 NEB ATATAT 0.07 none AT.vertline.TAAT
0.07 vspI 2 BRL G.vertline.CGCGC 0.08 BssHII 4 NEB C.vertline.AATTG
0.08 MunI 4 NEB GACGT.vertline.C 0.08 AatII 4 NEB TTATAA 0.09 none
TGC.vertline.GCA 0.10 FspI 0 NEB C.vertline.TCGAG 0.01 XhoI 4 NEB
GAT.vertline.ATC 0.01 EcoRV 0 NEB CA.vertline.TATG 0.10 NdeI 2 NEB
ATGCA.vertline.T 0.01 NsiI 4 NEB AGC.vertline.GCT 0.11 Eco47III 0
NEB AAT.vertline.ATT 0.11 SspI 0 NEB T.vertline.CCGGA 0.11 AccIII 4
Stratag ene TTT.vertline.AAA 0.12 DraI 0 NEB A.vertline.CATGT 0.12
BspLVII 4 CAC.vertline.GTG 0.12 Eco72I 0 Stratag ene
CCGC.vertline.GG 0.12 SacII 2 NEB
[0434]
4TABLE 4 THE LAST 24 6-MER RESTRICTION SITES CDS Sequence Frequency
RE Overhang Vendor GCATG.vertline.C 0.13 SphI 4 NEB TTGCAA 0.13
none A.vertline.AGCTT 0.13 HindIll 4 NEB G.vertline.TGCAC 0.13
ApaLI 4 NEB AAATTT 0.14 none AGT.vertline.ACT 0.15 ScaI 0 NEB
G.vertline.AATTC 0.15 EcoRI 4 NEB GGTAC.vertline.C 0.15 KpnI 4 NEB
T.vertline.GTACA 0.15 Bsp1407I 4 NEB C.vertline.GGCCG 0.15 EagI 4
NEB G.vertline.CCGGC 0.16 NgoMI 4 NEB GGC.vertline.GCC 0.16 NarI 0
NEB T.vertline.GATCA 0.16 BclI 4 NEB T.vertline.CATGA 0.17 BspHI 4
NEB C.vertline.CCGGG 0.19 SmaI 4 NEB G.vertline.GATCC 0.19 BamHI 4
NEB A.vertline.GATCT 0.20 BglII 4 NEB AGG.vertline.CCT 0.22 StuI 0
NEB GGGCC.vertline.C 0.24 ApaI 4 NEB C.vertline.CATGG 0.24 NcoI 4
NEB GAGCT.vertline.C 0.25 SacI 4 NEB TGG.vertline.CCA 0.33 MscI 0
NEB CAG.vertline.CTG 0.42 PvuII 0 NEB CTGCA.vertline.G 0.43 PstI 4
NEB
6.3. RNA EXTRACTION AND cDNA SYNTHESIS
RNA Preparation
[0435] RNA extraction is done using Triazol reagent from Life
Technologies (Gaithersburg, Md.) following the protocol of
Chomszynski et. al., 1987, Annal. Biochem. 162:156-59 and
Chomszynski et. al., 1993, Biotechniques, 15:532-34,536-37. Total
RNA is first extracted from tissues, treated with Rnase-free Dnase
I from Pharmacia Biotech (Uppsala, Sweden) to remove contaminating
genomic DNA, followed by messenger RNA purification using oligo
(dT) magnetic beads from Dynal Corporation (Oslo, Norway), and then
used for cDNA synthesis.
[0436] If desired, total cellular RNA can be separated into
sub-pools prior to cDNA synthesis. For example, a sup-pool of
endoplasmic reticulum associated RNA is enriched for RNA producing
proteins having an extra-cellular or receptor function.
[0437] Tissue Homogenization and Total RNA Extraction:
[0438] A voxel is used to describe the specific piece of tissue to
be analyzed. Most frequently it will refer to grid punches
corresponding to pathologically characterized tissue sections.
[0439] 1. It is important that tissue voxels be quick frozen in
liquid nitrogen immediately after dissection, and stored at
-70.degree. C. until processed.
[0440] 2. The weight of the frozen tissue voxel is measured and
recorded.
[0441] 3. Tissue voxels are pulverized and ground in liquid
nitrogen, either with a porcelain mortar and pestle, or by
stainless steel pulverizers, or alternative means. This tissue is
ground to a fine powder and is kept on liquid nitrogen.
[0442] 4. The tissue powder is transferred to a tube containing
Triazol reagent (Life Technologies, Gaithersburg, Md.) with 1 ml of
reagent per 100 mg of tissue and is dispersed in the Triazol using
a Polytron homogenizer from Brinkman Instruments (Westbury, N.Y.).
For small tissue voxels less than 100 mg, a minimum of 1 ml of
Triazol reagent should be used for efficient homogenization.
[0443] 5. Add 0.1 volumes BCP (1-bromo-3-chloropropane) (Molecular
Research, Cincinnati, Ohio) and mix by vortexing for 30 seconds.
Let the mixture stand at room temperature for 15 minutes.
[0444] 6. Centrifuge for 15 minutes at 4.degree. C. at 12,000 X
G.
[0445] 7. Remove the aqueous phase to a fresh tube and add 0.5
volumes isopropanol per original amount of Triazol reagent used and
mix by vortexing for 30 seconds. Let the mixture stand at room
temperature for 10 minutes.
[0446] 8. Centrifuge at room temperature for 10 minutes at 12,000X
G.
[0447] 9. Wash with 70% ethanol and centrifuge at room temperature
for 5 minutes at 12,000 X G.
[0448] 10. Remove the supernatant and let the centrifuge tube stand
to dry in an inverted position.
[0449] 11. Resuspend the RNA pellet in water (1 .mu.l per mg of
original tissue weight) and heat to 55.degree. C. until completely
dissolved.
[0450] DNase treatment:
[0451] 1. Add 0.2 volume of 5X reverse transcriptase buffer (Life
Technologies, Gaithersburg, Md.), 0.1 volumes of 0.1 M DTT, and 5
units RNAguard per 100 mg starting tissue from Pharmacia Biotech
(Uppsala, Sweden).
[0452] 2. Add 1 unit RNase-free DNase I, Pharmacia Biotech, per 100
mg starting tissue. Incubate at 37.degree. C. for 20 minutes.
[0453] The following additional steps are optional,
[0454] Opt 1. Repeat RNA extraction by adding 10 volumes of Triazol
reagent.
[0455] Opt 2. Repeat steps 5 through 11.
[0456] 3. Quantify the total RNA (from the RNA concentration
obtained by measuring OD.sub.260 of a 100 fold dilution). Store at
-20.degree.C.
[0457] Isolation of Poly A.sup.+ Messenger RNA:
[0458] Poly-adenylated mRNA is isolated from total RNA preparations
using magnetic bead mediated oligo-dT detection. Kits that can be
used include Dynabeads mRNA Direct Kit from Dynal (Oslo, Norway) or
MPG Direct mRNA Purification Kit from CPG (Lincoln Park, N.J.).
Protocols are used as directed by the manufacturer.
[0459] Less preferably, the following procedure can be used. The
Dynal oligo(dT) magnetic beads have a capacity of 1 ug
poly(A.sup.+) per 100 ug of beads (1 mg/ml concentration), assuming
2% of the total RNA has poly(A.sup.+) tails.
[0460] 1. Add 5 volumes of Lysis/Binding buffer (Dynal) and
sufficient beads to bind the estimated poly(A.sup.+) RNA.
[0461] 2. Incubate at 65.degree. C. for 2 minutes, then at room
temperature for 5 minutes.
[0462] 3. Wash beads with 1 ml Washing buffer/LiDS (Dynal)
[0463] 4. Wash beads with 1 ml Washing buffer (Dynal) 2 times.
[0464] 5. Elute poly(A.sup.+) RNA with 1 .mu.l water/ug beads 2
times.
[0465] For both methods, the poly-adenylated RNA is harvested in a
small volume of water, quantified as above, and stored at
-20.degree. C. Typical yields of poly-adenylated RNA range from 1%
to 4% of the input total RNA.
cDNA Synthesis
[0466] cDNA is synthesized using the Superscript.TM. Choice system
from Life Technologies, Inc. (Gaithersburg, Md.). if greater than 1
.mu.g of polyadenylated RNA is used, the manufacturer's protocols
are followed, using 50 ng of random hexamer primers per microgram
of polyadenylated RNA.
[0467] If tissue voxels are the source for the RNA, the
polyadenylated RNA is not quantified, and the entire yield of
polyadenylated RNA is concentrated by precipitation with ethanol.
The polyadenylated RNA is resuspended in 10 .mu.l of water, and 5
to 10 .mu.l are used for cDNA synthesis. The manufacturer's
protocols are followed for RNA amounts of less than 1 .mu.g, and
100 ng of random hexamers are used as primers. The resulting volume
of the cDNA solution is 150 .mu.l, but the amount is not
quantified. QEA test reactions are run using 1 .mu.l or 0.1 .mu.l
of cDNA solution in order to determine the appropriate amount of
cDNA to use for subsequence QEA reactions.
[0468] Alternative primers for first strand synthesis known in the
art can also be used for first strand synthesis. Such primers
include oligo(dT) primers, phasing primers, etc.
6.4. QEA PREFERRED RE METHOD
[0469] This protocol is designed to keep the number of individual
manipulations down, and thereby raise the reproducibility of the
QEA procedure. In a preferred method no buffer changes,
precipitations or organic (phenol/chloroform) extractions are used,
all of which lower the overall efficiency of the process and reduce
its utility for general use and more specifically for its use in
automated or robotic procedures.
6.4.1. cDNA PREPARATION
[0470] Terminal phosphate removal from cDNA is illustrated with the
use of Barents sea shrimp alkaline phosphatase ("SAP") (U.S.
Biochemical Corp.) and 2.5 .mu.g of cDNA. Substantially less
(<10 ng) or more (>20 .mu.g) of cDNA can be prepared at a
time with proportionally adjusted amounts of enzymes. Volumes are
maintained to preserve ease of handling. The quantities necessary
are consistent with using the method to analyze small tissue
samples from normal or diseased specimens.
[0471] 1. Mix the following reagents
[0472] 2.5 .mu.l 200 mM Tris-HCL
[0473] 23 .mu.l cDNA
[0474] 2 .mu.l 2 units/.mu.l Shrimp alkaline phosphatase
[0475] The final resulting cDNA concentration is 100 ng/.mu.l.
[0476] 2. Incubate at 37.degree. C. for 1 hour
[0477] 3. Incubate at 80.degree. C. 15 minutes to inactivate the
SAP.
6.4.2. PREFERRED RE/LIGASE AND AMPLIFICATION REACTIONS
[0478] Once the cDNA has been prepared, including terminal
phosphate removal, it is separated into a number of batches of from
10 ng to 200 ng each, equal to the desired number of individual
samples that need to be analyzed and the extent of the analysis.
For example, if six RE/ligase reactions and six analyses are needed
to generate all necessary signals, six batches are made. Shown by
example are 50 ng fractions.
[0479] RE/ligase reactions are performed as digestions by,
preferably, a pair of REs; alternatively, one or three or more REs
can be used provided the four base pair overhangs generated by each
RE differ and can each be ligated to a uniquely adapter and a
sufficiently resolved length distribution results. The amount of RE
enzyme specified is sufficient for complete digestion while
minimizing any other exo- or endo-nuclease activity that may be
present in the enzyme.
[0480] Adapters are chosen that are unique to each RE in a
reaction. Thus, one uses a linker complementary to each unique RE
sticky overhang and a primer which uniquely hybridized with that
linker. The primer/linker combination is an adapter, which will
preferably be uniquely and distinguishably labeled.
Adapter Annealing
[0481] Pairs of 12-mer linkers and 24-mer primers are pre-annealed
to form adapters before they are used in the QEA reactions, as
follows:
[0482] 1. Add to water linker and primer in a 2:1 concentration
ratio (12-mer:24-mer) with the primer at a total concentration of 5
pM per .mu.l.
[0483] 2. Incubate at 50.degree. C. for 10 minutes.
[0484] 3. Cool slowly to room temperature and store at -20.degree.
C.
Restriction-Digestion/Ligation Reaction
[0485] Reactions are prepared for use in a 96 well thermal cycler.
Add per reaction:
[0486] 1. 1 U of appropriate REs (New England Biolabs, Beverly,
Mass.) (preferred RE pair listing in .sctn. 6.9)
[0487] 2. 1 .mu.l of appropriate annealed adapter
[0488] 3. 1 .mu.l of Ligase/ATP (0.2 .mu.l T4 DNA ligase [1
U/.mu.l]/0.8 .mu.l 10 mM ATP from Life Technologies (Gaithersburg,
Md.))
[0489] 4. 0.5 .mu.l 50 mM MgCl.sub.2
[0490] 5. 10 ng of subject prepared cDNA
[0491] 6. 1 .mu.l 10X NEB2 buffer from New England Biolabs
(Beverly, Mass.)
[0492] 7. Water to bring total volume to 10 .mu.l
[0493] Then perform the RE/ligation reaction by following the
thermal profile in FIG. 16A using a PTC-100 Thermal Cycler from MJ
Research (Watertown, Mass.).
Amplification Reaction
[0494] Prepare the PCR reaction mix by combining:
[0495] 1. 10 .mu.l 5X E-Mg (300 mM Tris-Hcl pH 9.0, 75 mM
(NH.sub.4).sub.2SO.sub.4, no Mg ions))
[0496] 2. 100 pm of appropriate fluorescently labeled 24-mer
primers
[0497] 3. 1 .mu.l 10 mM dNTP mix (Life Technologies, Gaithersburg,
Md.)
[0498] 4. 2.5 U of 50:1 Taq polymerase (Life Technologies,
Gaithersburg, Md.) : Pfu polymerase (Stratagene, La Jolla,
Calif.)
[0499] 5. Water to bring volume to 40 .mu.l per PCR reaction
[0500] Then perform the following steps:
[0501] 1. Add 40 .mu.l of the PCR reaction mix to each RE/ligation
reaction
[0502] 2. Perform the PCR temperature profile of FIG. 16B using a
PTC-100 thermal cycler (MJ Research, Watertown, Mass.)
6.4.3. PREFERRED AUTOMATED RE/LIGASE REACTIONS
[0503] The reactions of the preceding section can be automated
according to the following protocol which requires intermediate
reagent additions or by a protocol note requiring such
additions.
Single Tube Protocol With Reagent Additions
[0504] Reactions are preformed in a standard 96 well thermal cycler
format using a Beckman Biomek 2000 robot (Beckman, Sunnyvale,
Calif.). Typically 4 cDNA samples are analyzed in duplicate with 12
different RE pairs, for a total of 96 reactions. All steps are
performed by the robot, including solution mixing, from user
provided stock reagents, and temperature profile control.
[0505] Pre-annealed adapters are prepared as in the preceding
section.
Restriction-Digestion/Ligation Reaction
[0506] Mix per reaction:
[0507] 1. 1 U of appropriate RE (New England Biolabs, Beverly,
Mass.)
[0508] 2. 1 .mu.l of appropriate annealed adapter (10 pmoles)
[0509] 3. 0.1 .mu.l T4 DNA ligase [1 U/.mu.l] (Life Technologies
(Gaithersburg, Md.)
[0510] 4. 1 .mu.l ATP (Life Technologies, Gaithersburg, Md.)
[0511] 5. 5 ng of subject prepared cDNA
[0512] 6. 1.5 .mu.l 10X NEB2 buffer from New England Biolabs
(Beverly, Mass.)
[0513] 7. 0.5 .mu.l of 50 mM MgCl.sub.2
[0514] 8. Water to bring total volume to 10 .mu.l and transfer to
thermal cycler
[0515] The robot requires 23 minutes total time to set up the
reactions. Then it performs the RE/ligation reaction by following
the temperature profile of FIG. 16C using a PTC-100 Thermal Cycler
equipped with a mechanized lid from MJ Research (Watertown,
Mass.).
Amplification Reaction
[0516] Prepare the PCR reaction mix by combining:
[0517] 1. 10 .mu.l 5X E-Mg (300 mM Tris-HCl pH 9.0, 75 mM
(NH.sub.4).sub.2SO.sub.4)
[0518] 2. 100 pm of appropriate fluorescently labeled 24-mer
primer
[0519] 3. 1 .mu.l 10 mm dNTP mix (Life Technologies, Gaithersburg,
Md.)
[0520] 4. 2.5 U of 50:1 Taq polymerase (Life Technologies,
Gaithersburg, Md.) : Pfu polymerase (Stratagene, La Jolla,
Calif.)
[0521] 5. Water to being volume to 35 .mu.l per PCR reaction
[0522] Preheat the PCR mix to 72.degree. C. and transfer 35 .mu.l
of the PCR mix to each digestion/ligation reaction and mix. The
robot requires 6 minutes for the transfer and mixing.
[0523] Then the robot performs the PCR amplification reaction by
following the temperature profile of FIG. 16B using a PTC-100
thermal cycler equipped with a mechanized lid (MJ Research,
Watertown, Mass.).
[0524] The total elapsed time for the digestion/ligation and PCR
amplification reactions is 179 minutes. No user intervention is
required after initial experimental design and reagent
positioning.
Single Tube Protocol Without Reagent Additions
[0525] First, add the PCR reaction mix by combining in the reaction
tube:
[0526] 1. 10 .mu.l 5X E-Mg (300 mM Tris-HCl pH 9.0, 75 mM
(NH.sub.4).sub.2SO.sub.4)
[0527] 2. 100 pm of appropriate fluorescently labeled 24-mer
primer
[0528] 3. 2 .mu.l 10 mM dNTP mix (Life Technologies, Gaithersburg,
Md.)
[0529] 4. 2.5 U of 50:1 Taq polymerase (Life Technologies,
Gaithersburg, Md.): Pfu polymerase (Stratagene, La Jolla,
Calif.)
[0530] 5. Water to bring volume to 40 .mu.l per PCR reaction
[0531] Second, add a bead of wax melting approximately at
72.degree. C. (Ampliwax, Perkin-Elmer, Norwalk, Conn.). Melt the
wax at 75.degree. C. for 5 minutes, and let the wax solidify at
25.degree. C. for 10 minutes with the lid open.
[0532] Third, add the RE/ligase reaction mix by combining in the
reaction tube:
[0533] 1. 0.1 .mu.l of the REs (New England Biolabs, Beverly,
Mass.)
[0534] 2. 1 .mu.l of appropriate annealed adapter (2:1 of 12:24 mer
at 50 pmoles/ml)
[0535] 3. 0.2 .mu.l T4 DNA ligase [1 U/.mu.l] (Life Technologies
(Gaithersburg, Md.)
[0536] 4. 1 .mu.l of 0.1 M ATP (Life Technologies, Gaithersburg,
Md.)
[0537] 5. 1 .mu.l of subject prepared cDNA (0.1-10 ng)
[0538] 6. 0.1 .mu.l 10X NEB 2 buffer from New England Biolabs
(Beverly, Mass.)
[0539] 7. 0.5 .mu.l of 50 mM MgCl.sub.2
[0540] 8. Water to bring total volume to 10 .mu.l and transfer to
thermal cycler
[0541] Then perform the RE/ligation and PCR reactions by following
the thermal profile in FIG. 16D using, for example, a PTC-100
Thermal Cycler from MJ Research (Watertown, Mass.).
6.4.4. ALTERNATIVE RE/LIGASE AND AMPLIFICATION REACTIONS
[0542] Once the cDNA has been prepared it is separated into a
number of batches of from 20 ng to 200 ng each equal to the desired
number of individual samples that need to be analyzed and the
extent of the analysis. For example, if six RE/ligase reactions and
six analyses are needed to generate all necessary signals, six
batches are made. Shown by example are 50 ng fractions.
[0543] RE/ligase reactions are performed as digestions by,
preferably, a pair of REs; alternatively, one or three or more REs
can be used provided the four base pair overhangs generated by each
RE differ and can each be ligated to a uniquely adapter and a
sufficiently resolved length distribution results. The amount of RE
enzyme specified is sufficient for complete digestion while
minimizing any other exo- or endo-nuclease activity that may be
present in the enzyme.
RE Digestion
[0544] Digest (with 50 ng of cDNA)
[0545] 1. Mix the following reagents
[0546] 0.5 .mu.l prepared cDNA (100 ng/.mu.l) mixture
[0547] 10 .mu.l New England Biolabs Buffer No. 2
[0548] 3 Units RE enzyme
[0549] 2. Incubate for 2 hours at 37.degree. C. Larger size digests
with higher concentrations of cDNA can be used and fractions of the
digest saved for additional sets of experiments.
Adapter Ligation
[0550] Since it is important to remove unwanted ligation products,
such as concatamers of fragments from different cDNAs resulting
from hybridization of RE sticky ends, the restriction enzyme is
left active during ligation. This leads to a continuing cutting of
unwanted concatamers and end ligation of the desired end
adapters.
[0551] The majority of restriction enzymes are active at the
16.degree. C. ligation temperature. Ligation profiles consisting of
optimum ligation conditions interspersed with optimum digestion
conditions can also be used to increase efficiency of this process.
An exemplary profile comprises periodically cycling between
37.degree. C. and 10.degree. C. and 16.degree. C. at a ramp of
1.degree. C./min.
[0552] One linker complementary to each 5 minutes overhang
generated by each RE is required. 100 pico moles ("pm") is a
sufficient molar excess for the protocol described. For each linker
a complementary uniquely labeled primer is added for ligation to
the cut ends of cDNAs. 100 pm is a sufficient molar excess for the
protocol described. If the amounts of RE cDNA is changed the linker
and primer amounts should be proportionately changed.
Ligation Reaction
[0553] (per 10 .mu.l and 50 ng cDNA)
[0554] 1. Mix the following reagents
5 Component Volume RE digested cDNA mixture 10 .mu.l 100 pM/.mu.l
each primer 1 .mu.l 100 pM/.mu.l each linker 1 .mu.l
[0555] 2. Thermally cycle from 50.degree. C. to 10.degree. C.
(-1.degree. C./minute) then back to 16.degree. C.
[0556] 3. Add 2 .mu.l 10 mM ATP with 0.2 .mu.l T4 DNA ligase
(Premix 0.1 .mu.l ligase 1 U/.mu.l per 1 .mu.l ATP) (E. Coli ligase
is a less preferred alternative ligase.)
[0557] 4. Incubate 12 hours at 16.degree. C. This step can be
shortened to less than 2 hours with proportionately higher ligase
concentration. Alternately the thermal cycling protocol described
can be used here.
[0558] 5. Incubate 2 hours 37.degree. C.
[0559] 6. Incubate 20 minutes at 65.degree. C. to heat inactivate
the ligase (last step should be RE cutting).
[0560] 7. Hold at 4.degree. C.
Amplification Of Fragments With Ligated Adapters
[0561] This step amplifies the fragments that have been cut twice
and ligated with adapters unique for each RE cut end. It is
designed for a very high amplification specificity. Multiple
amplifications are performed, with an increasing number of
amplification cycles. Use the minimum number of cycles to get the
desired signal. Amplifications above 20 cycles are not generally
reliably quantitative.
[0562] Mix the following to form the ligation mix:
6 Component Volume RE/Ligase cDNA mixture 5 .mu.l 10X PCR Buffer 5
.mu.l 25 mM MgCl.sub.2 3 .mu.l 10 mM dNTPs 1 .mu.l 100 pM/.mu.l
each primer 1 .mu.l
[0563] Mix the following to form 150 .mu.l PCR-Premix
[0564] 30 .mu.l Buffer E (ligation mix will contribute 0.3 mM
MgCl)
7 1 .mu.l (300 pmoles/.mu.l Rbuni24 Flour) 24 mer primer strand (50
pmoles/.mu.l NBuni24 Tamra) 0.6 .mu.l Tag polymerase (per 150
.mu.l) 3 .mu.l dNTP (10 mM) 106 .mu.l H.sub.2O
[0565] Amplification of fragments is more specific if the small
linker dissociates from the ligated primer-cDNA complex prior to
amplification. The following is an exemplary method for
amplification of the results of six RE/ligase reactions.
[0566] 1. Place three strips of six PCR tubes, marked 10, 15, and
20 cycles, into three rows on ice as shown.
8 20 cycles 1 2 3 4 5 6-Add 140 .mu.l PCR-premix 15 cycles 1 2 3 4
5 6 10 cycles 1 2 3 4 5 6-Add 10 .mu.l ligation mix
[0567] 2. Place 10 .mu.l ligation mix in each tube in 10 cycle
row
[0568] 3. Place 140 .mu.l PCR premix in each tube in 20 cycle
row
[0569] 4. Place into cycler and incubate for 5 minutes at
72.degree. C.
[0570] This melts linker which was not covalently ligated to the
second strand of a cDNA fragment and allows the PCR premix to come
to temperature.
[0571] 5. Move the 140 .mu.l PCR premix into the tubes in the 10
cycle row containing the 10 .mu.l ligation mix, then place 50 .mu.l
of result into corresponding tubes each in other rows.
[0572] 6. Incubate for 5 minutes at 72.degree. C. This finishes
incompletely double stranded cDNA ends into complete dsDNA, the top
primer being used as template for second strand completion.
[0573] The amplification cycle is designed to raise specificity and
reproducibility of the reaction. High temperature and long melting
times are used to reduce bias of amplification due to high G+C
content. Long extension times are used to reduce bias in favor of
smaller fragments.
[0574] 7. Thermally cycle 95.degree. C. for 1 minute followed by
68.degree. C. for 3 minutes. Long denaturing times reduce PCR bias
due to melting rates of fragments, and long extension time reduces
PCR bias on fragment sizes.
[0575] 8. Incubate at 72.degree. C. for 10 minutes at end of
reaction.
6.4.5. OPTIONAL POST-AMPLIFICATION STEPS
[0576] Several optional steps can improve the signal from the
detected bands. First, single strands produced as a result of
linear amplification from singly cut fragments can be removed by
the use of single strand specific exonuclease. Exo I is the
preferred nuclease.
[0577] 1. Incubate 2 units of nuclease with the product of each PCR
reaction for 60 minutes at 37.degree. C.
[0578] Second, the amplified products can be concentrated prior to
detection either by ethanol precipitation or column separation with
a hydroxyapatite column.
[0579] Several labeling methods are usable, including fluorescent
labeling as has been described, silver staining, radiolabelled end
primers, and intercalating dyes. Fluorescent end labeling is
preferred for high throughput analysis with silver staining
preferred if the individual bands are to be removed from the gel
for further processing, such as sequencing.
[0580] Finally, fourth, use of two primers allows direct sequencing
of separated strands by standard techniques. Also separated strands
can be directly cloned into vectors for use in RNA assays such as
in situ analysis. In that case, it is more preferred to use primers
containing T7 or other polymerase signals.
6.5. QEA BY THE PCR EMBODIMENT
[0581] This is an alternative QEA implementation based on PCR
amplification of fragments between target subsequences recognized
by PCR primers or sets of PCR primers. It is designed for the
preferred primers described with reference to FIG. 5. If other
primers are used, such as simple sets of degenerate
oligonucleotides, step 5, the first low stringency PCR cycle, is
omitted.
[0582] First strand cDNA synthesis is carried out according to
Example 6.3. PCR amplification with defined sets of primers is
performed according to the following protocol.
[0583] 1. Rnase treat the 1st strand mix with 1 .mu.l of RNase
Cocktail from Ambion, Inc. (Austin, Tex.) at 37.degree. C. for 30
minutes.
[0584] 2. Phenol/CHCl.sub.3 extract the mixture 2 times, and purify
it on a Centricon 100, Milipore Corporation (Bedford, Mass.) using
water as the filtrate.
[0585] 3. Bring the end volume of the cDNA to 50 .mu.l (starting
with 10 ng RNA/.mu.l).
[0586] 4. Set up the following PCR Reaction:
9 Component Volume cDNA (.about.10 ng/.mu.l) 1 .mu.l 10X PCR Buffer
2.5 .mu.l 25 mM MgCl.sub.2 1.5 .mu.l 10 mM dNTPs 0.5 .mu.l 20
pM/.mu.l primer1 2.5 .mu.l 20 pM/.mu.l primer2 2.5 .mu.l Taq Pol.
(5 U/.mu.l) 0.2 .mu.l water 14.3 .mu.l
[0587] 5. One low stringency cycle with the profile:
[0588] 40.degree. C. for 3 minutes (annealing)
[0589] 72.degree. C. for 1 minute (extension)
[0590] 6. Cycle using the following profile:
[0591] 95.degree. C. for 1 minute
[0592] 15-30 times:
[0593] 95.degree. C. for 30 seconds
[0594] 50.degree. C. for 1 minute
[0595] 72.degree. C. for 1 minute
[0596] 72.degree. C. for 5 minutes
[0597] 7. 4.degree. C. hold.
[0598] 8. Samples are precipitated, resuspended in denaturing
loading buffer, and analyzed.
6.6. EXAMPLE OF SIMULATED ANNEALING
[0599] From the October 1994 GenBank database containing human
coding sequences, 12,000 of the first continuous coding domain
sequences ("CDS") were selected. This selection resulted in a set
of sequences biased towards short sequences, having an average
length of 1000 compared to the average gene length of 1800-2000.
Frequency tables were then created that listed the occurrence
frequency of each nucleotide subsequence of lengths 4, 5, 6, 7, and
8. Test target subsequences were initially selected whose
probability of occurrence was near to 50%. This was feasible for
the 4-mers, as they bind relatively frequently, but as the
occurrence probability decreases with length, for longer sequences,
the occurrence probability was often substantially less than 50%.
These initially selected target subsequences were then optimized,
using the simulated annealing CC experimental design methods, to
pick the best 16 subsequences.
[0600] Tables 5, 6 and 7 present the results for target
subsequences of lengths 4, 5 and 6, respectively. Table 8 presents
the results for optimizing target subsequences of length 4 through
6 together. Simulated annealing generally produced an approximately
20% improvement over target subsequence selection guided only by
the occurrence and independence probability criteria. This level of
optimization is likely to improve with larger and less redundant
databases that represent longer genes. Longer sequences bind too
infrequently in this database to make useful hash codes.
10TABLE 5 AN OPTIMIZED SET OF 4-MER SEQUENCES CGTC GTTA ACTA CTAG
TTTT TGTA AATC GTTG TACC TTGT TTCG GATA CGGT CTCG AACG GGTA
[0601] The target subsequences in Table 5 were chosen from all
possible 256 4-mers. There are 2.41 CDSs per hash code on average.
There was 692 CDSs (out of 12000) which are not complementary to
any of these PNAs.
11TABLE 6 AN OPTIMIZED SET OF 5-MER SEQUENCES AGGCA ACTGT GTCTC
TGTGC CAACT GCCCC ACTAC GTGAC GCACC GTCTG GCCTC CAGGT AGGGG GGAAC
GCTCC GCTCT
[0602] The target subsequences in Table 6 were chosen from the 300
most frequently occurring 5-mers. There are 2.33 CDSs per hash code
on average. There was 829 CDSs (out of 12000) which are not
complementary to any of these PNAs.
12TABLE 7 AN OPTIMIZED SET OF 6-MER SEQUENCES TCCTCA CCAGGC AGCAGC
CTCCTG AGCTGG CTCTGG CCAGGG CAGAGA GCCTGG ACTGGA CACCAT GCTGTG
ACTGTG TCTGTG CCAAGG CCTGGA
[0603] The target subsequences in Table 7 were chosen from the 200
most frequently occurring 6-mers. There was 2.63 CDSs per hash code
on average. There are 1530 CDSs (out of 12000) which are not
complementary to any of these PNAs.
13TABLE 8 AN OPTIMIZED SET OF 4-, 5-, AND 6-MER SEQUENCES CTCG TTCG
GATA TTTT CTAG GGTA ACTGT ACTAC CAACT GTCTG AGGCA GCACC TGTGC GGAAC
AGGGG CTCCTG
[0604] The target subsequences in Table 8 were chosen from sets in
Tables 1-3. There was 2.22 CDSs per hash code on average. There are
715 CDSs (out of 12000) which are not complementary to any of these
PNAs.
[0605] The bias of the selected CDSs toward short sequences, on the
average less than the length of a typical gene, partially explains
the 5-10% of CDSs that were not complementary to any selected
target subsequence. Longer sequences would be expected to have more
hits as they have more variability. Also more target subsequences
can be chosen to improve coverage. The 2.2 to 2.6 CDSs per
individual hash code is partially explained by replication in the
selected database. No attempt was make to insure each CDS is unique
among the other selected CDSs.
6.7. QEA RESULTS
[0606] This subsection present results from QEA experiments
directed primarily to the query and tissue modes.
6.7.1. QUERY MODE QEA RESULTS
[0607] The pattern of gene expression differs from tissue to
tissue, and is modulated both during normal development and during
the progression of many diseases, including cancer. Query mode QEA
experiments were used to investigate differences in gene expression
between normal, hyperplastic, and adenocarcinomic glandular
tissues. We had at our disposal voxels containing all three types
of tissue, preserved in such a way that the adjacent tissue
sections were available for later in situ hybridization. The
following experiments were carried out with normal, hyperplastic,
and adenocarcinomic tissue, respectively, as a particular
gland.
RNA Extraction and cDNA Synthesis
[0608] Isolation of total RNA and poly(A).sup.+ RNA from
homogenized glandular tissue voxels was performed substantially as
described in .sctn. 6.3. cDNA was prepared substantially as
described in .sctn..sctn. 6.3 and 6.4.1.
Quantitative Expression Analysis
[0609] QEA reactions were performed by the preferred RE embodiment
substantially as described in .sctn. 6.4.2. This included the
following steps.
Adapter Annealing
[0610] Pairs of 12-base and 24-base primers were pre-annealed at a
ratio of 2:1 (12 mer:24 mer) at a concentration of 5 picomoles 24
mer per microliter in 1X NEB2 buffer. The oligonucleotide mixture
was heated to 50.degree. C. for 10 minutes, and allowed to cool
slowly to room temperature. For this experiment, 10 picomoles of
JC3 and 5 picomoles of JC24, and 10 picomoles of RC6 and 5
picomoles of RC24 were separately pre-annealed. The sequences of
JC3, JC24, RC6, and RC24 are listed in Table 10 of .sctn. 6.9,
infra.
Restriction-Digestion/Ligation Reaction
[0611] Reactions were prepared in for use in a 8-well thermal
cycler format. Glandular cDNA isolated from 10 separate voxels of
tissue was cut with HindIII and NgoMI, and pre-annealed linkers
were ligated onto the 4 base 5' overhangs that these enzymes
generated. Added per each QEA reaction were:
[0612] 1 Unit of HindIII (New England Biolabs, Beverly Mass.)
[0613] 1 Unit of NgoMI (New England Biolabs, Beverly Mass.)
[0614] 1 .mu.l of pre-annealed JC3/JC24
[0615] 1 .mu.l of pre-annealed RC6/RC24
[0616] 1 .mu.l Ligase/ATP (0.2 .mu.l T4 DNA Ligase [1
Unit/.mu.l]/0.8.mu.l 10 mM ATP--Life Technologies, Gaithersburg
Md.)
[0617] 0.5 .mu.l 50 mM MgCL.sub.2
[0618] 10 nanograms of glandular cDNA
[0619] 1 .mu.l 10x NEB2 Buffer (New England Biolabs, Beverly
Mass.)
[0620] Total volume of 10 .mu.l with H.sub.2O
[0621] The temperature profile of FIG. 16A was performed using a
PTC-100 Thermal Cycler (MJ Research, Watertown Mass.).
Amplification Reaction
[0622] The products of the RE/ligation reaction were then amplified
using RC24 and JC24 primers. The PCR reaction mix included:
[0623] 10 .mu.l 5X E-Mg (300 mM Tris-HCL pH 9.0, 75 mM (NH.sub.4)
.sub.2SO.sub.4)
[0624] 100 picomoles RC24
[0625] 100 picomoles JC24
[0626] 1 .mu.l 10 mM dNTP mix (Life Technologies, Gaithersburg
Md.)
[0627] 2.5 Units 50:1 Taq polymerase (Life Technologies,
Gaithersburg Md.): Pfu polymerase (Stratagene, La Jolla Calif.)
mix
[0628] The total volume was brought to 40 .mu.l per reaction with
H.sub.2O.
[0629] 40 .mu.l preheated PCR reaction mix was added to each
restriction-digestion/ligation reaction.
[0630] The temperature profile of FIG. 16B was performed using a
PTC-100 Thermal Cycler (MJ Research, Watertown Mass.).
QEA Analysis
[0631] The reaction products were separated on a 5% acrylamide
sequencing gel, and detected by silver staining. Lane-to-lane
comparisons were made both by visual inspection of the gel, and by
comparing computer enhanced images obtained from scanning the gel
using standard computer scanner equipment. One particular band of
length X bp was differentially expressed, being prominent in some
samples but absent in others. This band was picked from the gel,
PCR re-amplified, and sequenced.
[0632] QEA analysis was performed substantially as described in
.sctn. 5.4.1 using the CDS database constructed as described in
.sctn. 6.1. Four possible sequences in that database were found to
be possible contributors to a fragment of Y bp (note that Y bp=X-46
bp, where PCR primers add 46 bp to the fragment length), sequences
A, B, C, and D. Analysis of the sequencing of the picked band
confirmed that this DNA fragment was produced by sequence C, which
is presently entered in GenBank. This result confirms the correct
functioning of the integrated experimental and analysis
methods.
[0633] Further, analysis of sequence C predicted that a second
double-digest, using REs BspHI and BstYI, would yield a second,
non-overlapping restriction fragment at Z bp in length (plus the 46
bp of ligated primers). A second QEA reaction was performed using
these glandular cDNAs. The previously described experimental
condition were used, with the exception of substituting BspHI,
BstYI, RA5/RA24 and JC9/JC24 for HindIII, NgoMI, JC3/JC24 and
RC6/RC24 during the RE/ligation reaction and of substituting RA24
and JC24 during amplification reaction. Analysis of the results of
this second QEA experiment on silver-stained acrylamide gels, as
above, revealed the presence of a band of the predicted size, Z+46
bp, that was also differentially expressed in the same tissue
samples as the X bp fragment. This results confirms the correct
functioning of the mock digest prediction methods coupled with
subsequence actual experimental digest.
[0634] Additional hybrid primers were designed to facilitate direct
sequencing of the QEA products and the direct generation of RNA
probes for the in situ hybridization to the original tissue sample.
The M13-21 primer or the M13 reverse primer (in italics) were fused
to the first 23 nucleotides of JC24 and RC24 (in bold),
respectively, to allow direct sequencing of the double-digested QEA
products.
[0635] M13-21J+JA24: 5' GGC GCG CCT GTA AAA CGA CGG CCA GTA CCG ACG
TCG ACT ATC CAT GAA G 3' (SEQ ID NO:56)
[0636] M13revR+RA24: 5' AAA ACT GCA GGA AAC AGC TAT GAC CAG CAC TCT
CCA GCC TCT CAC CGA 3' (SEQ ID NO:57)
[0637] In order to enable direct generation of anti-sense RNA
probes for in situ hybridization, the phage T7 promotor (in
italics) was fused to the first 23 nucleotides of JA24/JC24 and
RA24/RC24 (in bold).
[0638] T7+JA24: 5' ACT TCG AAA TTA ATA CGA CTC ACT ATA GGG ACC GAC
GTC GAC TAT CCA TGA AG 3' (SEQ ID NO:58)
[0639] T7+RA24: 5' ACT TCG AAA TTA ATA CGA CTC ACT ATA GGG AGC ACT
CTC CAG CCT CTC ACC GA 3' (SEQ ID NO:59)
6.7.2. TISSUE MODE QEA RESULTS
Isolation of Human Placental Lactogen using QEA
[0640] Lactogen is one of the most highly expressed genes in the
human placenta and has a known sequence. The sequence of lactogen
was retrieved from GenBank and mock digestion reactions were
performed, substantially as described in .sctn. 5.4.1, with a wide
selection of possible RE pairs. These mock digestions showed that
digesting placental cDNA with the restriction enzymes BssHIII and
XbaI yields a lactogen fragment of 166 bp in length.
RNA Extraction and cDNA Synthesis
[0641] Isolation of total RNA and poly(A).sup.+ RNA from
homogenized human placenta tissue was performed substantially as
described in .sctn. 6.3. cDNA was prepared substantially as
described in .sctn..sctn. 6.3 and 6.4.1.
Quantitative Expression Analysis
[0642] QEA reactions were performed by the preferred RE embodiment
substantially as described in .sctn. 6.4.2. This included the
following steps.
Aadapter Annealing
[0643] Pairs of 12-base and 24-base primers were pre-annealed at a
ratio of 2:1 (12 mer:24 mer) at a concentration of 5 picomoles 24
mer per microliter in 1X NEB2 buffer. The oligonucleotide mixture
was heated to 50.degree. C. for 10 minutes, and allowed to cool
slowly to room temperature. For this experiment, 10 picomoles of
RC8 and 5 picomoles of RC24, and 10 picomoles of JC7 and 5
picomoles of JC24 were separately pre-annealed. The sequences of
RC8, RC24, JC7, and JC24 are set forth in Table 10 of .sctn. 6.9,
infra.
Restriction-Digestion/Ligation Reaction
[0644] Reactions were prepared for use in a 8-well thermal cycler
format. Placental cDNA was cut with BssHII and XbaI, and
pre-annealed adapters ligated onto the 4 base 5' overhangs that
these enzymes generated. Added per reaction were:
[0645] 1 Unit of BssHII (New England Biolabs, Beverly Mass.)
[0646] 1 Unit of XbaI (New England Biolabs, Beverly Mass.)
[0647] 1 .mu.l of pre-annealed RC8/RC24
[0648] 1 .mu.l of pre-annealed JC7/JC24
[0649] 1 .mu.l Ligase/ATP (0.2 .mu.l T4 DNA Ligase [1
Unit/.mu.l]/0.8 .mu.l 10 mM ATP--Life Technologies, Gaithersburg
Md.)
[0650] 0.5 .mu.l 50 mM MgCl.sub.2
[0651] 10 nanograms of placental cDNA
[0652] 1 .mu.l 10x NEB2 Buffer (New England Biolabs, Beverly
Mass.)
[0653] Total volume was brought to 10 .mu.l with H.sub.2O.
[0654] The temperature profile of FIG. 16A was performed using a
PTC-100 Thermal Cycler (MJ Research, Watertown Mass.).
Amplification Reaction
[0655] The products of the RE/ligation reaction were then amplified
using RC24 and JC24 primers (see Table 10, infra). The PCR reaction
mix included:
[0656] 10 .mu.l 5X E-Mg (300 mM Tris-HCl pH 9.0, 75 mM
(NH.sub.4).sub.2SO.sub.4)
[0657] 100 picomoles RC24
[0658] 100 picomoles JC24
[0659] 1 .mu.l 10 mM dNTP mix (Life Technologies, Gaithersburg
Md.)
[0660] 2.5 Units 50:1 Taq polymerase (Life Technologies,
Gaithersburg Md.): Pfu polymerase (Stratagene, La Jolla Calif.)
mix.
[0661] The total volume was brought to to 40 .mu.l per reaction
with H.sub.2O.
[0662] 40 .mu.l preheated PCR reaction mix was added to each
restriction-digestion/ligation reaction.
[0663] The temperature profile of FIG. 16B was performed using a
PTC-100 Thermal Cycler (MJ Research, Watertown Mass.).
QEA Analysis
[0664] The reaction products were separated on a 5% acrylamide
sequencing gel and detected by silver staining. A prominent band of
size 212 bp was seen. This was predicted to correspond to the 166
bp lactogen BssHII-XbaI fragment, with JC24 ligated to the BssHII
site, and RC24 ligated to the XbaI site. To prove that this band
did indeed correspond to lactogen, the 212 bp band was excised from
the gel, re-amplified using JC24 and RC24, and the fragment was
sequenced. Analysis of these sequencing results proved that the
fragment was from lactogen. Moreover, the lactogen sequence ended
at the expected 4 base remnant of the restriction site, immediately
followed by either JC24 (at the BssHII end) or RC24 (at the XbaI
end).
[0665] This result confirmed the experimental design methods of
.sctn. 5.4.2 applied to selection of a QEA experiment to identify
certain sequences of interest, in this case the human placental
lactogen sequence, in a tissue cDNA sample. These design methods
resulted in the selection of an experiment which successfully
identified the gene intended.
[0666] Further QEA experiments were done according to the protocols
of this section on human placental derived cDNA with differing
enzyme combinations. One unit of each enzyme of the enzyme
combinations listed in the first column of Table 9 were used in the
restriction-digestion/lig- ation reaction protocol. Primers and
linbers for each RE were chosen according to Table 10, with one
appropriate "J" series linker and primer and one appropriate "R"
series linker and primer used in each reaction. The reaction
products were separated by electrophoresis on a 5% acrylamide gel
and the bands detected by silver staining. Fragments from certain
bands, listed in the second column of Table 9, were removed from
the gel and sequenced. Sequencing identified the subsequences on
the ends of the fragments and the lengths of he fragments. Each
subsequence was characteristic of one of he REs used, confirming
correct action of the ligation and amplification protocols. These
end subsequences for each fragment are listed in the third column
of Table 9, where a "1" indicates digestion by RE "Enz1" and a "2"
indicated digestion by RE "Enz2". Multiple fragments with the same
length but differing end subsequences are placed in separate rows
in Table 9.
[0667] Mock digest reactions, as described in .sctn. 5.4.1, ere
performed using the CDS database selected according to .sctn. 6.1.
These mock digestion reactions searched this CDS database for
sequences having recognition sites for the REs and such that the
recognition sites are spaced apart in order to produce the
fragments of the determined lengths listed. This search identified
the database accession numbers listed in the fourth column of Table
9. The gene responsible for each accession number was determined
from a GenBank lookup and is listed in the fifth column of Table 9.
Table 9 is further grouped into one row for each such gene.
Multiple accession numbers associated with one gene reflect the
redundancy present in current CDS DNA sequence databases.
[0668] For all fragments recovered from the gel, the sequence for
the fragment corresponded to one of the genes identified by the
mock digestion reaction as causing that fragment. This particular
gene is indicated by displaying the gene name in underscore in the
fifth column of Table 9. That the gene determined by sequencing the
separated fragment matched the prediction of the database search
confirms the efficacy of the experimental protocols and the
computer implemented experimental analysis and ambiguity resolution
methods of .sctn..sctn. 5.4.1 and 5.4.2 for tissue mode QEA. In
fact, the mock digestion reactions provide a simple way of
identifying possible ambiguities in DNA sequence databases.
14TABLE 9 PLACENTA GENE CALLS Data- RE Frag- End base Combinations
ment Sub- Acc. Gene Causing (Enz1 & Enz2) Length seq. Numbers
Fragment BglII & BspE1 97 1,2 D23660, Ribosomal L20868, Protein
L4 X73974 97 1,1 X07767 cAMP-Dependent Protein-Kinase 97 1,2
J03278, PDGF Receptor M21616 97 2,2 M74096 Long Chain Acyl-CoA
Dehydrogenase BamH1 & BspE1 112 1,2 L26914, Nitric Oxide
M93718, Synthase M95296 112 1,2 L22453, Ribosomal M90054, Protein
L3a X73460 BglII & BspE1 115 1,2 M20496, Cathepsin L X05256
BglII & NgoM1 137 1,2 L18967 TRP2 Dopachrome Tautomerase 137
2,2 X55740 5'- Nucleotidase 137 1,2 L10386 Tranglutaminase E3 137
1,2 S69231 Tyrosinease- Related Protein 2 137 1,2 X56998, Ubiquitin
X56999 EcoR1 & Bcl1 139 1,2 U14967 Ribosomal Protein L21 Bcl1
& NgoM1 144 1,2 J02984 Ribosomal Protein S15 144 2,2 L12700
Engrailed-2 144 1,2 U04683, Olfactory X80391 Receptor OR17-40 BamH1
& BspE1 144 1,2 X97234 Ribosomal Protein L11 144 1,2 X14362
C3B/C4B Receptor EcoR1 & 146 1,2 M13932 Ribosomal HindIII
Protein S17 BssHII & Xba1 166 1,2 J00118, Lactogen V00573 Bcl1
& NgoM1 168 1,2 S56985, Ribosomal X63527 Protein L19 BamH1
& BspE1 173 1,1 S59493, Nuclear Factor U10323 NF45 BamH1 &
BspE1 173 1,2 M20882, Pregnancy Sp. M23575, Glycoprotein M31125,
beta 1 M33666, M34420, M37399, M69245, M93061 BglII & NgoM1 192
1,2 D29992, Tissue Factor L27624 Pathway Inhibitor 2 192 1,1 D26350
Inositol Triphosphatase Receptor 192 1,1 L27711, Protein L25876
Phosphatase CIP2/KAP1 BglII & Age1 215 1,2 M11353, Histone H3.3
M11354
6.8. COLONY CALLING
[0669] Colony calling comprises the principal steps of cDNA library
filter construction, PNA hybridization, and detection of
hybridization. Determination of the sequence in a sample is done by
the prior described computer implemented CC experimental analysis
methods. Alternatively, cDNA library filters may be obtained from
commercial sources in certain cases.
cDNA Library Filter Construction
[0670] This protocol comprises three steps: first, robotic picking
of colonies into microtiter plates, second, PCR amplification of
inserts, and third, spotting of amplified cDNA inserts onto
filters.
[0671] 1. Colony picking
[0672] a) Libraries are plated out at a density of 1,000-10,000
colonies per 100 mm Petri dish and are picked using a robot into
384 well microtiter plates containing 50 .mu.l of TB medium with
the appropriate antibiotic. There are several commercially
available robots to do this task. The preferable robot is from the
Washington University Human Genome Sequencing Center (St. Louis,
Mo.).
[0673] b) The picked colonies are grown for 8 hours at 37.degree.
C., and are frozen for archiving.
[0674] 2. PCR amplification--PCR primer pairs designed for insert
amplification are dispensed with a standard 25 .mu.l PCR mix into
96 well microtiter plates. A 96 prong transfer tool picks and
transfers samples to provide amplification templates from the 384
well colony into the 96 well PCR mixes. A standard 25 cycle
amplification protocol generates 100-500 ng of insert DNA.
[0675] 3. Spotting on filers--The PCR products are pooled back into
a 384 well format microtiter plates identical to the colony plates
above. Spotting onto filters is a service performed by Research
Genetics (Huntsville, Ala.).
PNA Hybridization and Detection
[0676] PNAs are commercially available from Perseptive Biosystems
(Bedford, Mass.). The protocol below uses 8 dyes on 16 different
degenerate sets of PNA 8-mers containing as common subsequences the
optimized 6-mer subsequences from Table 7. Thereby, complete
classification and determination of expressed genes in a human
tissue can be done with only 4 hybridizations generating a code of
length 32. Actual conditions for stringency may vary depending on
the PNA set used.
[0677] 1. Hybridization--A pool of 8 PNAs are used, labeled with 8
different fluorochromes made up at a concentration of 0.1 .mu.g/ml
in 10 mM Phosphate buffer, pH 7.0, 1X Denhardt's solution (20 mg/ml
Ficoll 400, polyvinylpyrollidone, and BSA). The arrayed filters are
hybridized for 16 hrs at 25.degree. C., and washed 3 times in the
above buffer without PNAs at a temperature which maximizes
signal/noise.
[0678] 2. Visualization--A fluorescent detection system, such as
used for DNA analysis, can be used to distinguish the dyes, and
thus the PNAs, present at each filter hybridization position. PNA
presence or absence defines a code for each hybridization position
on the filter.
6.9. PREFERRED QEA ADAPTERS AND REs PAIRS
[0679] Table 10 lists preferred primer-linker pairs that may be
used as adapters for the preferred RE embodiment of QEA. The
primers listed cover all possible double-digest RE combinations
involving approximately 56 available RE having a 5' 4 bp overhang.
There are 40 such REs available from New England Biolabs. For each
QEA double digest, one primer and one linker from the "R" series
and one primer and one linker from the "J" series are used
together. This choice satisfies all adapter constraints previously
described. Two pairs from the same series are not compatible during
amplification.
15TABLE 10 SAMPLE ADAPTERS Adapter: Primer (longer strand) Series
Linker (shorter strand) RE RA24 5' AGC ACT CTC CAG CCT CTC ACC GAA
3' (SEQ ID NO: 1) RA1 3' AG TGG CTT TTAA Tsp509I (SEQ ID NO: 2)
Mfe1 EcoRI RA5 3' AG TGG CTT GTAC NcoI (SEQ ID NO: 3) BspHI RA6 3'
AG TGG CTT GGCC XmaI (SEQ ID NO: 4) NgoMI BspEI RA7 3' AG TGG CTT
GCGC BssHII (SEQ ID NO: 5) AscI RA8 3' AG TGG CTT GATC AvrII (SEQ
ID NO: 6) NheI XbaI RA9 3' AG TGG CTT CTAG DpnhI (SEQ ID NO: 7)
BamHI BclI RA10 3' AG TGG CTT CGCG KasI (SEQ ID NO: 8) RA11 3' AG
TGG CTT CCGG EagI (SEQ ID NO: 9) Bsp120I NotI EaeI RA12 3' AG TGG
CTT CATG BsiWI (SEQ ID NO: 10) Acc65I BsrGI RA14 3' AG TGG CTT AGCT
XhoI (SEQ ID NO: 11) SalI RA15 3' AG TGG CTT ACGT ApaLI (SEQ ID NO:
12) RA16 3' AG TGG CTT AATT AflII (SEQ ID NO: 13) RA17 3' AG TGG
CTT AGCA BssSI (SEQ ID NO: 14) RC24 5' AGC ACT CTC CAG CCT CTC ACC
GAC 3' (SEQ ID NO: 15) RC1 3' AG TCG CTG TTAA Tsp509I (SEQ ID NO:
16) EcoRI ApoI RC3 3' AG TCG CTG TCGA HindIII (SEQ ID NO: 17) RC5
3' AG TCG CTG GTAC BspHI (SEQ ID NO: 18) RC6 3' AG TCG CTG GGCC
AgeI (SEQ ID NO: 19) NgoMI BspEI SgrAI BsrFI BsaWI RC7 3' AG TCG
CTG GCGC MluI (SEQ ID NO: 20) BssHII AscI RC8 3' AG TCG CTG GATC
SpeI (SEQ ID NO: 21) NheI XbaI RC9 3' AG TCG CTG CTAG DpnII (SEQ ID
NO: 22) BglII BamHI BclI BstYI RC10 3' AG TCG CTG CGCG KasI (SEQ ID
NO: 23) RC11 3' AG TCG CTG CCGG Bsp120I (SEQ ID NO: 24) NotI RC12
3' AG TCG CTG CATG Acc56I (SEQ ID NO: 25) BsrGT RC14 3' AG TCG CTG
AGCT SalI (SEQ ID NO: 26) RC15 3' AG TCG CTG ACGT Ppu10I (SEQ ID
NO: 27) ApaLI JA24 5' ACC GAC GTC GAC TAT CCA TGA AGA 3' (SEQ ID
NO: 28) JA1 3' GT ACT TCT TTAA Tsp509I (SEQ ID NO: 29) Mfe1 EcoRI
JA5 3' GT ACT TCT GTAC NcoI (SEQ ID NO: 30) BspHI JA6 3' GT ACT TCT
GGCC XmaI (SEQ ID NO: 31) NgoMI BspEI JA7 3' GT ACT TCT GCGC BssHII
(SEQ ID NO: 32) AscI JA8 3' GT ACT TCT GATC AvrII (SEQ ID NO: 33)
NheI XbaI JA9 3' GT ACT TCT CTAG DpnII (SEQ ID NO: 34) BamHI BclI
JA10 3' GT ACT TCT CGCG KasI (SEQ ID NO: 35) JA11 3' GT ACT TCT
CCGG EagI (SEQ ID NO: 36) Bsp120I NotI EaeI JA12 3' GT ACT TCT CATG
BsiWI (SEQ ID NO: 37) Acc65I BsrGI JA14 3' GT ACT TCT AGCT XhoI
(SEQ ID NO: 38) SalI JA15 3' GT ACT TCT ACGT ApaLI (SEQ ID NO: 39)
JA16 3' GT ACT TCT AATT AflII (SEQ ID NO: 40) JA17 3' GT ACT TCT
AGCA BssSI (SEQ ID NO: 41) JC24 5' ACC GAC GTC GAC TAT CCA TGA AGC
3' (SEQ ID NO: 42) JC1 3' GT ACT TCG TTAA Tsp509I (SEQ ID NO: 43)
EcoRI ApoI JC3 3' GT ACT TCG TCGA HindIII (SEQ ID NO: 44) JC5 3' GT
ACT TCG GTAC BspHI (SEQ ID NO: 45) JC6 3' GT ACT TCG GGCC AgeI (SEQ
ID NO: 46) NgoMI BspEI SgrAI BsrFI BsaWI JC7 3' GT ACT TCG GCGC
MluI (SEQ ID NO: 47) BssHII AscI JC8 3' GT ACT TCG GTAC SpeI (SEQ
ID NO: 48) NheI XbaI JC9 3' GT ACT TCG CTAG DpnII (SEQ ID NO: 49)
BglII BamHI BclI BstYI JC10 3' GT ACT TCG CGCG KasI (SEQ ID NO: 50)
JC11 3' GT ACT TCG CCGG Bsp120I (SEQ ID NO: 51) NotI JC12 3' GT ACT
TCG CATG Acc56I (SEQ ID NO: 52) BsrGI JC14 3' GT ACT TCG AGCT SalI
(SEQ ID NO: 53) JC15 3' GT ACT TCG ACGT Ppu10I (SEQ ID NO: 54)
ApaLI
[0680] Tables 11 and 12 list the RE combinations that have been
tested in QEA experiments on human placental and glandular cDNAs
samples. The preferred double digests are those that give more than
approximately 50 bands in the range of 100 to 700 bp. Table 11
lists the preferred RE combinations for human cDNA analyses.
16TABLE 11 PREFERRED RE COMBINATIONS FOR HUMAN cDNA ANALYSIS Acc56I
& HindIII Acc65I & NgoMI BamHI & EcoRI BglII &
HindIII BglII & NgoMI BsiWI & BspHI BspHI & BstYI BspHI
& NgoMI BsrGI & EcoRI EagI & EcoRI EagI & HindIII
EagI & NcoI HindIII & NgoMI NgoMI & NheI NgoMI &
SpeI BglII & BspHI Bsp120I & NcoI BssHII & NgoMI EcoRI
& HindIII NgoMI & XbaI
[0681] Table 12 lists other RE combinations tested and that can be
used for human cDNA analyses.
17TABLE 12 OTHER RE COMBINATIONS FOR HUMAN cDNA ANALYSIS AvrII
& NgoMI BamHI & Bsp120I BamHI & BspHI BamHI & NcoI
BclI & BspHI BclI & NcoI BglII & BspEI BglII &
EcoRI BglII & NcoI BssHII & BsrGI BstYI & NcoI BamHI
& HindIII BglII & Bsp120I BspHI & HindIII
[0682] Tables 13 and 14 list the RE combinations that have been
tested in QEA experiments on mouse cDNA samples. The preferred
double digests are those that give more than approximately 50 bands
in the range of 100 to 700 bp. Table 13 lists the preferred RE
combinations for mouse cDNA analyses.
18TABLE 13 PREFERRED RE COMBINATIONS FOR MOUSE cDNA ANALYSIS Acc56I
& NindIII Acc65I & NgoMI AscI & HindIII AvrII &
NgoMI BamHI & BspHI BamHI & HindIII BamHI & NcoI BclI
& NcoI BglII & BspHI BglII & HindIII BglII & NcoI
BglII & NgoMI Bsp120I & NcoI Acc65I & BspHI BspHI &
Bsp120I BspHI & BsrGI BspHI & EagI BspHI & NgoMI BspHI
& NotI BssHII & HindIII BstYI & HindIII HindIII &
NcoI HindIII & NgoMI NcoI & NotI NgoMI & NheI NgoMI
& SpeI NgoMI & XbaI BclI & HindIII
[0683] Table 14 lists other RE combinations tested and that can be
used for mouse cDNA analyses.
19TABLE 14 OTHER RE COMBINATIONS FOR MOUSE cDNA ANALYSIS Acc65I
& NcoI BclI & BspHI BsiWI & BspHI BsiWI & NcoI
BspHI & HindIII BsrGI & NcoI BssHII & NgoMI BstYI &
BspHII EagI & NcoI HindIII & MluI
[0684] Table 15 lists the data obtained from various RE
combinations using mouse cDNA samples. The number of bands was
observed from silver stained acrylamide separation gels.
20TABLE 15 MOUSE cDNA RE DIGESTION RESULTS Number of RE Combination
Bands AccG5I & HindIII 200 AccG5I & NgoMI 150 AscI &
HindIII 100 AvrII & NgoMI 50 BamHI & BspHI 200 BamHI &
HindIII 150 BamHI & NcoI 150 BclI & BspHI 5 BclI &
HindIII 150 BclI & NcoI 50 BglII & BspHI 50 BglII &
HindIII 150 BglII & NcoI 50 BglII & NgoMI 50 Bsp120I &
NcoI 50 BspHI & Acc65I 150 BspHI & Bsp120I 50 BspHI &
BsrGI 200 BspHI & EagI 150 BspHI & HindIII 0 BspHI &
NgoMI 150 BspHI & NotI 150 BsrGI & NcoI 10 BssHII &
HindIII 100 BssHII & NgoMI 20 BstYI & BspHI 20 BstYI &
HindIII 200 EagI & NcoI 10 HindIII & MluI 25 HindIII &
NcoI 50 HindIII & NgoMI 150 NcoI & NotI 200 NgoMI &
NheI 50 NgoMI & SpeI 200 NgoMI & XbaI 50 T0TAL # BANDS
3490
[0685] 31 available REs that recognize a 6 bp recognition sequence
and generate a 4 bp 5' overhang are: Acc65I, AflII, AgeI, ApaLI,
Apol, AscI, AvrI, BamHI, BclI, BglII, BsiWI, Bsp120I, BspEI, BspHI,
BsrGI, BssHII, BstYI, EagI, EcoRI, HindIII, MfeI, MluI, NcoI,
NgoMI, NheI, NotI, Ppu101, SalI, SpeI, XbaI, and XhoI.
[0686] All of these enzymes have been tested in QEA protocols with
the specified buffer conditions with the exception of AflII. All
were useable except for MfeI, Ppu101, SalI, and XhoI. All the other
26 enzymes have been tested and are usable in the RE implementation
of QEA.
[0687] However certain pairs of these enzymes are less informative
due to the fact that they produce identical overhangs, and thus
their recognition sequences cannot be distinguished by QEA
adapters. These pairs are Acc65I and (BsiWI or BsrGI); AgeI and
(BspEI or NcoMI); ApoI and EcoRI; AscI and (BssHII or MluI); AvrI
and (NheI, SpeI, or XbaI); BamHI and (BclI, BglII, or BstYI); BclI
and (BgLII or BstYI); BglII and BstYI; BsiWI and BsrGI; Bsp120I and
EagI; BspEI and NcoMI; BspHI and NcoI; BssHII and MluI; NheI and
(SpeI or XbaI); and SpeI and XbaI.
[0688] Thus 301 RE pairs have been tested and are useable in the RE
embodiments of QEA.
6.10. FLUORESCENT LABELS
[0689] Fluorochromes labels that can be used in the methods of the
present invention include the classic fluorochromes as well as more
specialized fluorochromes. The classic fluorochromes include
bimane, ethidium, europium (III) citrate, fluorescein, La Jolla
blue, methylcoumarin, nitrobenzofuran, pyrene butyrate, rhodamine,
terbium chelate, and tetramethylrhodamine. More specialized
fluorochromes are listed in Table 16 along with their
suppliers.
21TABLE 16 FLORESCENT LABELS Absorption Emission Fluorochrome
Vendor Maximum Maximum Bodipy Molecular Probes 493 503 493/503 Cy2
BDS 489 505 Bodipy FL Molecular Probes 508 516 FTC Molecular Probes
494 518 FluorX BDS 494 520 FAM Perkin-Elmer 495 535 Carboxy-
Molecular Probes 519 543 rhodamine EITC Molecular Probes 522 543
Bodipy Molecular Probes 530 550 530/550 JOE Perkin-Elmer 525 557
HEX Perkin-Elmer 529 560 Bodipy Molecular Probes 542 563 542/563
Cy3 BDS 552 565 TRITC Molecular Probes 547 572 LRB Molecular Probes
556 576 Bodipy LMR Molecular Probes 545 577 Tamra Perkin-Elmer 552
580 Bodipy Molecular Probes 576 589 576/589 Bodipy Molecular Probes
581 591 581/591 Cy3.5 BDS 581 596 XRITC Molecular Probes 570 596
ROX Perkin-Elmer 550 610 Texas Red Molecular Probes 589 615 Bodipy
TR Molecular Probes 596 625 (618?) Cy5 BDS 650 667 Cy5.5 BDS 678
703 DdCy5 Beckman 680 710 Cy7 BDS 443 767 DbCy7 Beckman 790 820
[0690] The suppliers listed in Table 16 are Molecular Probes
(Eugene, Oreg.), Biological Detection Systems ("BDS") (Pittsburgh,
Pa.) and Perkin-Elmer (Norwalk, Conn.).
[0691] Means of utilizing these fluorochromes by attaching them to
particular nucleotide groups are described in Kricka et al., 1995,
Molecular Probing, Blotting, and Sequencing, chap. 1, Academic
Press, New York. Preferred methods of attachment are by an amino
linker or phosophoramidite chemistry.
7. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES
[0692] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0693] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *