U.S. patent application number 08/892503 was filed with the patent office on 2002-04-11 for methods and compositions for detection or quantification of nucleic acid species.
Invention is credited to DRMANAC, RADOJE.
Application Number | 20020042048 08/892503 |
Document ID | / |
Family ID | 27419818 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020042048 |
Kind Code |
A1 |
DRMANAC, RADOJE |
April 11, 2002 |
METHODS AND COMPOSITIONS FOR DETECTION OR QUANTIFICATION OF NUCLEIC
ACID SPECIES
Abstract
The present invention provides a method for detecting a target
nucleic acid species including the steps of providing an array of
probes affixed to a substrate and a plurality of labeled probes
wherein each labeled probe is selected to have a first nucleic acid
sequence which is complementary to a first portion of a target
nucleic acid and wherein the nucleic acid sequence of at least one
probe affixed to the substrate is complementary to a second portion
of the nucleic acid sequence of the target, the second portion
being adjacent to the first portion; applying a target nucleic acid
to the array under suitable conditions for hybridization of probe
sequences to complementary sequences; introducing a labeled probe
to the array; hybridizing a probe affixed to the substrate to the
target nucleic acid; hybridizing the labeled probe to the target
nucleic acid; affixing the labeled probe to an adjacently
hybridized probe in the array; and detecting the labeled probe
affixed to the probe in the array.
Inventors: |
DRMANAC, RADOJE; (PALO ALTO,
CA) |
Correspondence
Address: |
SAMUEL B ABRAMS
PENNIE & EDMONDS, LLP
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
|
Family ID: |
27419818 |
Appl. No.: |
08/892503 |
Filed: |
July 14, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08892503 |
Jul 14, 1997 |
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08812951 |
Mar 4, 1997 |
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6297006 |
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08892503 |
Jul 14, 1997 |
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08784747 |
Jan 16, 1997 |
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Current U.S.
Class: |
435/6.12 ;
435/183; 435/91.1; 435/91.2; 436/94; 530/350; 536/23.1 |
Current CPC
Class: |
C12Q 1/6837 20130101;
C12Q 1/6869 20130101; Y10T 436/143333 20150115; C12Q 1/6874
20130101; C12Q 1/6874 20130101; C12Q 2561/125 20130101; C12Q
2535/131 20130101; C12Q 1/6874 20130101; C12Q 2537/113 20130101;
C12Q 2537/107 20130101; C12Q 2535/131 20130101; C12Q 1/6837
20130101; C12Q 2537/143 20130101; C12Q 2533/107 20130101; C12Q
1/6874 20130101; C12Q 2537/143 20130101; C12Q 1/6869 20130101; C12Q
2537/143 20130101; C12Q 2533/107 20130101; C12Q 2525/197
20130101 |
Class at
Publication: |
435/6 ; 435/91.1;
435/91.2; 435/183; 436/94; 536/23.1; 530/350 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; G01N 033/00; C12N 009/00; C07K 001/00; C07K
014/00; C12P 019/34; C07K 017/00 |
Claims
What is claimed is:
1. A method for confirming sequencing results, comprising the steps
of: obtaining a sequence from a nucleic acid using SBH; identifying
a set of probes that are complementary and not exactly
complementary to the sequence of the nucleic acid; hybridizing the
probes to the nucleic acid under conditions that allow the
differentiation of perfect matches from one base mismatches;
confirming that the probes do not form perfect matches with the
nucleic acid.
2. The method of claim 1, wherein the SBH is format I SBH.
3. The method of claim 1, wherein the SBH is format III SBH.
4. The method of claim 1, wherein the set of probes are not exactly
complementary to the sequence of the nucleic acid.
5. A method for confirming sequencing results, comprising the steps
of: obtaining a sequence from a nucleic acid using SBH; selecting
at least one primer for the nucleic acid; sequencing the nucleic
acid with the primer using Sanger-sequencing; comparing the
sequence of the nucleic acid derived from SBH to the sequence for
the nucleic acid derived from Sanger sequencing.
6. A method for ordering a plurality of Sfs from a nucleic acid
sequence, comprising the steps of: obtaining the sequence from the
nucleic acid using SBH; identifying a plurality of primers from the
sequence of the plurality of Sfs, whereby the primers can initiate
a replication reaction on the nucleic acid that will read through a
branch point; sequencing the nucleic acid with the primers using
Sanger-sequencing; comparing the sequence derived by
Sanger-sequencing of the nucleic acid around the branch point to
the sequences of the Sfs, whereby the order of the Sfs is
determined.
7. A plurality of probes for analyzing a nucleic acid wherein the
plurality of probes are used to interrogate the nucleic acid under
conditions whereby the plurality of probes can be differentiated
from each other.
8. The probes of claim 7, wherein the nucleic acid has a known
sequence and probes are labeled with a label.
9. The plurality of probes of claim 7, wherein the plurality of
probes are labeled with a plurality of different labels, whereby
the probes can be differentiated from each other by the different
labels attached to the probes.
10. A set of probes for analyzing a nucleic acid, comprising a
plurality of pools of probes wherein each pool is used to
interrogate a nucleic acid, and wherein the plurality of probes are
labeled with a plurality of different labels, whereby the probes in
each pool can be differentiated from each other by the different
labels attached to the probes.
11. The set of probes in claim 9, wherein the plurality of
different labels are a plurality of different radioisotopes.
12. The set of probes of claim 9, wherein the plurality of
different labels are a plurality of different flourescent
molecules.
13. The set of probes of claim 9, wherein the plurality of
different labels are a plurality of different EMLs.
14. The set of probes in claim 10, wherein the plurality of
different labels are a plurality of different radioisotopes.
15. The set of probes of claim 10, wherein the plurality of
different labels are a plurality of different flourescent
molecules.
16. The set of probes of claim 10, wherein the plurality of
different labels are a plurality of different EMLs.
17. A method for analyzing a nucleic acid, comprising the steps of:
providing an array of oligonucleotide probes; introducing a sample
nucleic acid to the array; adding a plurality of labeled probes to
the array under conditions that allow the differentiation of
perfect matches from one base mismatches,; adding ligase to the
array; incubating the ligase, labeled probes, sample nucleic acid
and array probes under conditions whereby labeled probe is ligated
to array probes when the labeled probe is adjacent to the array
probe on the sample nucleic acid; and detecting the labeled probes
that have been ligated to the array.
18. The method of claim 17, further comprising the step of removing
unligated labeled probe after the incubation step.
19. The method of claim 18, wherein the nucleic acid has a known
sequence and plurality of probes are labeled with a label.
20. The method of claim 19, wherein the label is selected from the
group consisting of a radioisotope, a flourescent molecule, and an
EML.
21. The method of claim 18, wherein the plurality of probes are
labeled with a plurality of different labels, whereby the probes
can be differentiated from each other by the different labels
attached to the probes.
22. The method of claim 21, wherein the plurality of different
labels are a plurality of different radioisotopes.
23. The method of claim 21, wherein the plurality of different
labels are a plurality of different flourescent molecules.
24. The method of claim 21, wherein the plurality of different
labels are a plurality of different EMLs.
25. A method for analyzing a plurality of nucleic acids, comprising
the steps of: obtaining a sample comprising the plurality of
nucleic acids, wherein a target nucleic acid is present at least in
a ratio of one part to ninety nine parts of a nucleic acid that is
homologous to the target and differs by at least one nucleotide
from the target; selecting a set of probes that will identify the
target nucleic acid; mixing the sample and the probes under
conditions that allow the differentiation of perfect matches from
one base mismatches; identifying whether the probes form a perfect
match with a nucleic acid in the sample.
26. An appartus for analyzing a nucleic acid, comprising: a first
array of nucleic acids; a second array of nucleic acids; a material
disposed between the first and second arrays that prevents the
mixing of the nucleic acids in the first array with the nucleic
acids in the second array.
27. The apparatus of claim 26, wherein the nucleic acids in the
second array are labeled oligonucleotide probes.
28. The apparatus of claim 27, wherein the nucleic acids in the
first array are a plurality of sample nucleic acids.
29. A method for analyzing a target nucleic acid, comprising the
steps of: providing an array of bound probes of known sequence
fixed to a substrate; providing an array of labeled probes of known
sequence; providing a material disposed between the arrays of bound
and labeled probes that prevents the mixing of the probes in the
bound and labeled probe arrays; adding the target nucleic acid to
the labeled probes; removing the material between the bound and
labeled probes so that the labeled probes bound probes and target
nucleic acids are mixed together under conditions that allow the
differentiation of perfect matches from one base mismatches;
joining the bound and labeled probes that are hybridized to
adjacent sites in the target nucleic acid; detecting the labeled
probe that has been joined to the bound probe array.
30. A method for analyzing a target nucleic acid, comprising the
steps of: providing an array of bound probes of known sequence
fixed to a substrate; providing an array of labeled probes of known
sequence; providing a material disposed between the arrays of bound
and labeled probes that prevents the mixing of the probes in the
bound and labeled probe arrays; removing the material between the
bound and labeled probes so that the labeled probes and bound
probes are mixed together; adding the target nucleic acid to the
labeled and bound probes under conditions that allow the
differentiation of perfect matches from one base mismatches;
ligating the bound and labeled probes that are hybridized to
adjacent sites in the target nucleic acid; detecting the labeled
probe that has been ligated to the bound probe array.
31. A method for analyzing a target nucleic acid, comprising the
steps of: providing an array of bound probes of known sequence
fixed to a substrate, wherein some of the bound probes are
complementary to a plurality of first portions of the target
nucleic acid; providing an array of labeled probes of known
sequence, wherein some of the labeled probes are complementary to a
plurality of second portions of the target nucleic acid and wherein
specific second portions are adjacent to specific first portions;
providing a material disposed between the arrays of bound and
labeled probes that prevents the mixing of the probes in the bound
and labeled probe arrays; adding the target nucleic acid to the
labeled probes; removing the material between the bound and labeled
probes so that the labeled probes bound probes and target nucleic
acids are mixed together under conditions that allow the
differentiation of perfect matches from one base mismatches;
joining the bound and labeled probes that are bound at the specific
first and second portions in the target nucleic acid; detecting the
labeled probe joined to the bound probe array.
32. A method for analyzing a target nucleic acid, comprising the
steps of: providing an array of bound probes of known sequence
fixed to a substrate, wherein some of the bound probes are
complementary to a plurality of first portions of the target
nucleic acid; providing an array of labeled probes of known
sequence, wherein some of the labeled probes are complementary to a
plurality of second portions of the target nucleic acid and wherein
specific second portions are adjacent to specific first portions;
providing a material disposed between the arrays of bound and
labeled probes that prevents the mixing of the probes in the bound
and labeled probe arrays; removing the material between the bound
and labeled probes so that the labeled probes and bound probes are
mixed together; adding the target nucleic acid to the labeled and
bound probes under conditions that allow the differentiation of
perfect matches from one base mismatches; joining the bound and
labeled probes that are bound at the specific first and second
portions in the target nucleic acid; detecting the labeled probe
joined to the bound probe array.
33. A method for analyzing a target nucleic acid, comprising the
steps of: providing an array of bound target nucleic acids;
providing an array of labeled probes of known sequence; providing a
material disposed between the arrays of bound target and labeled
probes that prevents the mixing of the target nucleic acid and
labeled probes; removing the material between the bound target and
labeled probes so that the labeled probes and bound target nucleic
acids are mixed together under conditions that allow the
differentiation of perfect matches from one base mismatches;
determining which labeled probes have formed perfect matches with
the target DNA.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation-in-part of U.S.
patent application Ser. No. 08/812,951, filed on Mar. 4, 1997, and
a continuation-in-part of U.S. patent application Ser. No.
08/784,747, filed on Jan. 16, 1997.
FIELD OF THE INVENTION
[0002] This invention relates in general to methods and apparatus
for nucleic acid analysis, and, in particular, to methods and
apparati for nucleic acid analysis.
BACKGROUND
[0003] The rate of determining the sequence of the four nucleotides
in nucleic acid samples is a major technical obstacle for further
advancement of molecular biology, medicine, and biotechnology.
Nucleic acid sequencing methods which involve separation of nucleic
acid molecules in a gel have been in use since 1978. The other
proven method for sequencing nucleic acids is sequencing by
hybridization (SBH).
[0004] The traditional method of determining a sequence of
nucleotides (i.e., the order of the A, G, C and T nucleotides in a
sample) is performed by preparing a mixture of randomly-terminated,
differentially labelled nucleic acid fragments by degradation at
specific nucleotides, or by dideoxy chain termination of
replicating strands. Resulting nucleic acid fragments in the range
of 1 to 500 bp are then separated on a gel to produce a ladder of
bands wherein the adjacent samples differ in length by one
nucleotide.
[0005] The array-based approach of SBH does not require single base
resolution in separation, degradation, synthesis or imaging of a
nucleic acid molecule. Using mismatch discriminative hybridization
of short oligonucleotides K bases in length, lists of constituent
K-mer oligonucleotides may be determined for target nucleic acid.
Sequence for the target nucleic acid may be assembled by uniquely
overlapping scored oligonucleotides.
[0006] There are several approaches available to achieve sequencing
by hybridization. In a process called SBH Format 1, nucleic acid
samples are arrayed, and labeled probes are hybridized with the
samples. Replica membranes with the same sets of sample nucleic
acids may be used for parallel scoring of several probes and/or
probes may be multiplexed. Nucleic acid samples may be arrayed and
hybridized on nylon membranes or other suitable supports. Each
membrane array may be reused many times. Format 1 is especially
efficient for batch processing large numbers of samples.
[0007] In SBH Format 2, probes are arrayed at locations on a
subsrate which correspond to their respective sequences, and a
labelled nucleic acid sample fragment is hybridized to the arrayed
probes. In this case, sequence information about a fragment may be
determined in a simultaneous hybridization reaction with all of the
arrayed probes. For sequencing other nucleic acid fragments, the
same oligonucleotide array may be reused. The arrays may be
produced by spotting or by in situ synthesis of probes.
[0008] In Format 3 SBH, two sets of probes are used. In one
embodiment, a set may be in the form of arrays of probes with known
positions, and another, labelled set may be stored in multiwell
plates. In this case, target nucleic acid need not be labelled.
Target nucleic acid and one or more labelled probes are added to
the arrayed sets of probes. If one attached probe and one labelled
probe both hybridize contiguously on the target nucleic acid, they
are covalently ligated, producing a detected sequence equal to the
sum of the length of the ligated probes. The process allows for
sequencing long nucleic acid fragments, e.g. a complete bacterial
genome, without nucleic acid subcloning in smaller pieces.
[0009] In the present invention, SBH is applied to the efficient
identification and sequencing of one or more nucleic acid samples.
The procedure has many applications in nucleic acid diagnostics,
forensics, and gene mapping. It also may be used to identify
mutations responsible for genetic disorders and other traits, to
assess biodiversity and to produce many other types of data
dependent on nucleic acid sequence.
SUMMARY OF THE INVENTION
[0010] The present invention provides a method for detecting a
target nucleic acid species including the steps of providing an
array of probes affixed to a substrate and a plurality of labeled
probes wherein each labeled probe is selected to have a first
nucleic acid sequence which is complementary to a first portion of
a target nucleic acid and wherein the nucleic acid sequence of at
least one probe affixed to the substrate is complementary to a
second portion of the nucleic acid sequence of the target, the
second portion being adjacent to the first portion; applying a
target nucleic acid to the array under suitable conditions for
hybridization of probe sequences to complementary sequences;
introducing a labeled probe to the array; hybridizing a probe
affixed to the substrate to the target nucleic acid; hybridizing
the labeled probe to the target nucleic acid;affixing the labeled
probe to an adjacently hybridized probe in the array; and detecting
the labeled probe affixed to the probe in the array. According to
preferred methods of the invention the array of probes affixed to
the substrate comprises a universal set of probes. According to
other preferred aspects of the invention at least two of the probes
affixed to the substrate define overlapping sequences of the target
nucleic acid sequence and more preferrably at least two of the
labelled probes define overlapping sequences of the target nucleic
acid sequences. Still further, according to another aspect of the
invention a method is provided for detecting a target nucleic acid
of known sequence comprising the steps of: contacting a nucleic
acid sample with a set of immobilized oligonucleotide probes
attached to a solid substrate under hybridizing conditions wherein
the immobilized probes are capable of specific hybridization with
different portions of said target nucleic acid sequence; contacting
the target nucleic acid with a set of labelled oligonucleotide
probes in solution under hybridizing conditions wherein the labeled
probes are capable of specific hybridization with different
portions of said target nucleic acid sequence adjacent to the
immobilized probes; covalently joining the immobilized probes to
labelled probes that are immediately adjacent to the immobilized
probe on the target sequence (e.g., with ligase); removing any
non-ligated labelled probes; detecting the presence of the target
nucleic acid by detecting the presence of said labelled probe
attached to the immobilized probes. The invention also provides a
method of determining expression of a member of a set of partially
or completely sequenced genes in a cell type, a tissue or a tissue
mixture comprising the steps of: defining pairs of fixed and
labeled probes specific for the sequenced gene; hybridizing
unlabeled nucleic acid sample and corresponding labeled probes to
one or more arrays of fixed probes; forming covalent bonds between
adjacent hybridized labeled and fixed probes; removing unligated
probes; and determining the presence of the sequenced gene by
detection of labeled probes bound to prespecified locations in the
array. In a preferred embodiment of this aspect of the invention,
the target nucleic acid will identify the presence of an infectious
agent.
[0011] Further, the present invention provides for an array of
oligonucleotide probes comprising a nylon membrane; a plurality of
subarrays of oligonucleotide probes on the nylon membrane, the
subarrays comprising a plurality of individual spots wherein each
spot is comprised of a plurality of oligonucleotide probes of the
same sequence; and a plurality of hydrophobic barriers located
between the subarrays on the nylon membrane, whereby the plurality
of hyydrophobic barriers prevents cross contamination between
adjacent subarrays.
[0012] Still further, the present invention provides a method for
sequencing a repetitive sequence, having a first end and a second
end, in a target nucleic acid comprising the steps of: (a)
providing a plurality of spacer oligonucleotides of varying lengths
wherein the spacer oligonucleotides comprise the repetitive
sequence; (b) providing a first oligonucleotide that is known to be
adjacent to the first end of the repetitive sequence; (c) providing
a plurality of second oligonucleotides one of which is adjacent to
the second end of the repetitive sequence, wherein the plurality of
second oligonucleotides is labeled; (d) hybridizing the first and
the plurality of second oligonucleotides, and one of the plurality
of spacer oligonucleotides to the target nucleic acid ; (e)
ligating the hybridized oligonucleotides; (f) separating ligated
oligonucleotides from unligated oligonucleotides; and (g) detecting
label in the ligated oligonucleotides.
[0013] Still further, the present invention provides a method for
sequencing a branch point sequence, having a first end and a second
end, in a target nucleic acid comprising the steps of: (a)
providing a first oligonucleotide that is complementary to a first
portion of the branch point sequence wherein the first
oligonucleotide extends from the first end of the branch point
sequence by at least one nucleotide; (b) providing a plurality of
second oligonucleotides that are labeled, and are complementary to
a second portion of the branch point sequence wherein the plurality
of second oligonucleotides extend from the second end of the branch
point sequence by at least one nucleotide, and wherein the portion
of the second oligonucleotides that extend from the second end of
the branch point sequence comprise sequences that are complementary
to a plurality of sequences that arise from the branch point
sequence; (c) hybridizing the first oligonucleotide, and one of the
plurality of second oligonucleotides to the target DNA; (d)
ligating the hybridized oligonucleotides; (e) separating ligated
oligonucleotides from unligated oligonucleotides; and (f) detecting
label in the ligated oligonucleotides.
[0014] Still further, the present invention provides a method for
confirming a sequence by using probes that are predicted to be
negative for the target nucleic acid. The sequence of a target is
then confirmed by hybridizing the target nucleic acid to the
"negative" probes to confirm that these probes do not form perfect
matches with the target nucleic acid.
[0015] Still further, the present invention provides a method for
analyzing a nucleic acid using oligonucleotide probes that are
complexed with different labels so that the probes may be
multiplexed in a hybridization reaction without a loss of sequence
information (i.e., different probes have different labels so that
hybridization of the different probes to the target can be
distinguished). In a preferred embodiment, the labels are
radioisotopes, or floursecent molecules, or enzymes, or
electrophore mass labels. In a more preferred embodiment, the
differently labeled oligonucleotides probes are used in format III
SBH, and multiple probes (more than two, with one probe being the
immobilized probe) are ligated together.
[0016] Still further, the present invention provides a method for
detecting the presence of a target nucleic acid having a known
sequence when the target is present in very small amounts compared
to homologous nucleic acids in a sample. In a preferred embodiment,
the target nucleic acid is an allele present at very low frequency
in a sample that has nucleic acids from a large number of sources.
In an alternative preferred embodiment, the target nucleic acid has
a mutated sequence, and is present at very low frequency within a
sample of nucleic acids.
[0017] Still further, the present invention provides a method for
confirming the sequence of a target nucleic acid by using single
pass gel sequencing. Primers for single pass gel sequencing are
derived from the seqeunce obtained by SBH, and these primers are
used in standard Sanger sequencing reactions to provide gel
sequence information for the target nucleic acid. The sequence
obtained by single pass gel sequencing is then compared to the SBH
derived sequence to confirm the sequence.
[0018] Still further, the present invention provides a method for
solving branch points by using single pass gel sequencing. Primers
for the single pass gel sequencing reactions are identified from
the ends of the Sfs obtained after a first round of SBH sequencing,
and these primers are used in standard Sanger-sequencing reactions
to provide gel sequencing information through the branch points of
the Sfs. Sfs are then aligned by comparing the Sanger-sequencing
results through the branch points to the Sfs to identify adjoining
Sfs.
[0019] Still further, the present invention provides for a method
of preparing a sample containing target nucleic acids by PCR,
without purifying the PCR products prior to the SBH reactions. In
Format I SBH, crude PCR products are applied to a substrate without
prior purification, and the substrate may be washed prior to
introduction of the labeled probes.
[0020] Still further, the present invention provides a method and
an apparatus for analyzing a target nucleic acid. The apparatus
comprises two arrays of nucleic acids that are mixed together at
the desired time. In a preferred embodiment, the nucleic acids in
one of the arrays are labeled. In a more preferred embodiment, a
material is disposed between the two arrays and this material
prevents the mixing of nucleic acids in the arrays. When this
material is removed, or rendered permeable, the nucleic acids in
the two arrays are mixed together. In an alternative preferred
embodiment, the nucleic acids in one array are target nucleic acids
and the nucleic acids in the other are oligonucleotide probes. In
another preferred embodiment, the nucleic acids in both arrays are
oligonucleotide probes. In another preferred embodiment, the
nucleic acids in one array are oligonucleotide probes and target
nucleic acids, and nucleic acids in the other array are
oligonucleotide probes. In another preferred embodiment, the
nucleic acids in both arrays are oligonucleotide probes and target
nucleic acids.
[0021] One method of the present invention using the apparatus
described above comprises the steps of providing an array of
nucleic acids fixed to a substrate, providing a second array of
nucleic acids, providing conditions that allow the nucleic acids in
the second array to come into contact with the nucleic acids of the
fixed array wherein one of the arrays of nucleic acids are target
nucleic acids and the other array is oligonucleotide probes, and
analyzing the hybridization results. In a preferred embodiment, the
fixed array is target nucleic acid and the second array is lableled
oligonucleotide probes. In a more preferred embodiment, there is a
material disposed between the two arrays that prevents mixing of
the nucleic acids until the material is removed or rendered
permeable to the nucleic acids.
[0022] In a second method of the present invention using the
apparatus described above comprises the steps of providing two
arrays of nucleic acid probes, providing conditions that allow the
two arrays of probes to come into contact with each other and a
target nucleic acid, ligating together probes that are adjacent on
the target nucleic acid, and analyzing the results. In a preferred
embodiment, the probes in one array are fixed and the probes in the
other array are labeled. In a more preferred embodiment, there is a
material disposed between the two arrays that prevents mixing of
the probes until the material is removed or rendered permeable to
the probes.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] Format 1 SBH is appropriate for the simultaneous analysis of
a large set of samples. Parallel scoring of thousands of samples on
large arrays may be performed in thousands of independent
hybridization reactions using small pieces of membranes. The
identification of DNA may involve 1-20 probes per reaction and the
identification of mutations may in some cases involve more than
1000 probes specifically selected or designed for each sample. For
identification of the nature of the mutated DNA segments, specific
probes may be synthesized or selected for each mutation detected in
the first round of hybridizations.
[0024] DNA samples may be prepared in small arrays which may be
separated by appropriate spacers, and which may be simultaneously
tested with probes selected from a set of oligonucleotides which
may be arrayed in multiwell plates. Small arrays may consist of one
or more samples. DNA samples in each small array may include
mutants or individual samples of a sequence. Consecutive small
arrays may be organized into larger arrays. Such larger arrays may
include replication of the same small array or may include arrays
of samples of different DNA fragments. A universal set of probes
includes sufficient probes to analyze a DNA fragment with
prespecified precision, e.g. with respect to the redundancy of
reading each base pair ("bp"). These sets may include more probes
than are necessary for one specific fragment, but may include fewer
probes than are necessary for testing thousands of DNA samples of
different sequence.
[0025] DNA or allele identification and a diagnostic sequencing
process may include the steps of:
[0026] 1) Selection of a subset of probes from a dedicated,
representative or universal set to be hybridized with each of a
plurality of small arrays;
[0027] 2) Adding a first probe to each subarray on each of the
arrays to be analyzed in parallel;
[0028] 3) Performing hybridization and scoring of the hybridization
results;
[0029] 4) Stripping off previously used probes;
[0030] 5) Repeating hybridizaton, scoring and stripping steps for
the remaining probes which are to be scored;
[0031] 5) Processing the obtained results to obtain a final
analysis or to determine additional probes to be hybridized;
[0032] 6) Performing additional hybridizations for certain
subarrays; and
[0033] 7) Processing complete sets of data and obtaining a final
analysis.
[0034] This approach provides fast identification and sequencing of
a small number of nucleic acid samples of one type (e.g. DNA, RNA),
and also provides parallel analysis of many sample types in the
form of subarrays by using a presynthesized set of probes of
manageable size. Two approaches have been combined to produce an
efficient and versatile process for the determination of DNA
identity, for DNA diagnostics, and for identification of
mutations.
[0035] For the identification of known sequences, a small set of
shorter probes may be used in place of a longer unique probe. In
this approach, although there may be more probes to be scored, a
universal set of probes may be synthesized to cover any type of
sequence. For example, a full set of 6-mers includes only 4,096
probes, and a complete set of 7-mers includes only 16,384
probes.
[0036] Full sequencing of a DNA fragment may be performed with two
levels of hybridization. One level is hybridization of a sufficient
set of probes that cover every base at least once. For this
purpose, a specific set of probes may be synthesized for a standard
sample. The results of hybridization with such a set of probes
reveal whether and where mutations (differences) occur in
non-standard samples. Further, this set of probes may include
"negative" probes to confirm the hybridization results of the
"positive" probes. To determine the identity of the changes,
additional specific probes may be hybridized to the sample. This
additional set of probes will have both "positive" (the mutant
sequence) and "negative" probes, and the sequence changes will be
identified by the positive probes and confirmed by the negative
probes.
[0037] In another embodiment, all probes from a universal set may
be scored. A universal set of probes allows scoring of a relatively
small number of probes per sample in a two step process without an
undesirable expenditure of time. The hybridization process may
involve successive probings, in a first step of computing an
optimal subset of probes to be hybridized first and, then, on the
basis of the obtained results, a second step of determining
additional probes to be scored from among those in a universal set.
Both sets of probes have "negative" probes that confirm the
positive probes in the set. Further, the sequence that is obtained
may then be confirmed in a spearate step by hybridizing the sample
with a set of "negative" probes identified from the SBH
results.
[0038] In SBH sequence assembly, K-1 oligonucleotides which occur
repeatedly in analyzed DNA fragments due to chance or biological
reasons may be subject to special consideration. If there is no
additional information, relatively small fragments of DNA may be
fully assembled in as much as every base pair is read several
times.
[0039] In the assembly of relatively longer fragments, ambiguities
may arise due to the repeated occurrence in a set of
positively-scored probes of a K-1 sequence (i.e., a sequence
shorter than the length of the probe). This problem does not exist
if mutated or similar sequences have to be determined (i.e., the
K-1 sequence is not identically repeated). Knowledge of one
sequence may be used as a template to correctly assemble a sequence
known to be similar (e.g. by its presence in a database) by
arraying the positive probes for the unknown sequence to display
the best fit on the template.
[0040] The use of an array of sample avoids consecutive scoring of
many oligonucleotides on a single sample or on a small set of
samples. This approach allows the scoring of more probes in
parallel by manipulation of only one physical object. Subarrays of
DNA samples 1000 bp in length may be sequenced in a relatively
short period of time. If the samples are spotted at 50 subarrays in
an array and the array is reprobed 10 times, 500 probes may be
scored. In screening for the occurrence of a mutation, enough
probes may be used to cover each base three times. If a mutation is
present, several covering probes will be affected. The use of
information about the identity of negative probes may map the
mutation with a two base precision. To solve a single base mutation
mapped in this way, an additional 15 probes may be employed. These
probes cover any base combination for two questionable positions
(assuming that deletions and insertions are not involved). These
probes may be scored in one cycle on 50 subarrays which contain a
given sample. In the implementation of a multiple label color
scheme (i.e., multiplexing), two to six probes, each having a
different label such as a different fluorescent dye, may be used as
a pool, thereby reducing the number of hybridization cycles and
shortening the sequencing process.
[0041] In more complicated cases, there may be two close mutations
or insertions. They may be handled with more probes. For example, a
three base insertion may be solved with 64 probes. The most
complicated cases may be approached by several steps of
hybridization, and the selecting of a new set of probes on the
basis of results of previous hybridizations.
[0042] If subarrays to be analyzed include tens or hundreds of
samples of one type, then several of them may be found to contain
one or more changes (mutations, insertions, or deletions). For each
segment where mutation occurs, a specific set of probes may be
scored. The total number of probes to be scored for a type of
sample may be several hundreds. The scoring of replica arrays in
parallel facilitates scoring of hundreds of probes in a relatively
small number of cycles. In addition, compatible probes may be
pooled. Positive hybridizations may be assigned to the probes
selected to check particular DNA segments because these segments
usually differ in 75% of their constituent bases.
[0043] By using a larger set of longer probes, longer targets may
be analyzed. These targets may represent pools of fragments such as
pools of exon clones.
[0044] A specific hybridization scoring method may be employed to
define the presence of mutants in a genomic segment to be sequenced
from a diploid chromosomal set. Two variations are where: i) the
sequence from one chromosome represents a known allele and the
sequence from the other represents a new mutant; or, ii) both
chromosomes contain new, but different mutants. In both cases, the
scanning step designed to map changes gives a maximal signal
difference of two-fold at the mutant position. Further, the method
can be used to identify which alleles of a gene are carried by an
individual and whether theindividual is homozygous or heterozygous
for that gene.
[0045] Scoring two-fold signal differences required in the first
case may be achieved efficiently by comparing corresponding signals
with homozygous and heterozygous controls. This approach allows
determination of a relative reduction in the hybridization signal
for each particular probe in a given sample. This is significant
because hybridization efficiency may vary more than two-fold for a
particular probe hybridized with different nucleic acid fragments
having the same full match target. In addition, different mutant
sites may affect more than one probe depending upon the number of
oligonucleotide probes. Decrease of the signal for two to four
consecutive probes produces a more significant indication of a
mutant site. Results may be checked by testing with small sets of
selected probes among which one or few probes selected to give a
full match signal which is on average eight-fold stronger than the
signals coming from mismatch-containing duplexes.
[0046] Partitioned membranes allow a very flexible organization of
experiments to accommodate relatively larger numbers of samples
representing a given sequence type, or many different types of
samples represented with relatively small numbers of samples. A
range of 4-256 samples can be handled with particular efficiency.
Subarrays within this range of numbers of dots may be designed to
match the configuration and size of standard multiwell plates used
for storing and labelling oligonucleotides. The size of the
subarrays may be adjusted for different number of samples, or a few
standard subarray sizes may be used. If all samples of a type do
not fit in one subarray, additional subarrays or membranes may be
used and processed with the same probes. In addition, by adjusting
the number of replicas for each subarray, the time for completion
of identification or sequencing process may be varied.
[0047] As used herein, "intermediate fragment" means an
oligonucleotide between 5 and 1000 bases in length, and preferably
between 10 and 40 bp in length.
[0048] In Format 3, a first set of oligonucleotide probes of known
sequence is immobilized on a solid support under conditions which
permit them to hybridize with nucleic acids having respectively
complementary sequences. A labeled, second set of oligonucleotide
probes is provided in solution. Both within the sets and between
the sets the probes may be of the same length or of different
lengths. A nucleic acid to be sequenced or intermediate fragments
thereof may be applied to the first set of probes in
double-stranded form (especially where a recA protein is present to
permit hybridization under non-denaturing conditions), or in
single-stranded form and under conditions which permit hybrids of
different degrees of complementarity (for example, under conditions
which allow discriminatation between full match and one base pair
mismatch hybrids). The nucleic acid to be sequenced or intermediate
fragments thereof may be applied to the first set of probes before,
after or simultaneously with the second set of probes. Probes that
bind to adjacent sites on the atrget are bound together (e.g., by
stacking interactions or by a ligase or other means of causing
chemical bond formation between the adjacent probes). After
permitting adjacent probes to be bound, fragments and probes which
are not immobilized to the surface by chemical bonding to a member
of the first set of probe are washed away, for example, using a
high temperature (up to 100 degrees C.) wash solution which melts
hybrids. The bound probes from the second set may then be detected
using means appropriate to the label employed (which may, for
example, be chemiluminescent, fluorescent, radioactive, enzymatic,
densitometric, or electrophore mass labels).
[0049] Herein, nucleotide bases "match" or are "complementary" if
they form a stable duplex by hydrogen bonding under specified
conditions. For example, under conditions commonly employed in
hybridization assays, adenine ("A") matches thymine ("T"), but not
guanine ("G") or cytosine ("C"). Similarly, G matches C, but not A
or T. Other bases which will hydrogen bond in less specific
fashion, such as inosine or the Universal Base ("M" base, Nichols
et al 1994), or other modified bases, such as methylated bases, for
example, are complementary to those bases for which they form a
stable duplex under specified conditions. A probe is said to be
"perfectly complementary" or is said to be a "perfect match" if
each base in the probe forms a duplex by hydrogen bonding to a base
in the nucleic acid to be sequenced according to the Watson and
Crick base paring rules (i.e. absent any surrounding sequence
effects, the duplex formed has the maximal binding energy for a
particular probe). "Perfectly complementary" and "perfect match"
are also meant to encompass probes which have analogs or modified
nucleotides. A "perfect match" for an analog or modified nucleotide
is judged according to a "perfect match rule" selected for that
analog or modified nucleotide (e.g. the binding pair that has
maximal binding energy for a particular analog or modified
nucleotide). Each base in a probe that does not form a binding pair
according to the "rules" is said to be a "mismatch" under the
specified hybridization conditions.
[0050] A list of probes may be assembled wherein each probe is a
perfect match to the nucleic acid to be sequenced. The probes on
this list may then be analyzed to order them in maximal overlap
fashion. Such ordering may be accomplished by comparing a first
probe to each of the other probes on the list to determine which
probe has a 3' end which has the longest sequence of bases
identical to the sequence of bases at the 5' end of a second probe.
The first and second probes may then be overlapped, and the process
may be repeated by comparing the 5' end of the second probe to the
3' end of all of the remaining probes and by comparing the 3' end
of the first probe with the 5' end of all of the remaining probes.
The process may be continued until there are no probes on the list
which have not been overlapped with other probes. Alternatively,
more than one probe may be selected from the list of positive
probes, and more than one set of overlapped probes ("sequence
nucleus") may be generated in parallel. The list of probes for
either such process of sequence assembly may be the list of all
probes which are perfectly complementary to the nucleic acid to be
sequenced or may be any subset thereof.
[0051] The 5' and 3' ends of the probes may be overlapped to
generate longer stretches of sequence.
[0052] This process of assembling probes continues until an
ambiguity arises because of a branch point (a probe is repeated in
the fragment), repetitive sequences longer than the probes, or an
uncloned segment. The streches of sequence between any two
ambiguities are referred to as fragment os a subclone sequence
(Sfs). Where ambiguities arise in sequence assembly due to the
availability of alternative proper overlaps with probes,
hybridization with longer probes spanning the site of overlap
alternatives, competitive hybridization, ligation of alternative
end to end pairs of probes spanning the site of ambiguity or single
pass gel analysis (to provide an unambiguous ordering of Sfs) may
be used.
[0053] By employing the above procedures, one may obtain any
desired level of sequence, from a pattern of hybridization (which
may be correlated with the identity of a nucleic acid sample to
serve as a signature for identifying the nucleic acid sample) to
overlapping or non-overlapping probes up through assembled Sfs and
on to complete sequence for an intermediate fragment or an entire
source DNA molecule (e.g. a chromosome).
[0054] Sequencing may generally comprise the following steps:
[0055] (a) contacting an array of immobilized oligonucleotide
probes with a nucleic acid fragment under conditions effective to
allow the fragment to form a primary complex with an immobilized
probe having a complementary sequence;
[0056] (b) contacting this primary complex with a set of labeled
oligonucleotide probes in solution under conditions effective to
allow the primary complex to hybridize to the labeled probe,
thereby forming secondary complexes wherein the fragment is
hybridized with both an immobilized probe and a labeled probe;
[0057] (c) removing from a secondary complex any labeled probe that
has not hybridized adjacent to an immobilized probe;
[0058] (d) detecting the presence of adjacent labeled and unlabeled
probes by detecting the presence of the label; and
[0059] (e) determining a nucleotide sequence of the fragment by
connecting the known sequence of the immobilized and labeled
probes.
[0060] Hybridization and washing conditions may be selected to
detect substantially perfect match hybrids (such as those wherein
the fragment and probe hybridize at six out of seven positions),
may be selected to allow differentiation of perfect matches and one
base pair mismatches, or may be selected to permit detection only
of perfect match hybrids.
[0061] Suitable hybridization conditions may be routinely
determined by optimization procedures or pilot studies. Such
procedures and studies are routinely conducted by those skilled in
the art to establish protocols for use in a laboratory. See e.g.,
Ausubel et al., Current Protocols in Molecular Biology, Vol. 1-2,
John Wiley & Sons (1989); Sambrook et al., Molecular Cloning A
Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Springs Harbor Press
(1989); and Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y.
(1982), all of which are incorporated by reference herein. For
example, conditions such as temperature, concentration of
components, hybridization and washing times, buffer components, and
their pH and ionic strength may be varied.
[0062] In embodiments wherein the labeled and immobilized probes
are not physically or chemically linked, detection may rely solely
on washing steps of controlled stringency. Under such conditions,
adjacent probes have increased binding affinity because of stacking
interactions between the adjacent probes. Conditions may be varied
to optimize the process as described above.
[0063] In embodiments wherein the immobilized and labeled probes
are ligated, ligation may be implemented by a chemical ligating
agent (e.g. water-soluble carbodiimide or cyanogen bromide), or a
ligase enzyme, such as the commercially available T.sub.4 DNA
ligase may be employed. The washing conditions may be selected to
distinguish between adjacent versus nonadjacent labeled and
immobilized probes exploiting the difference in stability for
adjacent probes versus nonadjacent probes.
[0064] Oligonucleotide probes may be labeled with fluorescent dyes,
chemiluminescent systems, radioactive labels (e.g., .sup.35S,
.sup.3H, .sup.=P or .sup.33 P) or with isotopes detectable by mass
spectrometry.
[0065] Where a nucleic acid molecule of unknown sequence is longer
than about 45 or 50 bp, the molecule may be fragmented and the
sequences of the fragments determined. Fragmentation may be
accomplished by restriction enzyme digestion, shearing or NaOH.
Fragments may be separated by size (e.g. by gel electrophoresis) to
obtain a preferred fragment length of about ten to forty bps.
[0066] Oligonucleotides may be immobilized, by a number of methods
known to those skilled in the art, such as laser-activated
photodeprotection attachment through a phosphate group using
reagents such as a nucleoside phosphoramidite or a nucleoside
hydrogen phosphorate. Glass, nylon, silicon and fluorocarbon
supports may be used.
[0067] Oligonucleotides may be organized into arrays, and these
arrays may include all or a subset of all probes of a given length,
or sets of probes of selected lengths. Hydrophobic partitions may
be used to separate probes or subarrays of probes. Arrays may be
designed for various applications (e.g. mapping, partial
sequencing, sequencing of targeted regions for diagnostic purposes,
mRNA sequencing and large scale sequencing). A specific chip may be
designed to be dedicated to a particular application by selecting a
combination and arrangement of probes on a substrate.
[0068] For example, 1024 immobilized probe arrays of all
oligonucleotide probes 5 bases in length (each array containing
1024 distinct probes) may be constructed. The probes in this
example are 5-mers in an informational sense (they may actually be
longer probes). A second set of 1024 5-mer probes may be labeled,
and one of each labeled probe may be applied to an array of
immobilized probes along with a fragment to be sequenced. In this
example, 1024 arrays would be combined in a large superarray, or
"superchip." In those instances where an immobilized probe and one
of the labeled probes hybridize end-to-end along a nucleic acid
fragment., the two probes are joined, for example by ligation, and,
after removing unbound label, 10-mers complementary to the sample
fragment are detected by the correlation of the presence of a label
at a point in an array having an immobilized probe of known
sequence to which was applied a labeled probe of known sequence.
The sequence of the sample fragment is simply the sequence of the
immobilized probe continued in the sequence of the labeled probe.
In this way, all one million possible 10-mers may be tested by a
combinatorial process which employs only 5-mers and which thus
involves one thousandth of the amount of effort for oligonucleotide
synthesis.
[0069] A nucleic acid sample to be sequenced may be fragmented or
otherwise treated (for example, by the use of recA) to avoid
hindrance to hybridization from secondary structure in the sample.
The sample may be fragmented by, for example, digestion with a
restriction enzyme such as Cvi JI, physical shearing (e.g. by
ultrasound), or by NaOH treatment. The resulting fragments may be
separated by gel electrophoresis and fragments of an appropriate
length, such as between about 10 bp and about 40 bp, may be
extracted from the gel.
[0070] A reusable Format 3 SBH array may be produced by introducing
a cleavable bond between the fixed and labeled probes and then
cleaving this bond after a round of Format 3 analyziz is finished.
The labeled probes may be ribonucleotides or a ribonucleotide may
be used as the joining base in the labeled probe so that this probe
may subsequently be removed, e.g., by RNAse or uracil-DNA
glycosylate treatment, or NaOH treatment. In addition, bonds
produced by chemical ligation may be selectively cleaved.
[0071] Other variations include the use of modified
oligonucleotides to increase specificity or efficiency, cycling
hybridizations to increase the hybridization signal, for example by
performing a hybridization cycle under conditions (e.g.
temperature) optimally selected for a first set of labeled probes
followed by hybridization under conditions optimally selected for a
second set of labeled probes. Shifts in reading frame may be
determined by using mixtures (preferably mixtures of equimolar
amounts) of probes ending in each of the four nucleotide bases A,
T, C and G.
[0072] Branch points produce ambiguities as to the ordered sequence
of a fragment. Although the sequence information is determined by
SBH, either: (i) long read length, single-pass gel sequencing at a
fraction of the cost of complete gel sequencing; or (ii) comparison
to related sequences, may be used to order hybridization data where
such ambiguities ("branch points") occur. Primers for single pass
gel sequencing through the branch points are identified from the
SBH sequence information or from known vector sequences, e.g., the
flanking sequences to the vector insert site, and standard
Sanger-sequencing reactions are performed on the sample nucleic
acid. The sequence obtained from this single pass gel sequencing is
compared to the Sfs that read into and out of the branch points to
identify the order of the Sfs. Alternatively, the Sfs may be
ordered by comparing the sequence of the Sfs to related sequences
and ordering the Sfs to produce a sequence that is closest to the
related sequence.
[0073] In addition, the number of tandem repetitive nucleic acid
segments in a target fragment may be determined by single-pass gel
sequencing. As tandem repeats occur rarely in protein-encoding
portions of a gene, the gel-sequencing step will be performed only
when one of these noncoding regions is identified as being of
particular interest (e.g., if it is an important regulatory
region).
[0074] Obtaining information about the degree of hybridization
exhibited for a set of only about 200 oligonucleotides probes
(about 5% of the effort required for complete sequencing) defines a
unique signature of each gene and may be used for sorting the cDNAs
from a library to determine if the library contains multiple copies
of the same gene. By such signatures, identical, similar and
different cDNAs can be distinguished and inventoried.
[0075] Nucleic acids and methods for isolating, cloning and
sequencing nucleic acids are well known to those of skill in the
art. See e.g., Ausubel et al., Current Protocols in Molecular
Biology, Vol. 1-2, John Wiley & Sons (1989); and Sambrook et
al., Molecular Cloning A Laboratory Manual, 2nd Ed., Vols. 1-3,
Cold Springs Harbor Press (1989), both of which are incorporated by
reference herein.
[0076] SBH is a well developed technology that may be practiced by
a number of methods known to those skilled in the art.
Specifically, techniques related to sequencing by hybridization of
the following documents is incorporated by reference herein:
Drmanac et al., U.S. Pat. No. 5,202,231 (hereby incorporated by
reference herein)--Issued Apr. 13, 1993; Drmanac et al., Genomics,
4, 114-128 (1989); Drmanac et al., Proceedings of the First Int'l.
Conf. Electrophoresis Supercomputing Human Genome Cantor et al.
eds, World Scientific Pub. Co., Singapore, 47-59 (1991); Drmanac et
al., Science, 260, 1649-1652 (1993); Lehrach et al., Genome
Analysis: Genetic and Physical Mapping, 1, 39-81 (1990), Cold
Spring Harbor Laboratory Press; Drmanac et al., Nucl. Acids Res.,
4691 (1986); Stevanovic et al., Gene, 79, 139 (1989); Panusku et
al., Mol. Biol. Evol., 1, 607 (1990); Nizetic et al., Nucl. Acids
Res., 19, 182 (1991); Drmanac et al., J. Biomol. Struct. Dyn., 5,
1085 (1991); Hoheisel et al., Mol Gen., 4, 125-132 (1991);
Strezoska et al., Proc. Nat'l. Acad. Sci. (USA), 88, 10089 (1991);
Drmanac et al., Nucl. Acids Res., 19, 5839 (1991); and Drmanac et
al., Int. J. Genome Res., 1, 59-79 (1992).
[0077] The present invention is illustrated in the following
examples. Upon consideration of the present disclosure, one of
skill in the art will appreciate that many other embodiments and
variations may be made in the scope of the present invention.
Accordingly, it is intended that the broader aspects of the present
invention not be limited to the disclosure of the following
examples.
EXAMPLE 1
Preparation of Sets of Probes
[0078] Two types of universal sets of probes may be prepared. The
first is a complete set (or at least a noncomplementary subset) of
relatively short probes, for example all 4096 (or about 2000
non-complementary) 6-mers, or all 16,384 (or about 8,000
non-complementary) 7-mers. Full noncomplementary subsets of 8-mers
and longer probes are less convenient inasmuch as they include
32,000 or more probes.
[0079] A second type of probe set is selected as a small subset of
probes still sufficient for reading every bp in any sequence with
at least with one probe. For example, 12 of 16 dimers are
sufficient. A small subset for 7-mers, 8-mer and 9-mers for
sequencing double stranded DNA may be about 3000, 10,000 and 30,000
probes, respectively.
[0080] Sets of probes may also be selected to identify a target
nucleic acid of known sequence, and/or to identify alleles or
mutants of a target nucleic acid with a known sequence. Such a set
of probes contains sufficient probes so that every nucleotide
position of the target nucleic acid is read at least once. Alleles
or mutants are identified by the loss of binding of one of the
"positive" probes. The specific sequence of these alleles or
mutants is then determined by interrogating the target nucleic acid
with sets of probes that contain every possible nucleotide change
and combination of changes at these probe positions.
[0081] Probes may be prepared using standard chemistry with one to
three non-specified (mixed A,T,C and G) or universal (e.g. M base
or inosine) bases at the ends. If radiolabelling is used, probes
may have an OH group at the 5' end for kinasing by radiolabelled
phosphorous groups.
[0082] Alternatively, probes labelled with any compatible system,
such as fluorescent dyes, may be employed. Other types of probes,
such as PNA (Protein Nucleic Acids)or probes containing modified
bases which change duplex stability also may be used.
[0083] Probes may be stored in bar-coded multiwell plates. For
small numbers of probes, 96-well plates may be used; for 10,000 or
more probes, storage in 384-or 864-well plates is preferred. Stacks
of 5 to 50 plates are enough to store all probes. Approximately 5
pg of a probe may be sufficient for hybridization with one DNA
sample. Thus, from a small synthesis of about 50 mg per probe, ten
million samples may be analyzed. If each probe is used for every
third sample, and if each sample is 1000 bp in length, then over 30
billion bases (10 human genomes) may be sequenced by a set of 5,000
probes.
EXAMPLE 2
Probes Having Modified Oligonucleotides
[0084] Modified oligonucleotides may be introduced into
hybridization probes and used under appropriate conditions
therefor. For example, pyrimidines with a halogen at the
C.sup.5-position may be used to improve duplex stability by
influencing base stacking. 2,6-diaminopurine may be used to provide
a third hydrogen bond in base pairing with thymine, thereby
thermally stabilizing DNA-duplexes. Using 2,5-diaminopurine may
increase duplex stability to allow more stringent conditions for
annealing, thereby improving the specificity of duplex formation,
suppressing background problems and permitting the use of shorter
oligomers.
[0085] The synthesis of the triphosphate versions of these modified
nucleotides is disclosed by Hoheisel & Lehrach (1990).
[0086] One may also use the non-discriminatory base analogue, or
universal base, as designed by Nichols et al. (1994). This new
analogue, 1-(2-deoxy-D-ribfuranosyl)-3-nitropyrrole (designated M),
was generated for use in oligonucleotide probes and primers for
solving the design problems that arise as a result of the
degeneracy of the genetic code, or when only fragmentary peptide
sequence data are available. This analogue maximizes stacking while
minimizing hydrogen-bonding interactions without sterically
disrupting a DNA duplex.
[0087] The M nucleoside analogue was designed to maximize stacking
interactions using aprotic polar substituents linked to
heteroaromatic rings, enhancing intra- and inter-strand stacking
interactions to lessen the role of hydrogen bonding in base-pairing
specificity. Nichols et al. (1994) favored 3-nitropyrrole
2-deoxyribonucleoside because of its structural and electronic
resemblance to p-nitroaniline, whose derivatives are among the
smallest known intercaltors of double-stranded DNA.
[0088] The dimethoxytrityl-protected phosphoramidite of nucleoside
M is also available for incorporation into nucleotides used as
primers for sequencing and polymerase chain reaction (PCR). Nichols
et al. (1994) showed that a substantial number of nucleotides can
be replaced by M without loss of primer specificity.
[0089] A unique property of M is its ability to replace long
strings of contiguous nucleosides and still yield functional
sequencing primers. Sequences with three, six and nine M
substitutions have all been reported to give readable sequencing
ladders, and PCR with three different M-containing primers all
resulted in amplification of the correct product (Nichols et al.,
1994).
[0090] The ability of 3-nitropyrrole-containing oligonucleotides to
function as primers strongly suggests that a duplex structure must
form with complementary strands. Optical thermal profiles obtained
for the oligonucleotide pairs d(5-C.sub.2-T.sub.5XT.sub.5G.sub.2-3)
and d(5-C.sub.2A.sub.5YA.sub.5G2-3) (where X and Y can be A, C, G,
T or M) were reported to fit the normal sigmoidal pattern observed
for the DNA double-to single strand transition. The Tm values of
the oligonucleotides containing X M base pairs (where X was A, C, G
or T, and Y was M) were reported to all fall within a 3.degree. C.
range (Nichols et al., 1994).
EXAMPLE 3
Selection and Labeling of Probes
[0091] When an array of subarrays is produced, the sets of probes
to be hybridized in each of the hybridization cycles on each of the
subarrays is defined. For example, a set of 384 probes may be
selected from the universal set, and 96 probings may be performed
in each of 4 cycles. Probes selected to be hybridized in one cycle
preferably have similar G+C contents.
[0092] Selected probes for each cycle are transferred to a 96-well
plate and then are labelled by kinasing or by other labelling
procedures if they are not labelled (e.g. with stable fluorescent
dyes) before they are stored.
[0093] On the basis of the first round of hybridizations, a new set
of probes may be defined for each of the subarrays for additional
cycles. Some of the arrays may not be used in some of the cycles.
For example, if only 8 of 64 patient samples exhibit a mutation and
8 probes are scored first for each mutation, then all 64 probes may
be scored in one cycle and 32 subarrays are not used. These unused
subarrays may then be treated with hybridization buffer to prevent
drying of the filters.
[0094] Probes may be retrieved from the storing plates by any
convenient approach, such as a single channel pipetting device, or
a robotic station, such as a Beckman Biomek 1000 (Beckman
Instruments, Fullerton, Calif.) or a Mega Two robot (Megamation,
Lawrenceville, N.J.). A robotic station may be integrated with data
analysis programs and probe managing programs. Outputs of these
programs may be inputs for one or more robotic stations.
[0095] Probes may be retrieved one by one and added to subarrays
covered by hybridization buffer. It is preferred that retrieved
probes be placed in a new plate and labelled or mixed with
hybridization buffer. The preferred method of retrieval is by
accessing stored plates one by one and pipetting (or transferring
by metal pins) a sufficient amount of each selected probe from each
plate to specific wells in an intermediary plate. An array of
individually addressable pipettes or pins may be used to speed up
the retrieval process.
EXAMPLE 4
Preparation of Labeled Probes
[0096] The oligonucleotide probes may be prepared by automated
synthesis, which is routine to those of skill in the art, for
example, using and Applied Biosystems system. Alternatively, probes
may be prepared using Genosys Biotechnologies Inc. Methods using
stacks of porous Teflon wafers.
[0097] Oligonucleotide probes may be labeled with, for example,
radioactive labels (.sup.35S, .sup.32p, .sup.33P, and preferably,
.sup.33P) for arrays with 100-200 um or 100-400 um spots;
non-radioactive isotopes (Jacobsen et al., 1990); or fluorophores
(Brumbaugh et al., 1988). All such labeling methods are routine in
the art, as exemplified by the relevant sections in Sambrook et al.
(1989) and by further references such as Schubert et al. (1990),
Murakami et al. (1991) and Cate et al. (1991), all articles being
specifically incorporated herein by reference.
[0098] In regard to radiolabelling, the common methods are
end-labeling using T4 polynucleotide kinase or high specific
activity labeling using Klenow or even T7 polymerase. These are
described as follows.
[0099] Synthetic oligonucleotides are synthesized without a
phosphate group at their 5 termini and are therefore easily labeled
by transfer of the -.sup.32P or -.sup.33P from [-.sup.32P]ATP or
[-.sup.33P]ATP using the enzyme bacteriophage T4 polynucleotide
kinase. If the reaction is carried out efficiently, the specificity
activity of such probes can be as high as the specific activity of
the [-.sup.32P]ATP or [-.sup.33P]ATP itself. The reaction described
below is designed to label 10 pmoles of an oligonucleotide to high
specific activity. Labeling of different amounts of oligonucleotide
can easily be achieved by increasing or decreasing the size of the
reaction, keeping the concentrations of all components
constant.
[0100] A reaction mixture would be created using 1.0 ul of
oligonucleotide (10 pmoles/ul); 2.0 ul of 10.times. bacteriophage
T4 polynucleotide kinase buffer; 5.0 ul of [-.sup.32P]ATP or
[-.sup.33P]ATP (sp. Act. 5000 Ci/mmole; 10 mCi/ml in aqueous
solution) (10 pmoles); and 11.4 ul of water. Eight (8) units
(.about.1 ul) of bacteriophage T4 polynucleotide kinase is added to
the reaction mixture, and incubated for 45 minutes at 37.degree. C.
The reaction is heated for 10 minutes at 68.degree. C. to
inactivate the bacteriophage T4 polynucleotide kinase.
[0101] The efficiency of transfer of .sup.32P or .sup..times.P to
the oligonucleotide and its specific activity is then determined.
If the specific activity of the probe is acceptable, it is
purified. If the specific activity is too low, an additional 8
units of enzyme is added and incubated for a further 30 minutes at
37.degree. C. before heating the reaction for 10 minutes at
68.degree. C. to inactivate the enzyme.
[0102] Purification of radiolabeled oligonucleotides can be
achieved by, e.g., precipitation with ethanol; precipitation with
cetylpyridinium bromide; by chromatography through bio-gel P-60; or
by chromatography on a Sep-Pak C.sub.18 column, or by
polyacrylamide gel electrophoresis.
[0103] Probes of higher specific activities can be obtained using
the Klenow fragment of E. coli. DNA polymerase I to synthesize a
strand of DNA complementary to the synthetic oligonucleotide. A
short primer is hybridized to an oligonucleotide template whose
sequence is the complement of the desired radiolabeled probe. The
primer is then extended using the Klenow fragment of E. coli DNA
polymerase I to incorporate [-.sup.32P] dNTPs or [-.sup.33P] dNTPs
in a template-directed manner. After the reaction, the template and
product are separated by denaturation followed by electrophoresis
through a polyacrylamide gel under denaturing conditions. With this
method, it is possible to generate oligonucleotide probes that
contain several radioactive atoms per molecule of
oligonucleotide.
[0104] To use this method, one would mix in a microfuge tube the
calculated amounts of [a-32P]dNTPs or [a-33P]dNTPs necessary to
achieve the desired specific activity and sufficient to allow
complete synthesis of all template strands. Then add to the tube
the appropriate amounts of primer and template DNAs, with the
primer being in three-to tenfold molar excess over the
template.
[0105] 0.1 volume of 10.times. Klenow buffer would then be added
and mixed well. 2-4 units of the Klenow fragment of E. coli DNA
polymerase I would then be added per 5 ul of reaction volume, mixed
and incubated for 2-3 hours at 4.degree. C. If desired, the process
of the reaction may be monitored by removing small (0.1 ul)
aliquots and measuring the proportion of radioactivity that has
become precipitable with 10% trichloroacetic acid (TCA).
[0106] The reaction would be diluted with an equal volume of
gel-loading buffer, heated to 80.degree. C. for 3 minutes, and then
the entire sample loaded on a denaturing polyacrylamide gel.
Following electrophoresis, the gel is autoradiographed, allowing
the probe to be localized and removed from the gel. Various methods
for fluorescent probe labeling are also available, e.g., Brumbaugh
et al. (1988) describe the synthesis of fluorescently labeled
primers. A deoxyuridine analog with a primary amine "linker arm" of
12 atoms attached at C-5 is synthesized. Synthesis of the analog
consists of derivatizing 2-deoxyuridine through organometallic
intermediates to give 5 (methyl propenoyl)-2-deoxyuridine. Reaction
with dimethoxytrityl-chloride produces the corresponding
5-dimethoxytrityl adduct. The methyl ester is hydrolyzed,
activated, and reacted with an appropriately monoacylated alkyl
diamine. After purification, the resultant linker arm nucleosides
are converted to nucleoside analogs suitable for chemical
oligonucleotide synthesis.
[0107] Oligonucleotides would then be made that include one or two
linker arm bases by using modified phosphoridite chemistry. To a
solution of 50 nmol of the linker arm oligonucleotide in 25 ul of
500 mM sodium biocarbonate (pH 9.4) is added 20 ul of 300 mM FITC
in dimethyl sulfoxide. The mixture is agitated at room temperature
for 6 hrs. The oligonucleotide is separated from free FITC by
elution form a 1.times.30 cm Sephadex G-25 column with 20 mM
ammonium acetate (pH 6), combining fractions in the first
UV-absorbing peak.
[0108] In general, fluorescent labeling of an oligonucleotide at
its 5'-end initially involved two steps. First, a N-protected
aminoalkyl phosphoramidite derivative is added to the 5'-end of an
oligonucleotide during automated nucleic acid synthesis. After
removal of all protecting groups, the NHS ester of an appropriate
fluorescent dye is coupled to the 5'-amino group overnight followed
by purification of the labeled oligonucleotide from the excess of
dye using reverse phase HPLC or PAGE.
[0109] Schubert et al. (1990) described the synthesis of a
phosphoramidite that enables oligonucleotides labeled with
fluorescein to be produced during automated DNA synthesis.
[0110] Murakami et al. also described the preparation of
flurescein-labeled oligonucleotides.
[0111] Cate et al. (1991) describe the use of oligonucleotide
probes directly conjugated to alkaline phosphatase in combination
with a direct chemiluminescent substrate (AMPPD) to allow probe
detection.
[0112] Labeled probes could readily be purchased form a variety of
commercial sources, including GENSET, rather then synthesized.
[0113] Other labels include ligands which can serve as specific
binding members to a labeled antibody, chemiluminescers, enzymes,
antibodies which can serve as a specific binding pair member for a
labeled ligand, and the like. A wide variety of labels have been
employed in immunoassays which can readily be employed. Still other
labels include antigens, groups with specific reactivity, and
electrochemically detectable moeities.
[0114] In general, labeling of nucleic acids with electrophore mass
labels ("EML") is described, for example, in Xu et al., J.
Chromatography 764:95-102 (1997). Electrophores are compounds that
can be detected with high sensitivity by electron capture mass
spectrometry (EC-MS). EMLs can be attached to a probe using
chemistry that is well known in the art for reversibly modifying a
nucleotide (e.g., well known nucleotide synthesis chemistry teaches
a variety of methods for attaching molecules to nucleotides as
protecting groups). EMLs are detected using a variety of well known
electron capture mass spectrometry devices (e.g., devices sold by
Finnigan Corporation). Further, techniques that may be used in the
detection of EMLs include, for example, fast atomic bombardment
mass spectrometry (see, e.g., Koster et al., Biomedical Environ.
Mass Spec. 14:111-116 (1987)); plasma desorption mass spectrometry;
electrospray/ionspray (see, e.g., Fenn et al., J. Phys. Chem.
88:4451-59 (1984), PCT Appln. No. WO 90/14148, Smith et al., Anal.
Chem. 62:882-89 (1990)); and matrix-assisted laser
desorption/ionization (Hillenkamp, et al., "Matrix Assisted
UV-Laser Desorption/Ionization: A New Approach to Mass Spectrometry
of Large Biomolecules," Biological Mass Spectrometry (Burlingame
and McCloskey, eds.), Elsevier Science Publishers, Amsterdam, pp.
49-60, 1990); Huth-Fehre et al., "Matrix Assisted Laser Desorption
Mass Spectrometry of Oligodeoxythymidylic Acids," Rapid
Communications in Mass Spectrometry, 6:209-13 (1992)).
[0115] In preferred embodmients, the EMLs are attached to a probe
by a covalent bond that is light sensitive. The EML is released
from the probe after hybridization with a target nucleic acid by a
laser or other light source emitting the desired wavelength of
light. The EML is then fed into a GC-MS (gas chromatogrph-mass
spectrometer) or other appropiate device, and identified by its
mass.
EXAMPLE 5
Preparation of Sequencing Chips and Arrays
[0116] A basic example is using 6-mers attached to 50 micron
surfaces to give a chip with dimensions of 3.times.3 mm which can
be combined to give an array of 20.times.20 cm. Another example is
using 9-mer oligonucleotides attached to 10.times.10 microns
surface to create a 9-mer chip, with dimensions of 5.times.5 mm.
4000 units of such chips may be used to create a 30.times.30 cm
array. In an array in which 4,000 to 16,000 oligochips are arranged
into a square array. A plate, or collection of tubes, as also
depicted, may be packaged with the array as part of the sequencing
kit.
[0117] The arrays may be separated physically from each other or by
hydrophobic surfaces. One possible way to utilize the hydrophobic
strip separation is to use technology such as the Iso-Grid
Microbiology System produced by QA Laboratories, Toronto,
Canada.
[0118] Hydrophobic grid membrane filters (HGMF) have been in use in
analytical food microbiology for about a decade where they exhibit
unique attractions of extended numerical range and automated
counting of colonies. One commercially-available grid is
ISO-GRID.TM. from QA Laboratories Ltd. (Toronto, Canada) which
consists of a square (60.times.60 cm) of polysulfone polymer
(Gelman Tuffryn HT-450, 0.45 u pore size) on which is printed a
black hydrophobic ink grid consisting of 1600 (40.times.40) square
cells. HGMF have previously been inoculated with bacterial
suspensions by vacuum filtration and incubated on the differential
or selective media of choice.
[0119] Because the microbial growth is confined to grid cells of
known position and size on the membrane, the HGMF functions more
like an MPN apparatus than a conventional plate or membrane filter.
Peterkin et al. (1987) reported that these HGMFs can be used to
propagate and store genomic libraries when used with a HGMF
replicator. One such instrument replicates growth from each of the
1600 cells of the ISO-GRID and enables many copies of the master
HGMF to be made (Peterkin et al., 1987).
[0120] Sharpe et al. (1989) also used ISO-GRID HGMF form QA
Laboratories and an automated HGMF counter (MI-100 Interpreter) and
RP-100 Replicator. They reported a technique for maintaining and
screening many microbial cultures.
[0121] Peterkin and colleagues later described a method for
screening DNA probes using the hydrophobic grid-membrane filter
(Peterkin et al., 1989). These authors reported methods for
effective colony hybridization directly on HGMFs. Previously, poor
results had been obtained due to the low DNA binding capacity of
the epoxysulfone polymer on which the HGMFs are printed. However,
Peterkin et al. (1989) reported that the binding of DNA to the
surface of the membrane was improved by treating the replicated and
incubated HGMF with polyethyleneimine, a polycation, prior to
contact with DNA. Although this early work uses cellular DNA
attachment, and has a different objective to the present invention,
the methodology described may be readily adapted for Format 3
SBH.
[0122] In order to identify useful sequences rapidly, Peterkin et
al. (1989) used radiolabeled plasmid DNA from various clones and
tested its specificity against the DNA on the prepared HGMFs. In
this way, DNA from recombinant plasmids was rapidly screened by
colony hybridization against 100 organisms on HGMF replicates which
can be easily and reproducibly prepared.
[0123] Manipulation with small (2-3 mm) chips, and parallel
execution of thousands of the reactions. The solution of the
invention is to keep the chips and the probes in the corresponding
arrays. In one example, chips containing 250,000 9-mers are
synthesized on a silicon wafer in the form of 8.times.8 mM plates
(15 uM/oligonucleotide, Pease et al., 1994) arrayed in 8.times.12
format (96 chips) with a 1 mM groove in between. Probes are added
either by multichannel pipette or pin array, one probe on one chip.
To score all 4000 6-mers, 42 chip arrays have to be used, either
using different ones, or by reusing one set of chip arrays several
times.
[0124] In the above case, using the earlier nomenclature of the
application, F=9; P=6; and F+P=15. Chips may have probes of formula
BxNn, where x is a number of specified bases B; and n is a number
of non-specified bases, so that x=4 to 10 and n=1 to 4. To achieve
more efficient hybridization, and to avoid potential influence of
any support oligonucleotides, the specified bases can be surrounded
by unspecified bases, thus represented by a formula such as
(N)nBx(N)m (FIG.4 +L).
EXAMPLE 6
Preparation of Support Bound Oligonucleotides
[0125] Oligonucleotides, i.e., small nucleic acid segments, may be
readily prepared by, for example, directly synthesizing the
oligonucleotide by chemical means, as is commonly practiced using
an automated oligonucleotide synthesizer.
[0126] Support bound oligonucleotides may be prepared by any of the
methods known to those of skill in the art using any suitable
support such as glass, polystyrene or Teflon. One strategy is to
precisely spot oligonucleotides synthesized by standard
synthesizers. Immobilization can be achieved using passive
adsorption (Inouye & Hondo, 1990); using UV light (Nagata et
al., 1985; Dahlen et al., 1987; Morriey & Collins, 1989) or by
covalent binding of base modified DNA (Keller et al., 1988; 1989);
all references being specifically incorporated herein.
[0127] Another strategy that may be employed is the use of the
strong biotin-streptavidin interaction as a linker. For example,
Broude et al. (1994) describe the use of Biotinylated probes,
although these are duplex probes, that are immobilized on
streptavidin-coated magnetic beads. Streptavidin-coated beads may
be purchased from Dynal, Oslo. Of course, this same linking
chemistry is applicable to coating any surface with streptavidin.
Biotinylated probes may be purchased from various sources, such as,
e.g., Operon Technologies (Alameda, Calif.).
[0128] Nunc Laboratories (Naperville, Ill.) is also selling
suitable material that could be used. Nunc Laboratories have
developed a method by which DNA can be covalently bound to the
microwell surface termed Covalink NH. CovaLink NH is a polystyrene
surface grafted with secondary amino groups (>NH) that serve as
bridge-heads for further covalent coupling. CovaLink Modules may be
purchased from Nunc Laboratories. DNA molecules may be bound to
CovaLink exclusively at the 5'-end by a phosphoramidate bond,
allowing immobilization of more than 1 pmol of DNA (Rasmussen et
al., 1991).
[0129] The use of CovaLink NH strips for covalent binding of DNA
molecules at the 5'-end has been described (Rasmussen et al.,
1991). In this technology, a phosphoramidate bond is employed (Chu
et al., 1983). This is beneficial as immobilization using only a
single covalent bond is preferred. The phosphoramidate bond joins
the DNA to the CovaLink NH secondary amino groups that are
positioned at the end of spacer arms covalently grafted onto the
polystyrene surface through a 2 nm long spacer arm. To link an
oligonucleotide to CovaLink NH via an phosphoramidate bond, the
oligonucleotide terminus must have a 5'-end phosphate group. It is,
perhaps, even possible for biotin to be covalently bound to
CovaLink and then streptavidin used to bind the probes.
[0130] More specifically, the linkage method includes dissolving
DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95.degree.
C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole,
pH 7.0 (1-MeIm.sub.7), is then added to a final concentration of 10
mM 1-MeIm.sub.7. A ss DNA solution is then dispensed into CovaLink
NH strips (75 ul/well) standing on ice.
[0131] Carbodiimide 0.2 M
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in
10 mM 1-MeIm.sub.7, is made fresh and 25 ul added per well. The
strips are incubated for 5 hours at 50.degree. C. After incubation
the strips are washed using, e.g., Nunc-Immuno Wash; first the
wells are washed 3 times, then they are soaked with washing
solution for 5 min., and finally they are washed 3 times (where in
the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50.degree.
C.).
[0132] It is contemplated that a further suitable method for use
with the present invention is that described in PCT Patent
Application WO 90/03382 (Southern & Maskos), incorporated
herein by reference. This method of preparing an oligonucleotide
bound to a support involves attaching a nucleoside 3'-reagent
through the phosphate group by a covalent phosphodiester link to
aliphatic hydroxyl groups carried by the support. The
oligonucleotide is then synthesized on the supported nucleoside and
protecting groups removed from the synthetic oligonucleotide chain
under standard conditions that do not cleave the oligonucleotide
from the support. Suitable reagents include nucleoside
phosphoramidite and nucleoside hydrogen phosphorate.
[0133] An on-chip strategy for the preparation of DNA probe for the
preparation of DNA probe arrays may be employed. For example,
addressable laser-activated photodeprotection may be employed in
the chemical synthesis of oligonucleotides directly on a glass
surface, as described by Fodor et al. (1991), incorporated herein
by reference. Probes may also be immobilized on nylon supports as
described by Van Ness et al. (1991); or linked to Teflon using the
method of Duncan & Cavalier (1988); all references being
specifically incorporated herein.
[0134] To link an oligonucleotide to a nylon support, as described
by Van Ness et al. (1991), requires activation of the nylon surface
via alkylation and selective activation of the 5'-amine of
oligonucleotides with cyanuric chloride.
[0135] One particular way to prepare support bound oligonucleotides
is to utilize the light-generated synthesis described by Pease et
al., (1994, incorporated herein by reference). These authors used
current photolithographic techniques to generate arrays of
immobilized oligonucleotide probes (DNA chips). These methods, in
which light is used to direct the synthesis of oligonucleotide
probes in high-density, miniaturized arrays, utilize photolabile
5'-protected N-acyl-deoxynucleoside phosphoramidites, surface
linker chemistry and versatile combinatorial synthesis strategies.
A matrix of 256 spatially defined oligonucleotide probes may be
generated in this manner and then used in the advantageous Format 3
sequencing, as described herein.
[0136] Of course, one could easily purchase a DNA chip, such as one
of the light-activated chips described above, from a commercial
source. In this regard, one may contact Affymetrix of Santa Clara,
Calif. 95051, and Beckman.
EXAMPLE 7
Preparation of Nucleic Acid Fragments
[0137] The nucleic acids to be sequenced may be obtained from any
appropriate source, such as cDNAs, genomic DNA, chromosomal DNA,
microdissected chromosome bands, cosmid or YAC inserts, and RNA,
including MRNA without any amplification steps. For example,
Sambrook et al. (1989) describes three protocols for the isolation
of high molecular weight DNA from mammalian cells (p.
9.14-9.23).
[0138] Target nucleic acid fragments may be prepared as clones in
M13, plasmid or lambda vectors and/or prepared directly from
genomic DNA or cDNA by PCR or other amplification methods. Samples
may be prepared or dispensed in multiwell plates. About 100-1000 ng
of DNA samples may be prepared in 2-500 ml of final volume. Target
nucleic acids prepared by PCR may be directly applied to a
substrate for Format I SBH without purification. Once the target
nucleic acids are fixed to the substrate, the substrate may be
washed or directly annealed with probes.
[0139] The nucleic acids would then be fragmented by any of the
methods known to those of skill in the art including, for example,
using restriction enzymes as described at 9.24-9.28 of Sambrook et
al. (1989), shearing by ultrasound and NaOH treatment.
[0140] Low pressure shearing is also appropriate, as described by
Schriefer et al. (1990, incorporated herein by reference). In this
method, DNA samples are passed through a small French pressure cell
at a variety of low to intermediate pressures. A lever device
allows controlled application of low to intermediate pressures to
the cell. The results of these studies indicate that low-pressure
shearing is a useful alternative to sonic and enzymatic DNA
fragmentation methods.
[0141] One particularly suitable way for fragmenting DNA is
contemplated to be that using the two base recognition
endonuclease, CviJI, described by Fitzgerald et al. (1992). These
authors described an approach for the rapid fragmentation and
fractionation of DNA into particular sizes that they contemplated
to be suitable for shotgun cloning and sequencing. The present
inventor envisions that this will also be particularly useful for
generating random, but relatively small, fragments of DNA for use
in the present sequencing technology.
[0142] The restriction endonuclease CviJI normally cleaves the
recognition sequence PuGCPy between the G and C to leave blunt
ends. Atypical reaction conditions, which alter the specificity of
this enzyme (CviJI**), yield a quasi-random distribution of DNA
fragments form the small molecule pUC19 (2688 base pairs).
Fitzgerald et al. (1992) quantitatively evaluated the randomness of
this fragmentation strategy, using a CviJI** digest of pUC19 that
was size fractionated by a rapid gel filtration method and directly
ligated, without end repair, to a lac Z minus M13 cloning vector.
Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy
and PuGCPu, in addition to PuGCPy sites, and that new sequence data
is accumulated at a rate consistent with random fragmentation.
[0143] As reported in the literature, advantages of this approach
compared to sonication and agarose gel fractionation include:
smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug);
and fewer steps are involved (no preligation, end repair, chemical
extraction, or agarose gel electrophoresis and elution are needed).
These advantages are also proposed to be of use when preparing DNA
for sequencing by Format 3.
[0144] Irrespective of the manner in which the nucleic acid
fragments are obtained or prepared, it is important to denature the
DNA to give single stranded pieces available for hybridization.
This is achieved by incubating the DNA solution for 2-5 minutes at
80-90.degree. C. The solution is then cooled quickly to 2.degree.
C. to prevent renaturation of the DNA fragments before they are
contacted with the chip. Phosphate groups must also be removed from
genomic DNA, as described in Example VI.
EXAMPLE 8
Preparation of DNA Arrays
[0145] Arrays may be prepared by spotting DNA samples on a support
such as a nylon membrane. Spotting may be performed by using arrays
of metal pins (the positions of which correspond to an array of
wells in a microtiter plate) to repeated by transfer of about 20 nl
of a DNA solution to a nylon membrane. By offset printing, a
density of dots higher than the density of the wells is achieved.
One to 25 dots may be accommodated in 1 mm.sup.2, depending on the
type of label used. By avoiding spotting in some preselected number
of rows and columns, separate subsets (subarrays) may be formed.
Samples in one subarray may be the same genomic segment of DNA (or
the same gene) from different individuals, or may be different,
overlapped genomic clones. Each of the subarrays may represent
replica spotting of the same samples. In one example, a selected
gene segment may be amplified from 64 patients. For each patient,
the amplified gene segment may be in one 96-well plate (all 96
wells containing the same sample). A plate for each of the 64
patients is prepared. By using a 96-pin device, all samples may be
spotted on one 8.times.12 cm membrane. Subarrays may contain 64
samples, one from each patient. Where the 96 subarrays are
identical, the dot span may be 1 mm.sup.2 and there may be a 1 mm
space between subarrays.
[0146] Another approach is to use membranes or plates (available
from NUNC, Naperville, Ill.) which may be partitioned by physical
spacers e.g. a plastic grid molded over the membrane, the grid
being similar to the sort of membrane applied to the bottom of
multiwell plates, or hydrophobic strips. A fixed physical spacer is
not preferred for imaging by exposure to flat phosphor-storage
screens or x-ray films.
EXAMPLE 9
Hybridization and Scoring Process
[0147] Labeled probes may be mixed with hybridization buffer and
pipetted, preferably by multichannel pipettes, to the subarrays. To
prevent mixing of the probes between subarrays (if there are no
hydrophobic strips or physical barriers imprinted in the membrane),
a corresponding plastic, metal or ceramic grid may be firmly
pressed to the membrane. Also, the volume of the buffer may be
reduced to about 1 ml or less per mm.sup.2. The concentration of
the probes and hybridization conditions used may be as described
previously except that the washing buffer may be quickly poured
over the array of subarrays to allow fast dilution of probes and
thus prevent significant cross-hybridization. For the same reason,
a minimal concentration of the probes may be used and hybridization
time extended to the maximal practical level. For DNA detection and
sequencing, knowledge of a "normal" sequence allows the use of the
continuous stacking interaction phenomenon to increase the signal.
In addition to the labelled probe, additional unlabelled probes
which hybridize back to back with a labelled one may be added in
the hybridization reaction. The amount of the hybrid may be
increased several times. The probes may be connected by ligation.
This approach may be important for resolving DNA regions forming
"compressions".
[0148] In the case of radiolabelled probes, images of the filters
may be obtained, preferably by phosphorstorage technology.
Fluorescent labels may be scored by CCD cameras, confocal
microscopy or otherwise. In order to properly scale and integrate
data from different hybridization experiments, raw signals are
normalized based on the amount of target in each dot. Differences
in the amount of target DNA per dot may be corrected for by
dividing signals of each probe by an average signal for all probes
scored on one dot. The normalized signals may be scaled, usually
from 1-100, to compare data from different experiments. Also, in
each subarray, several control DNAs may be used to determine an
average background signal in those samples which do not contain a
full match target. For samples obtained from diploid (polyploid)
scores, homozygotic controls may be used to allow recognition of
heterozygotes in the samples.
EXAMPLE 10
Hybridization With Oligonucleotides
[0149] Oligonucleotides were either purchased from Genosys Inc.,
Houston, Tex. or made on an Applied Biosystems 381A DNA
synthesizer. Most of the probes used were not purified by HPLC or
gel electrophoresis. For example, probes were designed to have both
a single perfectly complementary target in interferon, a M13 clone
containing a 921 bp Eco RI-Bgl II human B1 interferon fragment
(Ohno and Tangiuchi, Proc. Natl. Acad. Sci. 74: 4370-4374(1981)],
and at least one target with an end base mismatch in M13 vector
itself.
[0150] End labelling of oligonucleotides was performed as described
[Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Cold Spring Harbor, N.Y. (1982)] in 10 ml
containing T4-polynucleotide kinase (5 units Amersham),
g.sup.32-ATP (3.3 pM, 10 mCi Amersham 3000 Ci/mM) and
oligonucleotide (4 pM, 10 ng). Specific activities of the probes
were 2.5-5.times.10 9 cpm/nM.
[0151] Single stranded DNA (2 to 4 ml in 0.5 NaOH, 1.5 M NaCl) was
spotted on a Gene Screen membrane wetted with the same solution,
the filters were neutralized in 0.05 M Na.sub.2HPO.sub.4 pH 6.5,
baked in an oven at 80.degree. C. for 60 min. and UV irradiated for
1 min. Then, the filters were incubated in hybridization solution
(0.5 M Na.sub.2HPO.sub.4 pH 7.2, 7% sodium lauroyl sarcosine for 5
min at room temperature and placed on the surface of a plastic
Petri dish. A drop of hybridization solution (10 ml, 0.5 M
Na.sub.2HPO.sub.4 pH 7.2, 7% sodium lauroyl sarcosine) with a
.sup.32P end-labeled oligomer probe at 4 nM concentration was
placed over 1-6 dots per filter, overlaid with a square piece of
polyethylene (approximately 1.times.1 cm.), and incubated in a
moist chamber at the indicated temperatures for 3 hr. Hybridization
was stopped by placing the filter in 6.times. SSC washing solution
for 3.times.5 minute at 0.degree. C. to remove unhybridized probe.
The filter was either dried, or further washed for the indicated
times and temperatures, and autoradiographed. For discrimination
measurements, the dots were excised from the dried filters after
autoradiography [a phosphoimager (Molecular Dynamics, Sunnyvale,
Calif.) may be used] placed in liquid scintillation cocktail and
counted. The uncorrected ratio of cpms for IF and M13 dots is given
as D.
[0152] The conditions reported herein allow hybridization with very
short oligonucleotides but ensure discriminations between matched
and mismatched oligonucleotides that are complementary to and
therefore bind to a target nucleic acid. Factors which influence
the efficient detection of hybridization of specific short
sequences based on the degree of discriminations (D) between a
perfectly complementary target and an imperfectly complementary
target with a single mismatch in the hybrid are defined. In
experimental tests, dot blot hybridization of twenty-eight probes
that were 6 to 8 nucleotides in length to two M13 clones or to
model oligonucleotides bound to membrane filters was accomplished.
The principles guiding the experimental procedures are given
below.
[0153] Oligonucleotide hybridization to filter bound target nucleic
acids only a few nucleotides longer than the probe in conditions of
probe excess is a pseudo-first order reaction with respect to
target concentration. This reaction is defined by:
S.sub.t/S.sub.o=e.sup.-kh[OP]t
[0154] Wherein S.sub.t and S.sub.O are target sequence
concentrations at time t and t.sub.0, respectively. (OP) is probe
concentration and t is temperature. The rate constant for hybrid
formation, k.sub.h increases only slightly in the 0.degree. C. to
30.degree. C. range (Porschke and Eigen, J. Mol. Biol. 62: 361
(1971); Craig et al., J. Mol. Biol. 62: 383 (1971)]. Hybrid melting
is a first order reaction with respect to hybrid concentration
(here replaced by mass due to filter bound state) as shown in:
H.sub.t/H.sub.o=e.sup.-kmt
[0155] In this equation, H.sub.t and H.sub.o are hybrid
concentrations at times t and t.sub.0, respectively; k.sub.m is a
rate constant for hybrid melting which is dependent on temperature
and salt concentration [Ikuta et al., Nucl. Acids Res. 15: 797
(1987); Porsclike and Eigen, J. Mol. Biol. 62: 361 (1971); Craig et
al., J. Mol. Biol. 62: 303 (1971)]. During hybridization, which is
a strand association process, the back, melting, or strand
dissociation, reaction takes place as well. Thus, the amount of
hybrid formed in time is result of forward and back reactions. The
equilibrium may be moved towards hybrid formation by increasing
probe concentration and/or decreasing temperature. However, during
washing cycles in large volumes of buffer, the melting reaction is
dominant and the back reaction hybridization is insignificant,
since the probe is absent. This analysis indicates workable Short
Oligonucleotide Hybridization (SOH) conditions call be varied for
probe concentration or temperature.
[0156] D or discrimination is defined in equation four:
D=H.sub.p(t.sub.w)/H.sub.i(t.sub.w)
[0157] H.sub.p (t.sub.w) and H.sub.i (t.sub.w) are the amounts
hybrids remaining after a washing time, t.sub.w, for the identical
amounts of perfectly and imperfectly complementary duplex,
respectively. For a given temperature, the discrimination D changes
with the 10 length of washing time and reaches the maximal value
when H.sub.i=B which is equation five.
[0158] The background, B, represents the lowest hybridization
signal detectable in the system. Since any further decrease of
H.sub.i may not be examined, D increases upon continued washing.
Washing past t.sub.w just decreases H.sub.p relative to B, and is
seen as a decrease in D. The optimal washing time, t.sub.w, for
imperfect hybrids, from equation three and equation five is:
t.sub.w=-1n(B/H.sub.i(t.sub.0))/k.sub.m.i
[0159] Since H.sub.p is being washed for the same t.sub.w,
combining equations, one obtains the optimal discrimination
function:
D=e.sup.1n(B/Hi(t0))km,p/km,iXH.sub.p(t.sub.0)/B
[0160] The change of D as a function, of T is important because of
the choice of an optimal washing temperature. It is obtained by
substituting the Arhenius equation which is:
K-=Ae.sup.-Ea/RT
[0161] into the previous equation to form the final equation:
D=H.sub.p((t.sub.0)/BX(B/H.sub.i(t.sub.0))
.sup.(Ap/Ai)e(Ea,i.sup.-Ea,p.su- p.)/RT;
[0162] Wherein B is less than H.sub.i (t.sub.0).
[0163] Since the activation energy for perfect hybrids, E.sub.a,p,
and the activation energy for imperfect hybrids, E.sub.a,i, can be
either equal, or E.sub.a,i less than E.sub.a,p D is temperature
independent, or decreases with increasing temperature,
respectively. This result implies that the search for stringent
temperature conditions for good discrimination in SOH is
unjustified. By washing at lower temperatures, one obtains equal or
better discrimination, but the time of washing exponentially
increases with the decrease of temperature. Discrimination more
strongly decreases with T, if H.sub.i(t.sub.o) increases relative
to H.sub.p(t.sub.0).
[0164] D at lower temperatures depends to a higher degree on the
H.sub.p (t.sub.0)/B ratio than on the H.sub.p(t.sub.0)/H.sub.i
(t.sub.0) ratio. This result indicates that it is better to obtain
a sufficient quantity of H.sub.p in the hybridization regardless of
the discrimination that can be achieved in this step. Better
discrimination can then be obtained by washing, since the higher
amounts of perfect hybrid allow more time for differential melting
to show an effect. Similarly, using larger amounts of target
nucleic acid a necessary discrimination can be obtained even with
small differences between K.sub.m,p and K.sub.m,j.
[0165] Extrapolated to a more complex situation than covered in
this simple model, the result is that washing at lower temperatures
is even more important for obtaining discrimination in the case of
hybridization of a probe having many end-mismatches within a given
nucleic acid target.
[0166] Using the described theoretical principles as a guide for
experiments, reliable hybridizations have been obtained with probes
six to eight nucleotides in length. All experiments were performed
with a floating plastic sheet providing a film of hybridization
solution above the filter. This procedure allows maximal reduction
in the amount of probe, and thus reduced label costs in dot blot
hybridizations. The high concentration of sodium lauroyl sarcosine
instead of sodium lauroyl sulfate in the phosphate hybridization
buffer allows dropping the reaction from room temperature down to
12.degree. C. Similarly, the 4-6.times. SSC, 10% sodium lauroyl
sarcosine buffer allows hybridization at temperatures as low as
2.degree. C. The detergent in these buffers is for obtaining
tolerable background with up to 40 nM concentrations of labelled
probe. Preliminary characterization of the thermal stability of
short oligonucleotide hybrids was determined on a prototype octamer
with 50% G+C content, i.e. probe of sequence TGCTCATG. The
theoretical expectation is that this probe is among the less stable
octamers. Its transition enthalpy is similar to those of more
stable heptamers or, even to probes 6 nucleotides in length
(Bresslauer et al., Proc. Natl. Acad. Sci. U.S.A. 83: 3746 (1986)).
Parameter T.sub.d, the temperature at which 50% of the hybrid is
melted in unit time of a minute is 18.degree. C. The result shows
that T.sub.d is 15.degree. C. lower for the 8 bp hybrid than for an
11 bp duplex [Wallace et al., Nucleic Acids Res. 6: 3543
(1979)].
[0167] In addition to experiments with model oligonucleotides, an
M13 vector was chosen as a system for a practical demonstration of
short oligonucleotide hybridization. The main aim was to show
useful end-mismatch discrimination with a target similar to the
ones which will be used in various applications of the method of
the invention. Oligonucleotide probes for the M13 model were chosen
in such a way that the M13 vector itself contains the end
mismatched base. Vector IF, an M13 recombinant containing a 921 bp
human interferon gene insert, carries single perfectly matched
target. Thus, IF has either the identical or a higher number of
mismatched targets in comparison to the M13 vector itself.
[0168] Using low temperature conditions and dot blots, sufficient
differences in hybridization signals were obtained between tie dot
containing the perfect and the mismatched targets and the dot
containing the mismatched targets only. This was true for the 6-mer
oligonucleotides and was also true for the 7 and 8-mer
oligonucleotides hybridized to the large IF-M13 pair of nucleic
acids.
[0169] The hybridization signal depends on the amount of target
available on the filter for reaction with the probe. A necessary
control is to show that the difference in sign intensity is not a
reflection of varying amounts of nucleic acid in the two dots.
Hybridization with a probe that has the same number and kind of
targets in both IF and M13 shows that there is an equal amount of
DNA in the dots. Since the efficiency of hybrid formation increases
with hybrid length, the signal for a duplex having six nucleotides
was best detected with a high mass of oligonucleotide target bound
to the filter. Due to their lower molecular weight, a larger number
of oligonucleotide target molecules can be bound to a given surface
area when compared to large molecules of nucleic acid that serves
as target.
[0170] To measure the sensitivity of detection with unpurified DNA,
various amounts of phage supernatants were spotted on the filter
and hybridized with a .sup.32P-labelled octamer. As little as 50
million unpurified phage containing no more than 0.5 ng of DNA gave
a detectable signal indicating that sensitivity of the short
oligonucleotide hybridization method is sufficient. Reaction time
is short, adding to the practicality.
[0171] As mentioned in the theoretical section above, the
equilibrium yield of hybrid depends oil probe concentration and/or
temperature of reaction. For instance, the signal level for the
same amount of target with 4 nM octamer at 13.degree. C. is 3 times
lower than with a probe concentration of 40 nM, and is decreased
4.5-times by raising the hybridization temperature to 25.degree.
C.
[0172] The utility of the low temperature wash for achieving
maximal discrimination is demonstrated. To make the phenomenon
visually obvious, 50 times more DNA was put in the M13 dot than in
the IF dot using hybridization with a vector specific probe. In
this way, the signal after the hybridization step with the actual
probe was made stronger in the, mismatched that in the matched
case. The H.sub.p/H.sub.i ratio was 1:4. Inversion of signal
intensities after prolonged washing at 7.degree. C. was achieved
without a massive loss of perfect hybrid, resulting in a ratio of
2:1. In contrast, it is impossible to achieve any discrimination at
25.degree. C., since the matched target signal is already brought
down to the background level with 2 minute washing; at the same
time, the signal from the mismatched hybrid is still detectable.
The loss of discrimination at 13.degree. C. compared to 7.degree.
C. is not so great but is clearly visible. If one considers the 90
minute point at 7.degree. C. and the 15 minute point at 13.degree.
C. when, the mismatched hybrid signal is near the background level,
which represents optimal washing times for the respective
conditions, it is obvious that the amount of several times greater
at 7.degree. C. than at 13.degree. C. To illustrate this further,
the time course of the change discrimination with washing of the
same amount of starting hybrid at the two temperatures shows the
higher maximal D at the lower temperature. These results confirm
the trend in the change of D with temperature and the ratio of
amounts of the two types of hybrid at the start of the washing
step.
[0173] In order to show the general utility of the short
oligonucleotide hybridization conditions, we have looked
hybridization of 4 heptamers, 10 octamers and an additional 14
probes up to 12 nucleotides in length in our simple M13 system.
These include-the nonamer GTTTTTTAA and octamer GGCAGGCG
representing the two extremes of GC content. Although GC content
and sequence are expected to influence the stability of short
hybrids [Bresslauer et al., Proc. Natl. Acad. Sci. U.S.A. 83: 3746
(1986)], the low temperature short oligonucleotide conditions were
applicable to all tested probes in achieving sufficient
discrimination. Since the best discrimination value obtained with
probes 13 nucleotides in length was 20, a several fold drop due to
sequence variation is easily tolerated.
[0174] The M13 system has the advantage of showing the effects of
target DNA complexity on the levels of discrimination. For two
octamers having either none or five mismatched targets and
differing in only one GC pair the observed discriminations were
18.3 and 1.7, respectively.
[0175] In order to show the utility of this method, three probes 8
nucleotides in length were tested on a collection of 51 plasmid DNA
dots made from a library in Bluescript vector. One probe was
present and specific for Bluescript vector but was absent in M13,
while the other two probes had targets that were inserts of known
sequence. This system allowed the use of hybridization negative or
positive control DNAs with each probe. This probe sequence
(CTCCCTTT) also had a complementary target in the interferon
insert. Since the M13 dot is negative while the interferon insert
in either M13 or Bluescript was positive, the hybridization is
sequence specific. Similarly, probes that detect the target
sequence in only one of 51 inserts, or in none of the examined
inserts along with controls that confirm that hybridization would
have occurred if the appropriate targets were present in the
clones.
[0176] Thermal stability curves for very short oligonucleotide
hybrids that are 6-8 nucleotides in length are at least 15.degree.
C. lower than for hybrids 11-12 nucleotides in length [FIG. 1 and
Wallace et al., Nucleic Acids Res. 6: 3543-3557 (1979)]. However,
performing the hybridization reaction at a low temperature and with
a very practical 0.4-40 nM concentration of oligonucleotide probe
allows the detection of complementary sequence in a known or
unknown nucleic acid target. To determine an unknown nucleic acid
sequence completely, an entire set containing 65,535 8-mer probes
may be used. Sufficient amounts of nucleic acid for this purpose
are present in convenient biological samples such as a few
microliters of M13 culture, a plasmid prep from 10 ml of bacterial
culture or a single colony of bacteria, or less than 1 ml of a
standard PCR reaction.
[0177] Short oligonucleotides 6-10 nucleotides long give excellent
discrimination. The relative decrease in hybrid stability with a
single end mismatch is greater than for longer probes. Results with
the octamer TGCTCATG support this conclusion. In the experiments,
the target with a G/T end mismatch, hybridization to the target of
this type of mismatch is the most stable of all other types of
oligonucleotide. This discrimination achieved is the same as or
greater than an internal G/T mismatch in a 19 base paired duplex
greater than an internal G/T mismatch in a 19 paired duplex [Ikuta
et al., Nucl. Acids res. 15: 797 (1987)]. Exploiting these
discrimination properties using the described hybridization
conditions for short oligonucleotide hybridization allows a very
precise determination of oligonucleotide targets. In contrast to
the ease of detecting discrimination between perfect and imperfect
hybrids, a problem that may exist with using very short
oligonucleotides is the preparation of sufficient amounts of
hybrids. In practice, the need to discriminate H.sub.p and H.sub.i
is aided by increasing the amount of DNA in the dot and/or the
probe concentration, or by decreasing the hybridization
temperature. However, higher probe concentrations usually increase
background. Moreover, there are limits to the amounts of target
nucleic acid that are practical to use. This problems was solved by
the higher concentration of the detergent Sarcosyl which gave an
effective background with 4 nM of probe. Further improvements may
be effected either in the use of competitors for unspecific binding
of probe to filter, or by changing the hybridization support
material. Moreover, for probes having E.sub.a less than 45 Kcal/mol
(e.g. for many heptamers and a majority of hexamers, modified
oligonucleotides give a more stable hybrid [Asseline, et al., Proc.
Nat'l Acad. Sci. 81: 3297 (1984)] than their unmodified
counterparts. The hybridization conditions described in this
invention for short oligonucleotide hybridization using low
temperatures give better discriminating for all sequences and
duplex hybrid inputs. The only price paid in achieving uniformity
in hybridization conditions for different sequences is an increase
in washing time from minutes to up to 24 hours depending on the
sequence. Moreover, the washing time can be further reduced by
decreasing the salt concentration.
[0178] Although there is excellent discrimination of one matched
hybrid over a mismatched hybrids, in short oligonucleotide
hybridization, signals from mismatched hybrids exist, with the
majority of the mismatch hybrids resulting from end mismatch. This
may limit insert sizes that may be effectively examined by a probe
of a certain length.
[0179] The influence of sequence complexity on discrimination
cannot be ignored. However, the complexity effects are more
significant when defining sequence information by short
oligonucleotide hybridization for specific, nonrandom sequences,
and can be overcome by using an appropriate probe to target length
ratio. The length ratio is chosen to make unlikely, on statistical
grounds, the occurrence of specific sequences which have a number
of end-mismatches which would be able to eliminate or falsely
invert discrimination. Results suggest the use of oligonucleotides
6, 7, and 8 nucleotides in length on target nucleic acid inserts
shorter than 0.6, 2.5, and 10 kb, respectively.
EXAMPLE 11
DNA Sequencing
[0180] An array of subarrays allows for efficient sequencing of a
small set of samples arrayed in the form of replicated subarrays;
For example, 64 samples may be arrayed on a 8.times.8 mm subarray
and 16.times.24 subarrays may be replicated on a 15.times.23 cm
membrane with 1 mm wide spacers between the subarrays. Several
replica membranes may be made. For example, probes from a universal
set of three thousand seventy-two 7-mers may be divided in
thirty-two 96-well plates and labelled by kinasing. Four membranes
may be processed in parallel during one hybridization cycle. On
each membrane, 384 probes may be scored. All probes may be scored
in two hybridization cycles. Hybridization intensities may be
scored and the sequence assembled as described below.
[0181] If a single sample subarray or subarrays contains several
unknowns, especially when similar samples are used, a smaller
number of probes may be sufficient if they are intelligently
selected on the basis of results of previously scored probes. For
example, if probe AAAAAAA is not positive, there is a small chance
that any of 8 overlapping probes are positive. If AAAAAAA is
positive, then two probes are usually positive. The sequencing
process in this case consists of first hybridizing a subset of
minimally overlapped probes to define positive anchors and then to
successively select probes which confirms one of the most likely
hypotheses about the order of anchors and size and type of gaps
between them. In this second phase, pools of 2-10 probes may be
used where each probe is selected to be positive in only one DNA
sample which is different from the samples expected to be positive
with other probes from the pool.
[0182] The subarray approach allows efficient implementation of
probe competition (overlapped probes) or probe cooperation
(continuous stacking of probes) in solving branching problems.
After hybridization of a universal set of probes the sequence
assembly program determines candidate sequence subfragments (SFs).
For the further assembly of SFs, additional information has to be
provided (from overlapped sequences of DNA fragments, similar
sequences, single pass gel sequences, or from other hybridization
or restriction mapping data). Primers for single pass gel
sequencing through the branch points are identified from the SBH
sequence information or from known vector sequences, e.g., the
flanking sequences to the vector insert site, and standard
Sanger-sequencing reactions are performed on the sample DNA. The
sequence obtained from this single pass gel sequencing is compared
to the Sfs that read into and out of the branch points to identify
the order of the Sfs. Further, singel pass gel sequencing may be
combined with SBH to de novo sequence or re-sequence a nucleic
acid.
[0183] Competitive hybridization and continuous stacking
interactions can also be used to assemble Sfs. These approaches are
of limited commercial value for sequencing of large numbers of
samples by SBH wherein a labelled probe is applied to a sample
affixed to an array if a uniform array is used. Fortunately,
analysis of small numbers of samples using replica subarrays allows
efficient implementation of both approaches. On each of the replica
subarrays, one branching point may be tested for one or more DNA
samples using pools of probes similarly as in solving mutated
sequences in different samples spotted in the same subarray (see
above).
[0184] If in each of 64 samples described in this example, there
are about 100 branching points, and if 8 samples are analyzed in
parallel in each subarray, then at least 800 subarray probings
solve all branches. This means that for the 3072 basic probings an
additional 800 probings (25%) are employed. More preferably, two
probings are used for one branching point. If the subarrays are
smaller, less additional probings are used. For example, if
subarrays consist of 16 samples, 200 additional probings may be
scored (6%). By using 7-mer probes (N.sub.1-2B.sub.7N.sub.1-2) and
competitive or collaborative branching solving approaches or both,
fragments of about 1000 bp fragments may be assembled by about 4000
probings. Furthermore, using 8-mer probes (NB.sub.8N) 4 kb or
longer fragments may be assembled with 12,000 probings. Gapped
probes, for example, NB.sub.4NB.sub.3N or NB.sub.4NB.sub.4N may be
used to reduce the number of branching points.
EXAMPLE 12
DNA Analysis by Transient Attachment to Subarrays of Probes and
Ligation of Labelled Probes
[0185] Oligonucleotide probes having an informative length of four
to 40 bases are synthesized by standard chemistry and stored in
tubes or in multiwell plates. Specific sets of probes comprising
one to 10,000 probes are arrayed by deposition or in situ synthesis
on separate supports or distinct sections of a larger support. In
the last case, sections or subarrays may be separated by physical
or hydrophobic barriers. The probe arrays may be prepared by in
situ synthesis. A sample DNA of appropriate size is hybridized with
one or more specific arrays. Many samples may be interrogated as
pools at the same subarrays or independently with different
subarrays within one support. Simultaneously with the sample or
subsequently, a single labelled probe or a pool of labelled probes
is added on each of the subarrays. If attached and labelled probes
hybridize back to back on the complementary target in the sample
DNA they are ligated. Occurrence of ligation will be measured by
detecting a label from the probe.
[0186] This procedure is a variant of the described DNA analysis
process in which DNA samples are not permanently attached to the
support. Transient attachment is provided by probes fixed to the
support. In this case there is no need for a target DNA arraying
process. In addition, ligation allows detection of longer
oligonucleotide sequences by combining short labelled probes with
short fixed probes.
[0187] The process has several unique features. Basically, the
transient attachment of the target allows its reuse. After ligation
occur the target may be released and the label will stay covalently
attached to the support. This feature allows cycling the target and
production of detectable signal with a small quantity of the
target. Under optimal conditions, targets do not need to be
amplified, e.g. natural sources of the DNA samples may be directly
used for diagnostics and sequencing purposes. Targets may be
released by cycling the temperature between efficient hybridization
and efficient melting of duplexes. More preferablly, there is no
cycling. The temperature and concentrations of components may be
defined to have an equilibrium between free targets and targets
entered in hybrids at about 50:50% level. In this case there is a
continuous production of ligated products. For different purposes
different equilibrium ratios are optimal.
[0188] An electric field may be used to enhance target use. At the
beginning, a horizontal field pulsing within each subarray may be
employed to provide for faster target sorting. In this phase, the
equilibrium is moved toward hybrid formation, and unlabelled probes
may be used. After a target sorting phase, an appropriate washing
(which may be helped by a vertical electric field for restricting
movement of the samples) may be performed. Several cycles of
discriminative hybrid melting, target harvesting by hybridization
and ligation and removing of unused targets may be introduced to
increase specificity. In the next step, labelled probes are added
and vertical electrical pulses may be applied. By increasing
temperature, an optimal free and hybridized target ratio may be
achieved. The vertical electric field prevents diffusion of the
sorted targets.
[0189] The subarrays of fixed probes and sets of labelled probes
(specially designed or selected from a universal probe set) may be
arranged in various ways to allow an efficient and flexible
sequencing and diagnostics process. For example, if a short
fragment (about 100-500 bp) of a bacterial genome is to be
partially or completely sequenced, small arrays of probes (5-30
bases in length) designed on the bases of known sequence may be
used. If interrogated with a different pool of 10 labelled probes
per subarray, an array of 10 subarrays each having 10 probes,
allows checking of 200 bases, assuming that only two bases
connected by ligation are scored. Under the conditions where
mismatches are discriminated throughout the hybrid, probes may be
displaced by more than one base to cover the longer target with the
same number of probes. By using long probes, the target may be
interrogated directly without amplification or isolation from the
rest of DNA in the sample. Also, several targets may be analyzed
(screened for) in one sample simultaneously. If the obtained
results indicate occurrence of a mutation (or a pathogen),
additional pools of probes may be used to detect type of the
mutation or subtype of pathogen. This is a desirable feature of the
process which may be very cost effective in preventive diagnosis
where only a small fraction of patients is expected to have an
infection or mutation.
[0190] In the processes described in the examples, various
detection methods may be used, for example, radiolabels,
fluorescent labels, enzymes or antibodies (chemiluminescence),
large molecules or particles detectable by light scattering or
interferometric procedures.
EXAMPLE 13
Sequencing a Target Using Octamers and Nonamers
[0191] Data resulting from the hybridization of octamer and nonamer
oligonucleotides shows that sequencing by hybridization provides an
extremely high degree of accuracy. In this experiment, a known
sequence was used to predict a series of contiguous overlapping
component octamer and nonamer oligonucleotides.
[0192] In addition to the perfectly matching oligonucleotides,
mismatch oligonucleotides, mismatch oligonucleotides wherein
internal or end mismatches occur in the duplex formed by the
oligonucleotide and the target were examined. In these analyses,
the lowest practical temperature was used to maximize hybridization
formation. Washes were accomplished at the same or lower
temperatures to ensure maximal discrimination by utilizing the
greater dissociation rate of mismatch versus matched
oligonucleotide/target hybridization. These conditions are shown to
be applicable to all sequences although the absolute hybridization
yield is shown to be sequence dependent.
[0193] The least destabilizing mismatch that can be postulated is a
simple end mismatch, so that the test of sequencing by
hybridization is the ability to discriminate perfectly matched
oligonucleotide/target duplexes from end-mismatched
oligonucleotide/target duplexes.
[0194] The discriminative values for 102 of 105 hybridizing
oligonucleotides in a dot blot format were greater than 2 allowing
a highly accurate generation of the sequence. This system also
allowed an analysis of the effect of sequence on hybridization
formation and hybridization instability.
[0195] One hundred base pairs of a known portion of a
human-interferon genes prepared by PCR, i.e. a 100 bp target
sequence, was generated with data resulting from the hybridization
of 105 oligonucleotides probes of known sequence to the target
nucleic acid. The oligonucleotide probes used included 72 octamer
and 21 nonamer oligonucleotides whose sequence was perfectly
complementary to the target. The set of 93 probes provided
consecutive overlapping frames of the target sequence e displaced
by one or two bases.
[0196] To evaluate the effect of mismatches, hybridization was
examined for 12 additional probes that contained at least one end
mismatch when hybridized to the 100 bp test target sequence. Also
tested was the hybridization of twelve probes with target
end-mismatched to four other control nucleic acid sequences chosen
so that the 12 oligonucleotides formed perfectly matched duplex
hybrids with the four control DNAs. Thus, the hybridization of
internal mismatched, end-mismatched and perfectly matched duplex
pairs of oligonucleotide and target were evaluated for each
oligonucleotide used in the experiment. The effect of absolute DNA
target concentration on the hybridization with the test octamer and
nonamer oligonucleotides was determined by defining target DNA
concentration by detecting hybridization of a different
oligonucleotide probe to a single occurrence non-target site within
the co-amplified plasmid DNA.
[0197] The results of this experiment showed that all
oligonucleotides containing perfect matching complementary sequence
to the target or control DNA hybridized more strongly than those
oligonucleotides having mismatches. To come to this conclusion, we
examined H.sub.p and D values for each probe. H.sub.p defines the
amount of hybrid duplex formed between a test target and an
oligonucleotide probe. By assigning values of between 0 and 10 to
the hybridization obtained for the 105 probes, it was apparent that
68.5% of the 105 probes had an H.sub.p greater than 2.
[0198] Discrimination (D) values were obtained where D was defined
as the ratio of signal intensities between 1) the dot containing a
perfect matched duplex formed between test oligonucleotide and
target or control nucleic acid and 2) the dot containing a mismatch
duplex formed between the same oligonucleotide and a different site
within the target or control nucleic acid. Variations in the value
of D result from either 1) perturbations in the hybridization
efficiency which allows visualization of signal over background, or
2) the type of mismatch found between the test oligonucleotide and
the target. The D values obtained in this experiment were between 2
and 40 for 102 of the 105 oligonucleotide probes examined.
Calculations of D for the group of 102 oligonucleotides as a whole
showed the average D was 10.6.
[0199] There were 20 cases where oligonucleotide/target duplexes
exhibited an end-mismatch. In five of these, D was greater than 10.
The large D value in these cases is most likely due to
hybridization destabilization caused by other than the most stable
(G/T and G/A) end mismatches. The other possibility is there was an
error in the sequence of either the oligonucleotides or the
target.
[0200] Error in the target for probes with low H.sub.p was excluded
as a possibility because such an error would have affected the
hybridization of each of the other eight overlapping
oligonucleotides. There was no apparent instability due to sequence
mismatch for the other overlapping oligonucleotides, indicating the
target sequence was correct. Error in the oligonucleotide sequence
was excluded as a possibility after the hybridization of seven
newly synthesized oligonucleotides was re-examined. Only 1 of the
seven oligonucleotides resulted in a better D value. Low hybrid
formation values may result from hybrid instability or from an
inability to form hybrid duplex. An inability to form hybrid
duplexes would result from either 1) self complementarity of the
chosen probe or 2) target/target self hybridization.
Oligonucleotide/oligonucleo- tide duplex formation may be favored
over oligonucleotide/target hybrid duplex formation if the probe
was self-complementary. Similarly, target/target association may be
favored if the target was self-complementary or may form internal
palindromes. In evaluating these possibilities, it was apparent
from probe analysis that the questionable probes did not form
hybrids with themselves. Moreover, in examining the contribution of
target/target hybridization, it was determined that one of the
questionable oligonucleotide probes hybridized inefficiently with
two different DNAs containing the same target. The low probability
that two different DNAs have a self-complementary region for the
same target sequence leads to the conclusion that target/target
hybridization did not contribute to low hybridization formation.
Thus, these results indicate that hybrid instability and not the
inability to form hybrids was the cause of the low hybrid formation
observed for specific oligonucleotides. The results also indicate
that low hybrid formation is due to the specific sequences of
certain oligonucleotides. Moreover, the results indicate that
reliable results may be obtained to generate sequences if octamer
and nonamer oligonucleotides are used.
[0201] These results show that using the methods described long
sequences of any specific target nucleic acid may be generated by
maximal and unique overlap of constituent oligonucleotides. Such
sequencing methods are dependent on the content of the individual
component oligomers regardless of their frequency and their
position.
[0202] The sequence which is generated using the algorithm
described below is of high fidelity. The algorithm tolerates false
positive signals from the hybridization dots as is indicated from
the fact the sequence generated from the 105 hybridization values,
which included four less reliable values, was correct. This
fidelity in sequencing by hybridization is due to the "all or none"
kinetics of short oligonucleotide hybridization and the difference
in duplex stability that exists between perfectly matched duplexes
and mismatched duplexes. The ratio of duplex stability of matched
and end-mismatched duplexes increases with decreasing duplex
length. Moreover, binding energy decreases with decreasing duplex
length resulting in a lower hybridization efficiency. However, the
results provided show that octamer hybridization allows the
balancing of the factors affecting duplex stability and
discrimination to produce a highly accurate method of sequencing by
hybridization. Results presented in other examples show that
oligonucleotides that are 6, 7, or 8 nucleotides can be effectively
used to generate reliable sequence on targets that are 0.5 kb (for
hexamers) 2 kb (for septamers) and 6 kb (for octamers). The
sequence of long fragments may be overlapped to generate a complete
genome sequence.
EXAMPLE 14
Analyzing the Data Obtained
[0203] Image files are analyzed by an image analysis program, like
DOTS program (Drmanac et al., 1993), and scaled and evaluated by
statistical functions included, e.g., in SCORES program (Drmanac et
al. 1994). From the distribution of the signals an optimal
threshold is determined for transforming signal into +/- output.
From the position of the label detected, F+P nucleotide sequences
from the fragments would be determined by combining the known
sequences of the immobilized and labeled probes corresponding to
the labeled positions. The complete nucleic acid sequence or
sequence subfragments of the original molecule, such as a human
chromosome, would then be assembled from the overlapping F+P
sequence determined by computational deduction.
[0204] One option is to transform hybridization signals e.g.,
scores, into +/- output during the sequence assembly process. In
this case, assembly will start with a F+P sequence with a very high
score, for example F+P sequence AAAAAATTTTTT . Scores of all four
possible overlapping probes AAAAATTTTTTA, AAAAATTTTTT, AAAAATTTTTTC
and AAAAATTTTTTG and three additional probes that are different at
the beginning (TAAAAATTTTTT,; CAAAAATTTTTT, ; GAAAAATTTTTT, are
compared and three outcomes defined: (i) only the starting probe
and only one of the four overlapping proves have scores that are
significantly positive relatively to the other six probes, in this
case the AAAAAATTTTTT sequence will be extended for one nucleotide
to the right; (ii) no one probe except the starting probe has a
significantly positive score, assembly will stop, e.g., the
AAAAAATTTTT sequence is at the end of the DNA molecule that is
sequenced; (iii) more than one significantly positive probe among
the overlapped and/or other three probes is found; assembly is
stopped because of the error or branching (Drmanac et al.,
1989).
[0205] The processes of computational deduction would employ
computer programs using existing algorithms (see, e.g., Pevzner,
1989; Drmanac et al., 1991; Labat and Drmanac, 1993; each
incorporated herein by reference).
[0206] If, in addition to F+P, F (space 1)P, F (space 2)P, F(space
3)P or F(space 4)P are determined, algorithms will be used to match
all data sets to correct potential errors or to solve the situation
where there is a branching problem (see, e.g., Drmanac et al.,
1989; Bains et al., 1988; each incorporated herein by
reference).
EXAMPLE 15
Conducting Sequencing by Two Step Hybridization
[0207] Following the certain examples to describe the execution of
the sequencing methodology contemplated by the inventor. First, the
whole chip would be hybridized with mixture of DNA as complex as
100 million of bp (one human chromosome). Guidelines for conducting
hybridization can be found in papers such as Drmanac et al. (1990);
Khrapko et al. (1991); and Broude et al. (1994). These articles
teach the ranges of hybridization temperatures, buffers and washing
steps that are appropriate for use in the initial steps of Format 3
SBH.
[0208] The present inventor particularly contemplates that
hybridization is to be carried out for up to several hours in high
salt concentrations at a low temperature (-2.degree. C. to
5.degree. C.) because of a relatively low concentration of target
DNA that can be provided. For this purpose, SSC buffer is used
instead of sodium phosphate buffer (Drmanac et al, 1990), which
precipitates at 10.degree. C. Washing does not have to be extensive
(a few minutes) because of the second step, and can be completely
eliminated when the hybridization cycling is used for the
sequencing of highly complex DNA samples. The same buffer is used
for hybridization and washing steps to be able to continue with the
second hybridization step with labeled probes.
[0209] After proper washing using a simple robotic device on each
array, e.g., a 8.times.8 mm array, one labeled, probe, e.g., a
6-mer, would be added. A 96-tip or 96-pin device would be used,
performing this in 42 operations. Again, a range of discriminatory
conditions could be employed, as previously described in the
scientific literature.
[0210] The present inventor particularly contemplates the use of
the following conditions. First, after adding labeled probes and
incubating for several minutes only (because of the high
concentration of added oligonucleotides) at a low temperature
(0-5.degree. C.), the temperature is increased to 3-10.degree. C.,
depending on F+P length, and the washing buffer is added. At this
time, the washing buffer used is one compatible with any ligation
reaction (e.g., 100 mM salt concentration range). After adding
ligase, the temperate is increased again to 15-37.degree. C. to
allow fast ligation (less than 30 min) and further discrimination
of full match and mismatch hybrids.
[0211] The use of cationic detergents is also contemplated for use
in Format 3 SBH, as described by Pontius & Berg (1991,
incorporated herein by reference). These authors describe the use
of two simple cationic detergents, dodecy-and
cetyltrimethylammonium bromide (DTAB and CTAB) in DNA
renaturation.
[0212] DTAB and CTAB are variants of the quaternary amine
tetramethylammonium bromide (TMAB) in which one of the methyl
groups is replaced by either a 12-carbon (DTAB) or a 16-carbon
(CTAB) alkyl group. TMAB is the bromide salt of the
tetramethylammonium ion, a reagent used in nucleic acid
renaturation experiments to decrease the G-C content bias of the
melting temperature. DTAB and CTAB are similar in structure to
sodium dodecyl sulfate (SDS), with the replacement of the
negatively charged sulfate of SDS by a positively charged
quaternary amine. While SDS is commonly used in hybridization
buffers to reduce nonspecific binding and inhibit nucleases, it
does not greatly affect the rate of renaturation.
[0213] When using a ligation process, the enzyme could be added
with the labeled probes or after the proper washing step to reduce
the background. Although not previously proposed for use in any SBH
method, ligase technology is well established within the field of
molecular biology. For example, Hood and colleagues described a
ligase-mediated gene detection technique (Landegren et al., 1988),
the methodology of which can be readily adapted for use in Format 3
SBH. Wu & Wallace also describe the use of bacteriophage T4 DNA
ligase to join two adjacent, short synthetic olignucleotides. Their
oligo ligation reactions were carried out in 50 mM Tris HCl pH 7.6,
10 mM MgCl.sub.2, 1 mM ATP, 1 mM DTT, and 5% PEG. Ligation
reactions were heated to 100.degree. C. for 5-10 min followed by
cooling to 0.degree. C. prior to the addition of T4 DNA ligase (1
unit; Bethesda Research Laboratory). Most ligation reactions were
carried out at 30.degree. C. and terminated by heating to
100.degree. C. for 5 min.
[0214] Final washing appropriate for discriminating detection of
hybridized adjacent, or ligated, oligonucleotides of length (F+P),
is then performed. This washing step is done in water for several
minutes at 40-60.degree. C. to wash out all the non-ligated labeled
probes, and all other compounds, to maximally reduce background.
Because of the covalently bound labeled oligonucleotides, detection
is simplified (it does not have time and low temperature
constrains).
[0215] Depending on the label used, imaging of the chips is done
with different apparati. For radioactive labels, phosphor storage
screen technology and PhosphorImager as a scanner may be used
(Molecular Dynamics, Sunnyvale, Calif.). Chips are put in a
cassette and covered by a phosphorous screen. After 1-4 hours of
exposure, the screen is scanned and the image file stored at a
computer hard disc. For the detection of fluorescent labels, CCD
cameras and epifluorescent or confocal microscopy are used. For the
chips generated directly on the pixels of a CCD camera, detection
can be performed as described by Eggers et aL (1994, incorporated
herein by reference).
[0216] Charge-coupled device (CCD) detectors serve as active solid
supports that quantitatively detect and image the distribution of
labeled target molecules in probe-based assays. These devices use
the inherent characteristics of microelectronics that accommodate
highly parallel assays, ultrasensitive detection, high throughput,
integrated data acquisition and computation. Eggers et al. (1994)
describe CCDs for use with probe-based assays, such as Format 3 SBH
of the present invention, that allow quantitative assessment within
seconds due to the high sensitivity and direct coupling
employed.
[0217] The integrated CCD detection approach enables the detection
of molecular binding events on chips. The detector rapidly
generates a two-dimensional pattern that uniquely characterizes the
sample. In the specific operation of the CCD-based molecular
detector, distinct biological probes are immobilized directly on
the pixels of a CCD or can be attached to a disposable cover slip
placed on the CCD surface. The sample molecules can be labeled with
radioisotope, chemiluminescent or fluorescent tags.
[0218] Upon exposure of the sample to the CCD-based probe array,
photons or radioisotope decay products are emitted at the pixel
locations where the sample has bound, in the case of Format 3, to
two complementary probes. In turn, electron-hole pairs are
generated in the silicon when the charged particles, or radiation
from the labeled sample, are incident on the CCD gates. Electrons
are then collected beneath adjacent CCD gates and sequentially read
out on a display module. The number of photoelectrons generated at
each pixel is directly proportional to the number of molecular
binding events in such proximity. Consequently, molecular binding
can be quantitatively determined (Eggers et al., 1994).
[0219] By placing the imaging array in proximity to the sample, the
collection efficiency is improved by a factor of at least 10 over
lens-based techniques such as those found in conventional CCD
cameras. That is, the sample (emitter) is in near contact with the
detector (imaging array), and this eliminates conventional imaging
optics such as lenses and mirrors.
[0220] When radioisotopes are attached as reporter groups to the
target molecules, energetic particles are detected. Several
reporter groups that emit particles of varying energies have been
successfully utilized with the micro-fabricated detectors,
including .sup.32P, .sup.33P, .sup.35S, .sup.14C and .sup.125L. The
higher energy particles, such as from .sup.32P, provide the highest
molecular detection sensitivity, whereas the lower energy
particles, such as from .sup.35S, provide better resolution. Hence
the choice of the radioisotope reporter can be tailored as
required. Once the particular radioisotope label is selected, the
detection performance can be predicted by calculating the
signal-to-noise ration (SNR), as described by Eggers et al.
(1994).
[0221] An alternative luminescent detection procedure involves the
use of fluorescent or chemiluminescent reporter groups attached to
the target molecules. The fluorescent labels can be attached
covalently or through interaction. Fluorescent dyes, such as
ethidium bromide, with intense absorption bands in the near UV
(300-350 nm) range and principal emission bands in the visible
(500-650 nm) range, are most suited for the CCD devices employed
since the quantum efficiency is several orders of magnitude lower
at the excitation wavelength then at the fluorescent signal
wavelength.
[0222] From the perspective of detecting luminescence, the
polysilicon CCD gates have the built-in capacity to filter away the
contribution of incident light in the UV range, yet are very
sensitive to the visible luminescence generated by the fluorescent
reporter groups. Such inherently large discrimination against UV
excitation enables large SNRs (greater than 100) to be achieved by
the CCDs as formulated in the incorporated paper by Eggers et al.
(1994).
[0223] For probe immobilization on the detector, hybridization
matrices may be produced on inexpensive SiO.sub.2 wafers, which are
subsequently placed on the surface of the CCD following
hybridization and drying. This format is economically efficient
since the hybridization of the DNA is conducted on inexpensive
disposable SiO.sub.2 wafers, thus allowing reuse of the more
expensive CCD detector. Alternatively, the probes can be
immobilized directly on the CCD to create a dedicated probe
matrix.
[0224] To immobilize probes upon the SiO.sub.2 coating, a uniform
epoxide layer is linked to the film surface, employing an
epoxy-silane reagent and standard SiO.sub.2 modification chemistry.
Amine-modified oligonucleotide probes are then linked to the
SiO.sub.2 surface by means of secondary amine formation with the
epoxide ring. The resulting linkage provides 17 rotatable bonds of
separation between the 3 base of the oligonucleotide and the
SiO.sub.2 surface. To ensure complete amine deprotonation and to
minimize secondary structure formation during coupling, the
reaction is performed in 0.1 M KOH and incubated at 37.degree. C.
for 6 hours.
[0225] In Format 3 SBH in general, signals are scored per each of
billion points. It would not be necessary to hybridize all arrays,
e.g., 4000 5.times.5 mm, at a time and the successive use of
smaller number of arrays is possible.
[0226] Cycling hybridizations are one possible method for
increasing the hybridization signal. In one cycle, most of the
fixed probes will hybridize with DNA fragments with tail sequences
non-complementary for labeled probes. By increasing the
temperature, those hybrids will be melted. In the next cycle, some
of them (.about.0.1%) will hybridize with an appropriate DNA
fragment and additional labeled probes will be ligated. In this
case, there occurs a discriminative melting of DNA hybrids with
mismatches for both probe sets simultaneously.
[0227] In the cycle hybridization, all components are added before
the cycling starts, at the 37.degree. C. for T4, or a higher
temperature for a thermostable ligase. Then the temperature is
decreased to 15-37.degree. C. and the chip is incubated for up to
10 minutes, and then the temperature is increased to 37.degree. C.
or higher for a few minutes and then again reduced. Cycles can be
repeated up to 10 times.
[0228] In one variant, an optimal higher temperature (10-50.degree.
C.) can be used without cycling and longer ligation reaction can be
performed (1-3 hours).
[0229] The procedure described herein allows complex chip
manufacturing using standard synthesis and precise spotting of
oligonucleotides because a relatively small number of
oligonucleotides are necessary. For example, if all 7-mer oligos
are synthesized (16384 probes), lists of 256 million 14-mers can be
determined.
[0230] One important variant of the invented method is to use more
than one differently labeled probe per base array. This can be
executed with two purposes in mind; multiplexing to reduce number
of separately hybridized arrays; or to determine a list of even
longer oligosequences such as 3.times.6 or 3.times.7. In this case,
if two labels are used, the specificity of the 3 consecutive
oligonucleotides can be almost absolute because positive sites must
have enough signals of both labels.
[0231] A further and additional variant is to use chips containing
BxNy probes with y being from 1 to 4. Those chips allow sequence
reading in different frames. This can also be achieved by using
appropriate sets of labeled probes or both F and P probes could
have some unspecified end positions (i.e., some element of terminal
degeneracy). Universal bases may also be employed as part of a
linker to join the probes of defined sequence to the solid support.
This makes the probe more available to hybridization and makes the
construct more stable. If a probe has 5 bases, one may, e.g., use 3
universal bases as a linker (FIG. 4 +L).
EXAMPLE 16
Determining Sequence From Hybridization Data
[0232] Sequence assembly may be interrupted where ever a given
overlapping (N-1) mer is duplicated two or more times. Then either
of the two N-mers differing in the last nucleotide may be used in
extending the sequence. This branching point limits unambiguous
assembly of sequence.
[0233] Reassembling the sequence of known oligonucleotides that
hybridize to the target nucleic acid to generate the complete
sequence of the target nucleic acid may not be accomplished in some
cases. This is because some information may be lost if the target
nucleic acid is not in fragments of appropriate size in relation to
the size of oligonucleotide that is used for hybridizing. The
quantity of information lost is proportional to the length of a
target being sequenced. However, if sufficiently short targets are
used, their sequence msy be unambiguously determined.
[0234] The probable frequency of duplicated sequences that would
interfere with sequence assembly which is distributed along a
certain length of DNA may be calculated. This derivation requires
the introduction of the definition of a parameter having to do with
sequence organization: the sequence subfragment (SF). A sequence
subfragment results if any part of the sequence of a target nucleic
acid starts and ends with an (N-1)mer that is repeated two or more
times within the target sequence. Thus, subfragments are sequences
generated between two points of branching in the process of
assembly of the sequences in the method of the invention. The sum
of all subfragments is longer than the actual target nucleic acid
because of overlapping short ends. Generally, subfragments may not
be assembled in a linear order without additional information since
they have shared (N-1)mers at their ends and starts. Different
numbers of subfragments are obtained for each nucleic acid target
depending on the number of its repeated (N-1) mers. The number
depends on the value of N-1 and the length of the target.
[0235] Probability calculations can estimate the interrelationship
of the two factors. If the ordering of positive N-mers is
accomplished by using overlapping sequences of length N-1 or at an
average distance of A.sub.o, the N-1 of a fragment Lf bases long is
given by equation one:
N.sub.sf=1+A.sub.oXKXP(K,L.sub.f)
[0236] Where K greater than or=2, and P (K, L.sub.f) represents the
probability of an N-mer occurring K-times on a fragment L.sub.f
base long. Also, a computer program that is able to form
subfragments from the content of N-mers for any given sequence is
described below in Example 18.
[0237] The number of subfragments increases with the increase of
lengths of fragments for a given length of probe. Obtained
subfragments may not be uniquely ordered among themselves. Although
not complete, this information is very useful for comparative
sequence analysis and the recognition of functional sequence
characteristics. This type of information may be called partial
sequence. Another way of obtaining partial sequence is the use of
only a subset of oligonucleotide probes of a given length.
[0238] There may be relatively good agreement between predicted
sequence according to theory and a computer simulation for a random
DNA sequence. For instance, for N-1=7, [using an 8-mer or groups of
sixteen 10-mers of type 5' (A,T,C,G)B.sub.8 (A,T,C,G) 3'] a target
nucleic acid of 200 bases will have an average of three
subfragments. However, because of the dispersion around the mean, a
library of target nucleic acid should have inserts of 500 bp so
that less than 1 in 2000 targets have more than three subfragments.
Thus, in an ideal case of sequence determination of a long nucleic
acid of random sequence, a representative library with sufficiently
short inserts of target nucleic acid may be used. For such inserts,
it is possible to reconstruct the individual target by the method
of the invention. The entire sequence of a large nucleic acid is
then obtained by overlapping of the defined individual insert
sequences.
[0239] To reduce the need for very short fragments, e.g. 50 bases
for 8-mer probes. The information contained in the overlapped
fragments present in every random DNA fragmentation process like
cloning, or random PCR is used. It is also possible to use pools of
short physical nucleic acid fragments. Using 8-mers or 11-mers like
5' (A,T,C,G) N.sub.8 (A,T,C,G )3' for sequencing 1 megabase,
instead of needing 20,000 50 bp fragments only 2,100 samples are
sufficient. This number consists of 700 random 7 kb clones (basic
library), 1250 pools of 20 clones of 500 bp (subfragments ordering
library) and 150 clones from jumping (or similar) library. The
developed algorithm (see Example 18) regenerates sequence using
hybridization data of these described samples.
EXAMPLE 17
Algorithm
[0240] This example describes an algorithm for generation of a long
sequence written in a four letter alphabet from constituent k-tuple
words in a minimal number of separate, randomly defined fragments
of a starting nucleic acid sequence where K is the length of an
oligonucleotide probe. The algorithm is primarily intended for use
in the sequencing by hybridization (SBH) process. The algorithm is
based on subfragments (SF), informative fragments (IF) and the
possibility of using pools of physical nucleic sequences for
defining informative fragments.
[0241] As described, subfragments may be caused by branch points in
the assembly process resulting from the repetition of a K-1
oligomer sequence in a target nucleic acid. Subfragments are
sequence fragments found between any two repetitive words of the
length K-1 that occur in a sequence. Multiple occurrences of K-1
words are the cause of interruption of ordering the overlap of
K-words in the process of sequence generation. Interruption leads
to a sequence remaining in the form of subfragments. Thus, the
unambiguous segments between branching points whose order is not
uniquely determined are called sequence subfragments.
[0242] Informative fragments are defined as fragments of a sequence
that are determined by the nearest ends of overlapped physical
sequence fragments.
[0243] A certain number of physical fragments may be pooled without
losing the possibility of defining informative fragments. The total
length of randomly pooled fragments depends on the length of
k-tuples that are used in the sequencing process.
[0244] The algorithm consists of two main units. The first part is
used for generation of subfragments from the set of k-tuples
contained in a sequence. Subfragments may be generated within the
coding region of physical nucleic acid sequence of certain sizes,
or within the informative fragments defined within long nucleic
acid sequences. Both types of fragments are members of the basic
library. This algorithm does not describe the determination of the
content of the k-tuples of the informative fragments of the basic
library, i.e. the step of preparation of informative fragments to
be used in the sequence generation process.
[0245] The second part of the algorithm determines the linear order
of obtained subfragments with the purpose of regenerating the
complete sequence of the nucleic acid fragments of the basic
library. For this purpose a second, ordering library is used, made
of randomly pooled fragments of the starting sequence. The
algorithm does not include the step of combining sequences of basic
fragments to regenerate an entire, megabase plus sequence. This may
be accomplished using the link-up of fragments of the basic library
which is a prerequisite for informative fragment generation.
Alternatively, it may be accomplished after generation of sequences
of fragments of the basic library by this algorithm, using search
for their overlap, based on the presence of common
end-sequences.
[0246] The algorithm requires neither knowledge of the number of
appearances of a given k-tuple in a nucleic acid sequence of the
basic and ordering libraries, nor does it require the information
of which k-tuple words are present on the ends of a fragment. The
algorithm operates with the mixed content of k-tuples of various
length. The concept of the algorithm enables operations with the
k-tuple sets that contain false positive and false negative
k-tuples. Only in specific cases does the content of the false
k-tuples primarily influence the completeness and correctness of
the generated sequence. The algorithm may be used for optimization
of parameters in simulation experiments, as well as for sequence
generation in the actual SBH experiments e.g. generation of the
genomic DNA sequence. In optimization of parameters, the choice of
the oligonucleotide probes (k-tuples) for practical and convenient
fragments and/or the choice of the optimal lengths and the number
of fragments for the defined probes are especially important.
[0247] This part of the algorithm has a central role in the process
of the generation of the sequence from the content of k-tuples. It
is based on the unique ordering of k-tuples by means of maximal
overlap. The main obstacles in sequence generation are specific
repeated sequences and false positive and/or negative k-tuples. The
aim of this part of the algorithm is to obtain the minimal number
of the longest possible subfragments, with correct sequence. This
part of the algorithm consists of one basic, and several control
steps. A two-stage process is necessary since certain information
can be used only after generation of all primary subfragments.
[0248] The main problem of sequence generation is obtaining a
repeated sequence from word contents that by definition do not
carry information on the number of occurrences of the particular
k-tuples. The concept of the entire algorithm depends on the basis
on which this problem is solved. In principle, there are two
opposite approaches: 1) repeated sequences may be obtained at the
beginning, in the process of generation of pSFs, or 2) repeated
sequences can be obtained later, in the process of the final
ordering of the subfragments. In the first case, pSFs contain an
excess of sequences and in the second case, they contain a deficit
of sequences. The first approach requires elimination of the excess
sequences generated, and the second requires permitting multiple
use of some of the subfragments in the process of the final
assembling of the sequence.
[0249] The difference in the two approaches in the degree of
strictness of the rule of unique overlap of k-tuples. The less
severe rule is: k-tuple X is unambiguously maximally overlapped
with k-tuple Y if and only if, the rightmost k-1 end of k-tuple X
is present only on the leftmost end of k-tuple Y. This rule allows
the generation of repetitive sequences and the formation of surplus
sequences.
[0250] A stricter rule which is used in the second approach has an
addition caveat: k-tuple X is unambiguously maximally overlapped
with k-tuple Y if and only if, the rightmost K-1 end of k-tuple X
is present only on the leftmost end of k-tuple Y and if the
leftmost K-I end of k-tuple Y is not present on the rightmost end
of any other k-tuple. The algorithm based on the stricter rule is
simpler, and is described herein.
[0251] The process of elongation of a given subfragment is stopped
when the right k-1 end of the last k-tuple included is not present
on the left end of any k-tuple or is present on two or more
k-tuples. If it is present on only one k-tuple the second part of
the rule is tested. If in addition there is a k-tuple which differs
from the previously included one, the assembly of the given
subfragment is terminated only on the first leftmost position. If
this additional k-tuple does not exist, the conditions are met for
unique k-1 overlap and a given subfragment is extended to the right
by one element.
[0252] Beside the basic rule, a supplementary one is used to allow
the usage of k-tuples of different lengths. The maximal overlap is
the length of k-1 of the shorter k-tuple of the overlapping pair.
Generation of the pSFs is performed starting from the first k-tuple
from the file in which k-tuples are displayed randomly and
independently from their order in a nucleic acid sequence. Thus,
the first k-tuple in the file is not necessarily on the beginning
of the sequence, nor on the start of the particular subfragment.
The process of subfragrnent generation is performed by ordering the
k-tuples by means of unique overlap, which is defined by the
described rule. Each used k-tuple is erased from the file. At the
point when there are no further k-tuples unambiguously overlapping
with the last one included, the building of subfragment is
terminated and the buildup of another pSF is started. Since
generation of a majority of subfragments does not begin from their
actual starts, the formed pSF are added to the k-tuple file and are
considered as a longer k-tuple. Another possibility is to form
subfragments going in both directions from the starting k-tuple.
The process ends when further overlap, i.e. the extension of any of
the subfragments, is not possible.
[0253] The pSFs can be divided in three groups: 1) Subfragments of
the maximal length and correct sequence in cases of exact k-tuple
set; 2) short subfragments, formed due to the used of the maximal
and unambiguous overlap rule on the incomplete set, and/or the set
with some false positive k-tuples; and 3) pSFs of an incorrect
sequence. The incompleteness of the set in 2) is caused by false
negative results of a hybridization experiment, as well as by using
an incorrect set of k-tuples. These are formed due to the false
positive and false negative k-tuples and can be: a) misconnected
subfragments; b) subfragments with the wrong end; and c) false
positive k-tuples which appears as false minimal subfragments.
[0254] Considering false positive k-tuples, there is the
possibility for the presence of a k-tuple containing more than one
wrong base or containing one wrong base somewhere in the middle, as
well as the possibility for a k-tuple with a wrong base on the end.
Generation of short, erroneous or misconnected subfragments is
caused by the latter k-tuples. The k-tuples of the former two kinds
represent wrong pSFs with length equal to k-tuple length.
[0255] In the case of one false negative k-tuple, pSFs are
generated because of the impossibility of maximal overlapping. In
the case of the presence of one false positive k-tuple with the
wrong base on its leftmost or rightmost end, pSFs are generated
because of the impossibility of unambiguous overlapping. When both
false positive and false negative k-tuples with a common k-1
sequence are present in the file, pSFs are generated, and one of
these pSFs contains the wrong k-tuple at the relevant end.
[0256] The process of correcting subfragments with errors in
sequence and the linking of unambiguously connected pSF is
performed after subfragment generation and in the process of
subfragment ordering. The first step which consists of cutting the
misconnected pSFs and obtaining the final subfragments by
unambiguous connection of pSFs is described below.
[0257] There are two approaches for the formation of misconnected
subfragments. In the first a mistake occurs when an erroneous
k-tuple appears on the points of assembly of the repeated sequences
of lengths k-1. In the second, the repeated sequences are shorter
than k-1. These situations can occur in two variants each. In the
first variant, one of the repeated sequences represents the end of
a fragment. In the second variant, the repeated sequence occurs at
any position within the fragment. For the first possibility, the
absence of some k-tuples from the file (false negatives) is
required to generate a misconnection. The second possibility
requires the presence of both false negative and false positive
k-tuples in the file. Considering the repetitions of k-1 sequence,
the lack of only one k-tuple is sufficient when either end is
repeated internally. The lack of two is needed for strictly
internal repetition. The reason is that the end of a sequence can
be considered informatically as an endless linear array of false
negative k-tuples. From the "smaller than k-1 case", only the
repeated sequence of the length of k-2, which requires two or three
specific erroneous k-tuples, will be considered. It is very likely
that these will be the only cases which will be detected in a real
experiment, the others being much less frequent.
[0258] Recognition of the misconnected subfragments is more
strictly defined when a repeated sequence does not appear at the
end of the fragment. In this situation, one can detect further two
subfragments, one of which contains on its leftmost, and the other
on its rightmost end k-2 sequences which are also present in the
misconnected subfragment. When the repeated sequence is on the end
of the fragment, there is only one subfragment which contains k-2
sequence causing the mistake in subfragment formation on its
leftmost or rightmost end.
[0259] The removal of misconnected subframents by their cutting is
performed according to the common rule: If the leftmost or
rightmost sequence of the length of k-2 of any subfragments is
present in any other subfragment, the subfragment is to be cut into
two subfragments, each of them containing k-2 sequence. This rule
does not cover rarer situations of a repeated end when there are
more than one false negative k-tuple on the point of repeated k-1
sequence. Misconnected subfragments of this kind can be recognized
by using the information from the overlapped fragments, or
informative fragments of both the basic and ordering libraries. In
addition, the misconnected subfragment will remain when two or more
false negative k-tuples occur on both positions which contain the
identical k-1 sequence. This is a very rare situation since it
requires at least 4 specific false k-tuples. An additional rule can
be introduced to cut these subfragments on sequences of length k if
the given sequence can be obtained by combination of sequences
shorter than k-2 from the end of one subfragment and the start of
another.
[0260] By strict application of the described rule, some
completeness is lost to ensure the accuracy of the output. Some of
the subfragments will be cut although they are not misconnected
since they fit into the pattern of a misconnected subfragment.
There are several situations of this kind. For example, a fragment,
beside at least two identical k-1 sequences, contains any k-2
sequence from k-1 or a fragment contains k-2 sequence repeated at
least twice and at least one false negative k-tuple containing
given k-2 sequence in the middle, etc.
[0261] The aim of this part of the algorithm is to reduce the
number of pSFs to a minimal number of longer subfragments with
correct sequence. The generation of unique longer subfragments or a
complete sequence is possible in two situations. The first
situation concerns the specific order of repeated k-1 words. There
are cases in which some or all maximally extended pSFs (the first
group of pSFs) can be uniquely ordered. For example, in fragment
S-R1-a-R2-b-R1-c-R2-E where S and E are the start and end of a
fragment, a, b , and c are different sequences specific to
respective subfragments and R1 and R2 are two k-1 sequences that
are tandemly repeated, five subfragments are generated
(S-R1,R1-a-R2, R2-b-R1, R1-c-R2, and R-E). They may be ordered in
two ways; the original sequence above or S-R1-c-R-b-R1-a-R-E. In
contrast, in a fragment with the same number and types of repeated
sequences but ordered differently, i.e. S-R1-a-R1-b-R-c-R-E, there
is no other sequence which includes all subfragments. Examples of
this type can be recognized only after the process of generation of
pSFs. They represent the necessity for two steps in the process of
pSF generation. The second situation of generation of false short
subfragments on positions of nonrepeated k-1 sequences when the
files contain false negative and/or positive k-tuples is more
important.
[0262] The solution for both pSF groups consists of two parts.
First, the false positive k-tuples appearing as the nonexisting
minimal subfragments are eliminated. All k-tuple subfragments of
length k which do not have an overlap on either end, of the length
of longer than k-a on one end and longer than k-b on the other end,
are eliminated to enable formation of the maximal number of
connections. In our experiments, the values for a and b of 2 and 3,
respectively, appeared to be adequate to eliminate a sufficient
number of false positive k-tuples.
[0263] The merging of subfragments that can be uniquely connected
is accomplished in the second step. The rule for connection is: two
subfragments may be unambiguously connected if, and only if, the
overlapping sequence at the relevant end or start of two
subfragments is not present at the start and/or end of any other
subfragment.
[0264] The exception is if one subfragment from the considered pair
has the identical beginning and end. In that case connection is
permitted, even if there is another subfragrnent with the same end
present in the file. The main problem here is the precise
definition of overlapping sequence. The connection is not permitted
if the overlapping sequence unique for only one pair of
subfragments is shorter than k-2, of it is k-2 or longer but an
additional subfragment exists with the overlapping sequence of any
length longer than k-4. Also, both the canonical ends of pSFs and
the ends after omitting one (or few) last bases are considered as
the overlapping sequences.
[0265] After this step some false positive k-tuples (as minimal
subfragments) and some subfragments with a wrong end may survive.
In addition, in very rare occasions where a certain number of some
specific false k-tuples are simultaneously present, an erroneous
connection may take place. These cases will be detected and solved
in the subfragment ordering process, and in the additional control
steps along with the handling of uncut "misconnected"
subfragments.
[0266] The short subfragments that are obtained are of two kinds.
In the common case, these subfragments may be unambiguously
connected among themselves because of the distribution of repeated
k-1 sequences. This may be done after the process of generation of
pSFs and is a good example of the necessity for two steps in the
process of pSF generation. In the case of using the file containing
false positive and/or false negative k-tuples, short pSFs are
obtained on the sites of nonrepeated k-1 sequences. Considering
false positive k-tuples, a k-tuple may contain more than one wrong
base (or containing one wrong base somewhere in the middle), as
well as k-tuple on the end. Generation of short and erroneous (or
misconnected) subfragments is caused by the latter k-tuples. The
k-tuples of the former kind represent wrong pSFs with length equal
to k-tuple length.
[0267] The aim of merging pSF part of the algorithm is the
reduction of the number of pSFs to the minimal number of longer
subfragments with the correct sequence. All k-tuple subfragments
that do not have an overlap on either end, of the length of longer
than k-a on one, and longer than k-b on the other end, are
eliminated to enable the maximal number of connections. In this
way, the majority of false positive k-tuples are discarded. The
rule for connection is: two subfragments can be unambiguously
connected if, and only if the overlapping sequence of the relevant
end or start of two subfragments is not present on the start and/or
end of any other subfragment. The exception is a subfragment with
the identical beginning and end. In that case connection is
permitted, provided that there is another subfragment with the same
end present in the file. The main problem here is of precise
definition of overlapping sequence. The presence of at least two
specific false negative k-tuples on the points of repetition of k-1
or k-2 sequences, as well as combining of the false positive and
false negative k-tuples may destroy or "mask" some overlapping
sequences and can produce an unambiguous, but wrong connection of
pSFs. To prevent this, completeness must be sacrificed on account
of exactness: the connection is not permitted on the end-sequences
shorter than k-2, and in the presence of an extra overlapping
sequence longer than k-4. The overlapping sequences are defined
from the end of the pSFs, or omitting one, or few last bases.
[0268] In the very rare situations, with the presence of a certain
number of some specific false positive and false negative k-tuples,
some subfragments with the wrong end can survive, some false
positive k-tuples (as minimal subfragments) can remain, or the
erroneous connection can take place. These cases are detected and
solved in the subfragments ordering process, and in the additional
control steps along with the handling of uncut, misconnected
subfragments.
[0269] The process of ordering of subfragments is similar to the
process of their generation. If one considers subfragments as
longer k-tuples, ordering is performed by their unambiguous
connection via overlapping ends. The informational basis for
unambiguous connection is the division of subfragments generated in
fragments of the basic library into groups representing segments of
those fragments. The method is analogous to the biochemical
solution of this problem based on hybridization with longer
oligonucleotides with relevant connecting sequence. The connecting
sequences are generated as subfragments using the k-tuple sets of
the appropriate segments of basic library fragments. Relevant
segments are defined by the fragments of the ordering library that
overlap with the respective fragments of the basic library. The
shortest segments are informative fragments of the ordering
library. The longer ones are several neighboring informative
fragments or total overlapping portions of fragments corresponding
of the ordering and basic libraries. In order to decrease the
number of separate samples, fragments of the ordering library are
randomly pooled, and the unique k-tuple content is determined.
[0270] By using the large number of fragments in the ordering
library very short segments are generated, thus reducing the chance
of the multiple appearance of the k-1 sequences which are the
reasons for generation of the subfragments. Furthermore, longer
segments, consisting of the various regions of the given fragment
of the basic library, do not contain some of the repeated k-1
sequences. In every segment a connecting sequence (a connecting
subfragment) is generated for a certain pair of the subfragments
from the given fragment. The process of ordering consists of three
steps: (1) generation of the k-tuple contents of each segment; (2)
generation of subfragments in each segment; and (3) connection of
the subfragments of the segments. Primary segments are defined as
significant intersections and differences of k-tuple contents of a
given fragment of the basic library with the k-tuple contents of
the pools of the ordering library. Secondary (shorter) segments are
defined as intersections and differences of the k-tuple contents of
the primary segments.
[0271] There is a problem of accumulating both false positive and
negative k-tuples in both the differences and intersections. The
false negative k-tuples from starting sequences accumulate in the
intersections (overlapping parts), as well as false positive
k-tuples occurring randomly in both sequences, but not in the
relevant overlapping region. On the other hand, the majority of
false positives from either of the starting sequences is not taken
up into intersections. This is an example of the reduction of
experimental errors from individual fragments by using information
from fragments overlapping with them. The false k-tuples accumulate
in the differences for another reason. The set of false negatives
from the original sequences are enlarged for false positives from
intersections and the set of false positives for those k-tuples
which are not included in the intersection by error, i.e. are false
negative in the intersection. If the starting sequences contain 10%
false negative data, the primary and secondary intersections will
contain 19% and 28% false negative k-tuples, respectively. On the
other hand, a mathematical expectation of 77 false positives may be
predicted if the basic fragment and the pools have lengths of 500
bp and 10,000 bp, respectively. However, there is a possibility of
recovering most of the "lost" k-tuples and of eliminating most of
the false positive k-tuples.
[0272] First, one has to determine a basic content of the k-tuples
for a given segment as the intersection of a given pair of the
k-tuple contents. This is followed by including all k-tuples of the
starting k-tuple contents in the intersection, which contain at one
end k-1 and at the other end k-+ sequences which occur at the ends
of two k-tuples of the basic set. This is done before generation of
the differences thus preventing the accumulation of false positives
in that process. Following that, the same type of enlargement of
k-tuple set is applied to differences with the distinction that the
borrowing is from the intersections. All borrowed k-tuples are
eliminated from the intersection files as false positives.
[0273] The intersection, i.e. a set of common k-tuples, is defined
for each pair (a basic fragment) X (a pool of ordering library). If
the number of k-tuples in the set is significant it is enlarged
with the false negatives according to the described rule. The
primary difference set is obtained by subtracting from a given
basic fragment the obtained intersection set. The false negative
k-tuples are appended to the difference set by borrowing from the
intersection set according to the described rule and, at the same
time, removed from the intersection set as false positive k-tuples.
When the basic fragment is longer than the pooled fragments, this
difference can represent the two separate segments which somewhat
reduces its utility in further steps. The primary segments are all
generated intersections and differences of pairs (a basic fragment)
X (a pool of ordering library) containing the significant number of
k-tuples. K-tuple sets of secondary segments are obtained by
comparison of k-tuple sets of all possible pairs of primary
segments. The two differences are defined from each pair which
produces the intersection with the significant number of k-tuples.
The majority of available information from overlapped fragments is
recovered in this step so that there is little to be gained from
the third round of forming intersections, and differences.
[0274] (2) Generation of the subfragments of the segments is
performed identically as described for the fragments of the basic
library.
[0275] (3) The method of connection of subfragments consists of
sequentially determining the correctly linked pairs of subfragments
among the subfragments from a given basic library fragment which
have some overlapped ends. In the case of 4 relevant subfragments,
two of which contain the same beginning and two having the same
end, there are 4 different pairs of subfragments that can be
connected. In general 2 are correct and 2 are wrong. To find
correct ones, the presence of the connecting sequences of each pair
is tested in the subfragments generated from all primary and
secondary segments for a given basic fragment. The length and the
position of the connecting sequence are chosen to avoid
interference with sequences which occur by chance. They are k+2 or
longer, and include at least one element 2 beside overlapping
sequence in both subfragments of a given pair. The connection is
permitted only if the two connecting sequences are found and the
remaining two do not exist. The two linked subfragments replace
former subfragments in the file and the process is cyclically
repeated.
[0276] Repeated sequences are generated in this step. This means
that some subfragments are included in linked subfragments more
than once. They will be recognized by finding the relevant
connecting sequence which engages one subfragment in connection
with two different subfragments.
[0277] The recognition of misconnected subfragments generated in
the processes of building pSFs and merging pSFs into longer
subfragments is based on testing whether the sequences of
subfragments from a given basic fragment exist in the sequences of
subfragments generated in the segments for the fragment. The
sequences from an incorrectly connected position will not be found
indicating the misconnected subfragments.
[0278] Beside the described three steps in ordering of subfragments
some additional control steps or steps applicable to specific
sequences will be necessary for the generation of more complete
sequence without mistakes.
[0279] The determination of which subfragment belongs to which
segment is performed b comparison of contents of k-tuples in
segments and subfragments. Because of the errors in the k-tuple
contents (due to the primary error in pools and statistical errors
due to the frequency of occurrences of k-tuples) the exact
partitioning of subfragments is impossible. Thus, instead of "all
or none" partition, the chance of coming from the given segment
(P(sf,s)) is determined for each subfragment. This possibility is
the function of the lengths of k-tuples, the lengths of
subfragments, the lengths of fragments of ordering library, the
size of the pool, and of the percentage of false k-tuples in the
file:
P(sf,s)=(Ck-F)/Lsf,
[0280] where Lsf is the length of subfragment, Ck is the number of
common k-tuples for a given subfragment/segment pair, and F is the
parameter that includes relations between lengths of k-tuples,
fragments of basic library, the size of the pool, and the error
percentage.
[0281] Subfragments attributed to a particular segment are treated
as redundant short pSFs and are submitted to a process of
unambiguous connection. The definition of unambiguous connection is
slightly different in this case, since it is based on a probability
that subfragments with overlapping end(s) belong to the segment
considered. Besides, the accuracy of unambiguous connection is
controlled by following the connection of these subfragments in
other segments. After the connection in different segments, all of
the obtained subfragments are merged together, shorter subfragments
included within longer ones are eliminated, and the remaining ones
are submitted to the ordinary connecting process. If the sequence
is not regenerated completely, the process of partition and
connection of subfragments is repeated with the same or less severe
criterions of probability of belonging to the particular segment,
followed by unambiguous connection.
[0282] Using severe criteria for defining unambiguous overlap, some
information is not used. Instead of a complete sequence, several
subfragments that define a number of possibilities for a given
fragment are obtained. Using less severe criteria an accurate and
complete sequence is generated. In a certain number of situations,
e.g. an erroneous connection, it is possible to generate a
complete, but an incorrect sequence, or to generate "monster"
subfragments with no connection among them. Thus, for each fragment
of the basic library one obtains: a) several possible solutions
where one is correct and b) the most probable correct solution.
Also, in a very small number of cases, due to the mistake in the
subfragment generation process or due to the specific ratio of the
probabilities of belonging, no unambiguous solution is generated or
one, the most probable solution. These cases remain as incomplete
sequences, or the unambiguous solution is obtained by comparing
these data with other, overlapped fragments of basic library.
[0283] The described algorithm was tested on a randomly generated,
50 kb sequence, containing 40% GC to simulate the GC content of the
human genome. In the middle part of this sequence were inserted
various All, and some other repetitive sequences, of a total length
of about 4 kb. To simulate an in vitro SBH experiment, the
following operations were performed to prepare appropriate
data.
[0284] Positions of sixty 5 kb overlapping "clones" were randomly
defined, to simulate preparation of a basic library:
[0285] Positions of one thousand 500 bp "clones" were randomly
determined to simulate making the ordering library. These fragments
were extracted from the sequence. Random pools of 20 fragments were
made, and k-tuple sets of pools were determined and stored on the
hard disk. These data are used in the subfragment ordering phase:
For the same density of clones 4 million clones in basic library
and 3 million clones in ordering library are used for the entire
human genome. The total number of 7 million clones is several fold
smaller than the number of clones a few kb long for random cloning
of almost all of genomic DNA and sequencing by a gel-based
method.
[0286] From the data on the starts and ends of 5 kb fragments, 117
"informative fragments" were determined to be in the sequence. This
was followed by determination of sets of overlapping k-tuples of
which the single "informative fragment" consist. Only the subset of
k-tuples matching a predetermined list were used. The list
contained 65% 8-mers, 30% 9-mers, and 5% 10-12-mers. Processes of
generation and the ordering of subfragments were performed on these
data.
[0287] The testing of the algorithm was performed on the simulated
data in two experiments. The sequence of 50 informative fragments
was regenerated with the 100% correct data set (over 20,000 bp),
and 26 informative fragments (about 10,000 bp) with 10% false
k-tuples (5% positive and 5% negative ones).
[0288] In the first experiment, all subfragments were correct and
in only one out of 50 informative fragments the sequence was not
completely regenerated but remained in the form of 5 subfragments.
The analysis of positions of overlapped fragments of ordering
library has shown that they lack the information for the unique
ordering of the 5 subfragments. The subfragments may be connected
in two ways based on overlapping ends, 1-2-3-4-5 and 1-4-3-2-5. The
only difference is the exchange of positions of subfragments 2 and
4. Since subfragments 2, 3, and 4 are relatively short (total of
about 100 bp), the relatively greater chance existed, and occurred
in this case, that none of the fragments of ordering library
started or ended in the subfragment 3 region.
[0289] To simulate real sequencing, some false ("hybridization")
data was included as input in a number of experiments. In oligomer
hybridization experiments, under proposed conditions, the only
situation producing unreliable data is the end mismatch versus full
match hybridization. Therefore, in simulation only those k-tuples
differing in a single element on either end from the real one were
considered to be false positives. These "false" sets are made as
follows. On the original set of a k-tuples of the informative
fragment, a subset of 5% false positive k-tuples are added. False
positive k-tuples are made by randomly picking a k-tuple from the
set, copying it and altering a nucleotide on its beginning or end.
This is followed by subtraction of a subset of 5% randomly chosen
k-tuples. In this way the statistically expected number of the most
complicated cases is generated in which the correct k-tuple is
replaced with a k-tuple with the wrong base on the end.
[0290] Production of k-tuple sets as described leads to up to 10%
of false data. This value varies from case to case, due to the
randomness of choice of k-tuples to be copied, altered, and erased.
Nevertheless, this percentage 3-4 times exceeds the amount of
unreliable data in real hybridization experiments. The introduced
error of 10% leads to the two fold increase in the number of
subfragments both in fragments of basic library (basic library
informative fragments) and in segments. About 10% of the final
subfragments have a wrong base at the end as expected for the
k-tuple set which contains false positives (see generation of
primary subfragments). Neither the cases of misconnection of
subfragments nor subfragments with the wrong sequence were
observed. In 4 informative fragments out of 26 examined in the
ordering process the complete sequence was not regenerated. In all
4 cases the sequence was obtained in the form of several longer
subfragments and several shorter subfragments contained in the same
segment. This result shows that the algorithmic principles allow
working with a large percentage of false data.
[0291] The success of the generation of the sequence from its
k-tuple content may be described in terms of completeness and
accuracy. In the process of generation, two particular situations
can be defined: 1) Some part of the information is missing in the
generated sequence, but one knows where the ambiguities are and to
which type they belong, and 2) the regenerated sequence that is
obtained does not match the sequence from which the k-tuple content
is generated, but the mistake can not be detected. Assuming the
algorithm is developed to its theoretical limits, as in the use of
the exact k-tuple sets, only the first situation can take place.
There the incompleteness results in a certain number of
subfragments that may not be ordered unambiguously and the problem
of determination of the exact length of monotonous sequences, i.e.
the number of perfect tandem repeats.
[0292] With false k-tuples, incorrect sequence may be generated.
The reason for mistakes does not lie in the shortcomings of the
algorithm, but in the fact that a given content of k-tuples
unambiguously represents the sequence that differs from the
original one. One may define three classes of error, depending on
the kind of the false k-tuples present in the file. False negative
k-tuples (which are not accompanied with the false positives)
produce "deletions". False positive k-tuples are producing
"elongations (unequal crossing over)". False positives accompanied
with false negatives are the reason for generation of "insertions",
alone or combined with "deletions". The deletions are produced when
all of the k-tuples (or their majority) between two possible starts
of the subfragments are false negatives. Since every position in
the sequence is defined by k k-tuples, the occurrence of the
deletions in a common case requires k consecutive false negatives.
(With 10% of the false negatives and k=8, this situation takes
place after every 108 elements). This situation is extremely
infrequent even in mammalian genome sequencing using random
libraries containing ten genome equivalents.
[0293] Elongation of the end of the sequence caused by false
positive k-tuples is the special case of "insertions", since the
end of the sequence can be considered as the endless linear array
of false negative k-tuples. One may consider a group of false
positive k-tuples producing subfragments longer than one k-tuple.
Situations of this kind may be detected if subfragments are
generated in overlapped fragments, like random physical fragments
of the ordering library. An insertion, or insertion in place of a
deletion, can arise as a result of specific combinations of false
positive and false negative k-tuples. In the first case, the number
of consecutive false negatives is smaller than k. Both cases
require several overlapping false positive k-tuples. The insertions
and deletions are mostly theoretical possibilities without sizable
practical repercussions since the requirements in the number and
specificity of false k-tuples are simply too high.
[0294] In every other situation of not meeting the theoretical
requirement of the minimal number an the kind of the false positive
and/or negatives, mistakes in the k-tuples content may produce only
the lesser completeness of a generated sequence.
[0295] SBH, a sample nucleic acid is sequenced by exposing the
sample to a support-bound probe of known sequence and a labeled
probe or probes in solution. Wherever the probes ligase is
introduced into the mixture of probes and sample, such that,
wherever a support has a bound probe and a labeled probe hybridized
back to back along the sample, the two probes will be chemically
linked by the action of the ligase. After washing, only chemically
linked support-bound and labeled probes are detected by the
presence of the labeled probe. By knowing the identity of the
support-bound probe at a particular location in an array, and the
identity of the labeled probe, a portion of the sequence of the
sample may be determined by the presence of a label at a point in
an array on a Format with a sample of three substrate. And not
chances not working are maximally overlapping sequences of all of
the ligated probe pairs, the sequence of the sample may be
reconstructed. Not of the sample to be sequenced may be a nucleic
acid fragment or oligonucleotide of ten base pairs ("bp"). The
sample is preferably four to one thousand bases in length.
[0296] The length of the probe is a fragment less than ten bases in
length, and, preferably, is between four and nine bases in length.
In this way, arrays of support-bound probes may include all
oligonucleotides of a given length or may include only
oligonucleotides selected for a particular test. Where all
oligonucleotides of a given length are used, the number of central
oligonucleotides may be calculated by 4.sup.N where N is the length
of the probe.
EXAMPLE 18
Re-using Sequencing Chips
[0297] When ligation is employed in the sequencing process, then
the ordinary oligonucleotide chip cannot be immediately reused. The
inventor contemplates that this may be overcome in various
ways.
[0298] One may employ ribonucleotides for the second probe, probe
P, so that this probe may subsequently be removed by RNAse
treatment. RNAse treatment may utilize RNAse A an endoribonuclease
that specifically attacks single-stranded RNA 3 to pyrimidine
residues and cleaves the phosphate linkage to the adjacent
nucleotide. The end products are pyrimidiie 3 phosphates and
oligonucleotides with terminal pyrimidine 3 phosphates. RNAse A
works in the absence of cofactors and divalent cations.
[0299] To utilize an RNAse, one would generally incubate the chip
in any appropriate RNAse-containing buffer, as described by
Sambrook et al. (1989; incorporated herein by reference). The use
of 30-50 ul of RNAse-containing buffer per 8.times.8 mm or
9.times.9 mm array at 37.degree. C. for between 10 and 60 minutes
is appropriate. One would then wash with hybridization buffer.
[0300] Although not widely applicable, one could also use the
uracil base, as described by Craig et al. (1989), incorporated
herein by reference, in specific embodiments. Destruction of the
ligated probe combination, to yield a re-usable chip, would be
achieved by digestion with the E. Coli repair enzyme, uracil-DNA
glycosylase which removes uracil from DNA.
[0301] One could also generate a specifically cleavable bond
between the probes and then cleave the bond after detection. For
example, this may be achieved by chemical ligation as described by
Shabarova et al., (1991) and Dolinnaya et al., (1988), both
references being specifically incorporated herein by reference.
[0302] Shabarova et al. (1991) describe the condensation of
oligodeoxyribo nucleotides with cyanogen bromide as a condensing
agent. In their one step chemical ligation reaction, the
oligonucleotides are heated to 97.degree. C., slowly cooled to
0.degree. C., then 1 ul 10 mM BrCN in acetonitrile is added.
[0303] Dolinnaya et al. (1988) show how to incorporate
phosphoramidiate and pyrophosphate intemucleotide bonds in DNA
duplexes. They also use a chemical ligation method for modification
of the sugar phosphate backbone of DNA, with a water-soluble
carbodiimide (CDI) as a coupling agent. The selective cleavage of a
phosphoamide bond involves contact with 15% CH.sub.3COOH for 5 min
at 95.degree. C. The selective cleavage of a pyrophosphate bond
involves contact with a pyridine-water mixture (9:1) and freshly
distilled (CF.sub.3CO).sub.2O.
EXAMPLE 19
Diagnostics--Scoring Known Mutations or Full Gene Resequencing
[0304] In a simple case, the goal may be to discover whether
selected, known mutations occur in a DNA segment. Less than 12
probes may suffice for this purpose, for example, 5 probes positive
for one allele, 5 positive for the other, and 2 negative for both.
Because of the small number of probes to be scored per sample,
large numbers of samples may be analyzed in parallel. For example,
with 12 probes in 3 hybridization cycles, 96 different genomic loci
or gene segments from 64 patient may be analyzed on one 6.times.9
in membrane containing 12.times.24 subarrays each with 64 dots
representing the same DNA segment from 64 patients. In this
example, samples may be prepared in sixty-four 96-well plates. Each
plate may represent one patient, and each well may represent one of
the DNA segments to be analyzed. The samples from 64 plates may be
spotted in four replicas as four quarters of the same membrane.
[0305] A set of 12 probes may be selected by single channel
pipetting or by a single pin transferring device (or by an array of
individually-controlled pipets or pins) for each of the 96
segments, and the selected probes may be arrayed in twelve 96-well
plates. Probes may be labelled, if they are not prelabelled, and
then probes from four plates may be mixed with hybridization buffer
and added to the subarrays preferentially by a 96-channel pipeting
device. After one hybridization cycle it is possible to strip off
previously-applied probes by incubating the membrane at 37.degree.
to 55.degree. C. in the preferably undiluted hybridization or
washing buffer.
[0306] The likelihood that probes positive for one allele are
positive and probes positive for the other allele are negative may
be used to determine which of the two alleles is present. In this
redundant scoring scheme, some level (about 10%) of errors in
hybridization of each probe may be tolerated.
[0307] An incomplete set of probes may be used for scoring most of
the alleles, especially if the smaller redundancy is sufficient,
e.g. one or two probes which prove the presence or absence in a
sample of one of the two alleles. For example, with a set of four
thousand 8-mers there is a 91% chance of finding at least one
positive probe for one of the two alleles for a randomly selected
locus. The incomplete set of probes may be optimized to reflect G+C
content and other biases in the analyzed samples.
[0308] For full gene sequencing, genes may be amplified in an
appropriate number of segments. For each segment, a set of probes
(about one probe per 2-4 bases) may be selected and hybridized.
These probes may identify whether there is a mutation anywhere in
the analyzed segments. Segments (i.e., subarrays which contain
these segments) where one or more mutated sites are detected may be
hybridized with additional probes to find the exact sequence at the
mutated sites. If a DNA sample is tested by every second 6-mer, and
a mutation is localized at the position that is surrounded by
positively hybridized probes TGCAAA and TATTCC and covered by three
negative probes: CAAAAC, AAACTA and ACTATT, the mutated nucleotides
must be A and/or C occurring in the normal sequence at that
position. They may be changed by a single base mutation, or by a
one or two nucleotide deletion and/or insertion between bases AA,
AC or CT.
[0309] One approach is to select a probe that extends the
positively hybridized probe TGCAAA for one nucleotide to the right,
and which extends the probe TATTCC one nucleotide to the left. With
these 8 probes (GCAAAA, GCAAAT, GCAAAC, GCAAAG and ATATTC, TTATTC,
CTATTC, GTATTC) two questionable nucleotides are determined.
[0310] The most likely hypothesis about the mutation may be
determined. For example, A is found to be mutated to G. There are
two solutions satisfied by these results. Either replacement of A
with G is the only change or there is in addition to that change an
insertion of some number of bases between newly determined G and
the following C. If the result witth bridging probes is negativ
these options may then be checked first by at least one bridging
probe comprising the mutated position (AAGCTA) and with an
additional 8 probes: CAAAGA, CAAAGT, CAAAGC, CAAAGG and ACTATT,
TCTATT, CCTATT, GCTATT, I There are many other ways to select
mutation-solving probes.
[0311] In the case of diploid, particular comparisons of scores for
the test samples and homozygotic control may be performed to
identify heterozygotes (see above). A few consecutive probes are
expected to have roughly twice smaller signals if the segment
covered by these probes is mutated on one of the two
chromosomes.
EXAMPLE 20
Identification of Genes (Mutations) Responsible for
GeneticDisorders and Other Traits
[0312] Using universal sets of longer probes (8-mers or 9-mers) on
immobilized arrays of samples, DNA fragments as long as 5-20 kb may
be sequenced without subcloning. Furthermore, the speed of
sequencing readily may be about 10 million bp/day/hybridization
instrument. This performance allows for resequencing a large
fraction of human genes or the human genome repeatedly from
scientifically or medically interesting individuals. To resequence
50% of the human genes, about 100 million bp is checked. That may
be done in a relatively short period of time at an affordable
cost.
[0313] This enormous resequencing capability may be used in several
ways to identify mutations and/or genes that encode for disorders
or any other traits. Basically, mRNAs (which may be converted into
cDNAs) from particular tissues or genomic DNA of patients with
particular disorders may be used as starting materials. From both
sources of DNA, separate genes or genomic fragments of appropriate
length may be prepared either by cloning procedures or by in vitro
amplification procedures (for example by PCR). If cloning is used,
the minimal set of clones to be analyzed may be selected from the
libraries before sequencing. That may be done efficiently by
hybridization of a small number of probes, especially if a small
number of clones longer than 5 kb is to be sorted. Cloning may
increase the amount of hybridization data about two times, but does
not require tens of thousands of PCR primers.
[0314] In one variant of the procedure, gene or genomic fragments
may be prepared by restriction cutting with enzymes like Hga I
which cuts DNA in following way: GACGC(N5')/CTGCG(N10'). Protruding
ends of five bases are different for different fragments. One
enzyme produces appropriate fragments for a certain number of
genes. By cutting cDNA or genomic DNA with several enzymes in
separate reactions, every gene of interest may be excised
appropriately. In one approach, the cut DNA is fractionated by
size. DNA fragments prepared in this way (and optionally treated
with Exonuclease III which individually removes nucleotides from
the 3' end and increases length and specificity of the ends) may be
dispensed in the tubes or in multiwell plates. From a relatively
small set of DNA adapters with a common portion and a variable
protruding end of appropriate length, a pair of adapters may be
selected for every gene fragment that needs to be amplified. These
adapters are ligated and then PCR is performed by universal
primers. From 1000 adapters, a million pairs may be generated, thus
a million different fragments may be specifically amplified in the
identical conditions with a universal pair of primers complementary
to the common end of the adapters.
[0315] If a DNA difference is found to be repeated in several
patients, and that sequence change is nonsense or can change
function of the corresponding protein, then the mutated gene may be
responsible for the disorder. By analyzing a significant number of
individuals with particular traits, functional allelic variations
of particular genes could be associated by specific traits.
[0316] This approach may be used to eliminate the need for very
expensive genetic mapping on extensive pedigrees and has special
value when there is no such genetic data or material.
EXAMPLE 21
[0317] Scoring Single Nucleotide Polymorphisms in Genetic
Mapping
[0318] Techniques disclosed in this application are appropriate for
an efficient identification of genomic fragments with single
nucleotide polymorphisms (SNUPs). In 10 individuals by applying the
described sequencing process on a large number of genomic fragments
of known sequence that may be amplified by cloning or by in vitro
amplification, a sufficient number of DNA segments with SNUPs may
be identified. The polymorphic fragments are further used as SNUP
markers. These markers are either mapped previously (for example
they represent mapped STSS) or they may be mapped through the
screening procedure described below.
[0319] SNUPs may be scored in every individual from relevant
families or populations by amplifying markers and arraying them in
the form of the array of subarrays. Subarrays contain the same
marker amplified from the analyzed individuals. For each marker, as
in the diagnostics of known mutations, a set of 6 or less probes
positive for one allele and 6 or less probes positive for the other
allele may be selected and scored. From the significant association
of one or a group of the markers with the disorder, chromosomal
position of the responsible gene(s) may be determined. Because of
the high throughput and low cost, thousands of markers may be
scored for thousands of individuals. This amount of data allows
localization of a gene at a resolution level of less than one
million bp as well as localization of genes involved in polygenic
diseases. Localized genes may be identified by sequencing
particular regions from relevant normal and affected individuals to
score a mutation(s).
[0320] PCR is preferred for amplification of markers from genomic
DNA. Each of the markers require a specific pair of primers. The
existing markers may be convertible or new markers may be defined
which may be prepared by cutting genomic DNA by Hga I type
restriction enzymes, and by ligation with a pair of adapters.
[0321] SNUP markers can be amplified or spotted as pools to reduce
the number of independent amplification reactions. In this case,
more probes are scored per one sample. When 4 markers are pooled
and spotted on 12 replica membranes, then 48 probes (12 per marker)
may be scored in 4 cycles.
EXAMPLE 22
Detection and Verification of Identity of DNA Fragments
[0322] DNA fragments generated by restriction cutting, cloning or
in vitro amplification (e.g. PCR) frequently may be identified in a
experiment. Identification may be performed by verifying the
presence of a DNA band of specific size on gel electrophoresis.
Alternatively, a specific oligonucleotide may be prepared and used
to verify a DNA sample in question by hybridization. The procedure
developed here allows for more efficient identification of a large
number of samples without preparing a specific oligonucleotide for
each fragment. A set of positive and negative probes may be
selected from the universal set for each fragment on the basis of
the known sequences. Probes that are selected to be positive
usually are able to form one or a few overlapping groups and
negative probes are spread over the whole insert.
[0323] This technology may be used for identification of STSs in
the process of their mapping on the YAC clones. Each of the STSs
may be tested on about 100 YAC clones or pools of YAC clones. DNAs
from these 100 reactions possibly are spotted in one subarray.
Different STSs may represent consecutive subarrays. In several
hybridization cycles, a signature may be generated for each of the
DNA samples, which signature proves or disproves existence of the
particular STS in the given YAC clone with necessary
confidence.
[0324] To reduce the number of independent PCR reactions or the
number of independent samples for spotting, several STSs may be
amplified simultaneously in a reaction or PCR samples may be mixed,
respectively. In this case more probes have to be scored per one
dot. The pooling of STSs is independent of pooling YACs and may be
used on single YACs or pools of YACs. This scheme is especially
attractive when several probes labelled with different colors are
hybridized together.
[0325] In addition to confirmation of the existence of a DNA
fragment in a sample, the amount of DNA may be estimated using
intensities of the hybridization of several separate probes or one
or more pools of probes. By comparing obtained intensities with
intensities for control samples having a known amount of DNA, the
quantity of DNA in all spotted samples is determined
simultaneously. Because only a few probes are necessary for
identification of a DNA fragment, and there are N possible probes
that may be used for DNA N bases long, this application does not
require a large set of probes to be sufficient for identification
of any DNA segment. From one thousand 8-mers, on average about 30
full matching probes may be selected for a 1000 bp fragment.
EXAMPLE 23
Identification of Infectious Disease Organisms and Their
Variants
[0326] DNA-based tests for the detection of viral, bacterial,
fungal and other parasitic organisms in patients are usually more
reliable and less expensive than alternatives. The major advantage
of DNA tests is to be able to identify specific strains and
mutants, and eventually be able to apply more effective treatment.
Two applications are described below.
[0327] The presence of 12 known antibiotic resistance genes in
bacterial infections may be tested by amplifying these genes. The
amplified products from 128 patients may be spotted in two
subarrays and 24 subarrays for 12 genes may then be repeated four
times on a 8.times.12 cm membrane. For each gene, 12 probes may be
selected for positive and negative scoring. Hybridizations may be
performed in 3 cycles. For these tests, a much smaller set of
probes is most likely to be universal. For example, from a set of
one thousand 8-mers, on average 30 probes are positive in 1000 bp
fragments, and 10 positive probes are usually sufficient for a
highly reliable identification. As described in Example 9, several
genes may be amplified and/or spotted together and the amount of
the given DNA may be determined. The amount of amplified gene may
be used as an indicator of the level of infection.
[0328] Another example involves possible sequencing of one gene or
the whole genome of an HIV virus. Because of rapid diversification,
the virus poses many difficulties for selection of an optimal
therapy. DNA fragments may be amplified from isolated viruses from
up to 64 patients and resequenced by the described procedure. On
the basis of the obtained sequence the optimal therapy may be
selected. If there is a mixture of two virus types of which one has
the basic sequence (similar to the case of heterozygotes), the
mutant may be identified by quantitative comparisons of its
hybridization scores with scores of other samples, especially
control samples containing the basic virus type only. Scores twice
as small may be obtained for three to four probes that cover the
site mutated in one of the two virus types present in the sample
(see above).
EXAMPLE 24
Forensic and Parental Identification
[0329] Sequence polymorphisms make an individual genomic DNA
unique. This permits analysis of blood or other body fluids or
tissues from a crime scene and comparison with samples from
criminal suspects. A sufficient number of polymorphic sites are
scored to produce a unique signature of a sample. SBH may easily
score single nucleotide polymorphisms to produce such
signatures.
[0330] A set of DNA fragments (10-1000) may be amplified from
samples and suspects. DNAs from samples and suspects representing
one fragment are spotted in one or several subarrays and each
subarray may be replicated 4 times. In three cycles, 12 probes may
determine the presence of allele A or B in each of the samples,
including suspects, for each DNA locus. Matching the patterns of
samples and suspects may lead to discovery of the suspect
responsible for the crime.
[0331] The same procedure may be applicable to prove or disprove
the identity of parents of a child. DNA may be prepared and
polymorphic loci amplified from the child and adults; patterns of A
or B alleles may be determined by hybridization for each.
Comparisons of the obtained patterns, along with positive and
negative controls, aide in the determination of familial
relationships. In this case, only a significant portion of the
alleles need match with one parent for identification. Large
numbers of scored loci allow for the avoidance of statistical
errors in the procedure or of masking effects of de novo
mutations.
EXAMPLE 25
Assessing Genetic Diversity of Populations or Species and
Biological Diversity of Ecological Niches
[0332] Measuring the frequency of allelic variations on a
significant number of loci (for example, several genes or entire
mitochondrial DNA) permits development of different types of
conclusions, such as conclusions regarding the impact of the
environment on the genotypes, history and evolution of a population
or its susceptibility to diseases or extinction, and others. These
assessments may be performed by testing specific known alleles or
by full resequencing of some loci to be able to define de novo
mutations which may reveal fine variations or presence of mutagens
in the environment.
[0333] Additionally, biodiversity in the microbial world may be
surveyed by resequencing evolutionarily conserved DNA sequences,
such as the genes for ribosomal RNAs or genes for highly
conservative proteins. DNA may be prepared from the environment and
particular genes amplified using primers corresponding to
conservative sequences. DNA fragments may be cloned preferentially
in a plasmid vector (or diluted to the level of one molecule per
well in multiwell plates and than amplified in vitro). Clones
prepared this way may be resequenced as described above. Two types
of information are obtained. First of all, a catalogue of different
species may be defined as well as the density of the individuals
for each species. Another segment of information may be used to
measure the influence of ecological factors or pollution on the
ecosystem. It may reveal whether some species are eradicated or
whether the abundance ratios among species is altered due to the
pollution. The method also is applicable for sequencing DNAs from
fossils.
EXAMPLE 26
Detection or Quantification of Nucleic Acid Species
[0334] DNA or RNA species may be detected and quantified by
employing a probe pair including an unlabeled probe fixed to a
substrate and a labeled probe in a solution. The species may be
detected and quantified by exposure to the unlabeled probe in the
presence of the labeled probe and ligase. Specifically, the
formation of an extended probe by ligation of the labeled and
unlabeled probe on the sample nucleic acid backbone is indicative
of the presence of the species to be detected. Thus, the presence
of label at a specific point in the array on the substrate after
removing unligated labeled probe indicates the presence of a sample
species while the quantity of label indicates the expression level
of the species.
[0335] Alternatively, one or more unlabeled probes may be arrayed
on a substrate as first members of pairs with one or more labeled
probes to be introduced in solution. According to one method,
multiplexing of the label on the array may be carried out by using
dyes which fluoresce at distinguishable wavelengths. In this
manner, a mixture of cDNAs applied to an array with pairs of
labeled and unlabeled probes specific for species to be identified
may be examined for the presence of and expression level of cDNA
species. According to a preferred embodiment this approach may be
carried out to sequence portions of cDNAs by selecting pairs of
unlabeled and labeled probes pairs comprising sequences which
overlap along the sequence of a cDNA to be detected.
[0336] Probes may be selected to detect the presence and quantity
of particular pathogenic organisms genome by including in the
composition selected probe pairs which appear in combination only
in target pathogenic genome organisms. Thus, while no single probe
pair may necessarily be specific for the pathogenic organism
genome, the combination of pairs is. Similarly, in detecting or
sequencing cDNAs, it might occur that a particular probe is not be
specific for a cDNA or other type of species. Nevertheless, the
presence and quantity of a particular species may be determined by
a result wherein a combination of selected probes situated at
distinct array locations is indicative of the presence of a
particular species.
[0337] An infectious agent with about 10 kb or more of DNA may be
detected using a support-bound detection chip without the use of
polymerase chain reaction (PCR) or other target amplification
procedures. According to other methods, the genomes of infectious
agents including bacteria and viruses are assayed by amplification
of a single target nucleotide sequence through PCR and detection of
the presence of target by hybridization of a labelled probe
specific for the target sequence. Because such an assay is specific
for only a single target sequence it therefore is necessary to
amplify the gene by methods such as PCR to provide sufficient
target to provide a detectable signal.
[0338] According to this example, an improved method of detecting
nucleotide sequences characteristic of infectious agents through a
Format 3-type reaction is provided wherein a solid phase detection
chip is prepared which comprises an array of multiple different
immobilized oligonucleotide probes specific for the infectious
agent of interest. A single dot comprising a mixture of many
unlabeled probes complementary to the target nucleic acid
concentrates the label specific to a species at one location
thereby improving sensitivity over diffuse or single probe
labeling. Such multiple probes may be of overlapping sequences of
the target nucleotide sequence but may also be non-overlapping
sequences as well as non-adjacent. Such probes preferably have a
length of about 5 to 12 nucleotides.
[0339] A nucleic acid sample exposed to the probe array and target
sequences present in the sample will hybridize with the multiple
immobilized probes. A pool of multiple labeled probes selected to
specifically bind to the target sequences adjacent to the
immobilized probes is then applied with the sample to an array of
unlabeled oligonucleotide probe mixtures. Ligase enzyme is then
applied to the chip to ligate the adjacent probes on the sample.
The detection chip is then washed to remove unhybridized and
unligated probe and sample nucleic acids and the presence of sample
nucleic acid may be determined by the presence or absence of label.
This method provides reliable sample detection with about a
1000-fold reduction of molarity of the sample agent.
[0340] As a further aspect of the invention, the signal of the
labelled probes may be amplified by means such as providing a
common tail to the free probe which itself comprises multiple
chromogenic, enzymatic or radioactive labels or which is itself
susceptible to specific binding by a further probe agent which is
multiply labelled. In this way, a second round of signal
amplification may be carried out. Labeled or unlabeled probes may
be used in a second round of amplification. In this second round of
amplification, a lengthy DNA sample with multiple labels may result
in an increased amplification intensity signal between 10 to 100
fold which may result in a total signal amplification of 100,000
fold. Through the use of both aspects of this example, an intensity
signal approximately 100,000 fold may give a positive result of
probe-DNA ligation without having to employ PCR or other
amplification procedures.
[0341] According to a further aspect of the invention an array or
super array may be prepared which consists of a complete set of
probes, for example 4096 6-mer probes. Arrays of this type are
universal in a sense that they can be used for detection or partial
to complete sequencing of any nucleic acid species. Individual
spots in an array may contain single probe species or mixtures of
probes, for example N(1-3) B(4-6) N(1-3) type of mixtures that are
synthesized in the single reaction (N represents all four
nucleotides, B one specific nucleotide and where the associated
numbers are a range of numbers of bases i.e., 1-3 means "from one
to three bases".) These mixtures provide stronger signal for a
nucleic acid species present at low concentration by collecting
signal from different parts of the same long nucleic acid species
molecule. The universal set of probes may be subdivided in many
subsets which are spotted as unit arrays separated by barriers that
prevent spreading of hybridization buffer with sample and labeled
probe(s).
[0342] For detection of a nucleic acid species with a known
sequence one of more oligonucleotide sequences comprising both
unlabelled fixed and labeled probes in solution may be selected.
Labeled probes are synthesized or selected from the presynthesized
complete sets of, for example, 7-mers. The labeled probes are added
to corresponding unit arrays of fixed probes such that a pair of
fixed and labeled probes will adjacently hybridize to the target
sequence such that upon administration of ligase the probes will be
covalently bound.
[0343] If a unit array contains more than one fixed probe (as
separated spots or within the same spot) that are positive in a
given nucleic acid species all corresponding labeled probes may be
mixed and added to the same unit array. The mixtures of labeled
probes are even more important when mixtures of nucleic acid
species are tested. One example of a complex mixture of nucleic
acid species are mRNAs in one cell or tissue.
[0344] According to one embodiment of the invention unit arrays of
fixed probes allow use of every possible immobilized probe with
cocktails of a relatively small number of labeled probes. More
complex cocktails of labeled probes may be used if a multiplex
labeling scheme is implemented. Preferred multiplexing methods may
use different fluorescent dyes or molecular tags that may be
separated by mass spectroscopy.
[0345] Alternatively, according to a preferred embodiment of the
invention, relatively short fixed probes may be selected which
frequently hybridize to many nucleic acid sequences. Such short
probes are used in combination with a cocktail of labeled probes
which may be prepared such that at least one labeled probe
corresponds to each of the fixed proves. Preferred cocktails are
those in which none of the labeled probes corresponds to more than
one fixed probe.
EXAMPLE 27
Interrogation of Segments of the HIV Virus with All Possible
10-mers
[0346] In this example of Format III SBH, an array was generated on
nylon membranes (e.g., Gene Screen) of all possible bound 5-mers
(1024 possible pentamers). The bound 5-mer oligonucleotides were
synthesized with 5' tails of 5'-TTTTTT-NNN-3' (N=all four bases A,
C, G, T, at this step in the synthesis equal molar amounts of all
four bases are added). These oligonucleotides were precisely
spotted onto the nylon membrane, the spots were allowed to dry, and
the oligonucleotides were immobilized by treating the dried spots
with UV light. Oligonucleotide densities of up to 18
oligonucleotides per square nanometer were obtained using this
method. After the UV treatment, the nylon membranes were treated
with a detergent containing buffer at 60-80.degree. C. The spots of
oligonucleotides were gridded in subarrays of 10 by 10 spots, and
each subarray has 64 5-mer spots and 36 control spots. 16 subarrays
give 1024 5-mers which encompasses all possible 5-mers.
[0347] The subarrays in the array were partitioned from each other
by physical barriers, e.g., a hydrophobic strip, that allowed each
subarray to be hybridized to a sample without cross-contamination
from adjacent subarrays. In a preferred embodiment, the hydrophobic
strip is made from a solution of silicone (e.g., household silicone
glue and seal paste) in an appropriate solvent (such solvents are
well known in the art). This solution of silicone grease is applied
between the subarrays to form lines which after the solvent
evaporates act as hydrophobic strips separating the cells.
[0348] In this Format III example, the free or solution (nonbound)
5-mers were synthesized with 3' tails of 5'-NN-3' (N=all four bases
A, C, G, T). In this embodiment, the free 5-mers and the bound
5-mers are combined to produce all possible 10-mers for sequencing
a known DNA sequence of less than 20 kb. 20 kb of double stranded
DNA is denatured into 40 kb of single-stranded DNA. This 40 kb of
ss DNA hybridizes to about 4% of all possible 10-mers. This low
frequency of 10-mer binding and the known target sequence allow the
pooling of free or solution (nonbound) 5-mers for treatment of each
subarray, without a loss of sequence information. In a preferred
embodiment, 16 probes are pooled for each subarray, and all
possible 5-mers are represented in 64 total pools of free 5-mers.
Thus, all possible 10-mers may be probed against a DNA sample using
1024 subarrays (16 subarrays for each pool of free 5-mers).
[0349] The target DNA in this embodiment represents two-600 bp
segments of the HIV virus. These 600 bp segments are represented by
pools of 60 overlapping 30-mers (the 30-mers overlap each adjacent
30 mer by 20 nucleotides). The pools of 30-mers mimic a target DNA
that has been treated using techniques well known in the art to
shear, digest, and/or random PCR the target DNA to produce a random
pool of very small fragments.
[0350] As described above in the previous Format III examples, the
free 5-mers are labeled with radioactive isotopes, biotin,
fluorescent dyes, etc. The labeled free 5-mers are then hybridized
along with the bound 5-mers to the target DNA, and ligated. In a
preferred embodiment, 300-1000 units of ligase are added to the
reaction. The hybridization conditions were worked out following
the teachings of the previous examples. Following ligation and
removal of the target DNA and excess free probe, the array is
assayed to determine the location of labeled probes (using the
techniques described in the examples above).
[0351] The known DNA sequence of the target, and the known free and
bound 5-mers in each subarray, predict which bound 5-mers will be
ligated to a labeled free 5-mer in each subarray. The signal from
20 of these predicted dots were lost and 20 new signals were gained
for each change in the target DNA from the predicted sequence. The
overlapping sequence of the bound 5-mers in these ten new dots
identifies which free, labeled 5-mer is bound in each new dot.
[0352] Using the described methods, arrays and pools of free,
labeled 5-mers, the test HIV DNA sequence was probed with all
possible 10-mers. Using this Format III approach, we properly
identified the "wild-type" sequence of the segments tested, as well
as several sequence "mutants" that were introduced into these
segments.
EXAMPLE 28
Sequencing of Repetitive DNA Sequences
[0353] In one embodiment, repetitive DNA sequences in the target
DNA are sequenced with "spacer oligonucleotides" in a modified
Format III approach. Spacer oligonucleotides of varying lengths of
the repetitive DNA sequence (the repeating sequence is identified
on a first SBH run) are hybridized to the target DNA along with a
first known adjoining oligonucleotide and a second known, or group
of possible oligonucleotides adjoining the other side of the spacer
(known from the first SBH run). When a spacer matching the length
of the repetitive DNA segment is hybridized to the target, the two
adjacent oligonucleotides can be ligated to the spacer. If the
first known oligonucleotide is fixed to a substrate, and the second
known or possible oligonucleotide(s) is labeled, a bound ligation
product including the labeled second known or possible
oligonucleotide(s) is formed when a spacer of the proper length is
hybridized to the target DNA.
EXAMPLE 29
Sequencing Through Branch Points with Format III SBH
[0354] In one embodiment, branch points in the target DNA are
sequenced using a third set of oligonucleotides and a modified
Format III approach. After a first SBH run, several branch points
may be identified when the sequence is compiled. These can be
solved by hybridizing oligonucleotide(s) that overlap partially
with one of the known sequences leading into the branch point and
then hybridizing to the target an additional oligonucleotide that
is labeled and corresponds to one of the sequences that comes out
of the branch point. When the proper oligonucleotides are
hybridized to the target DNA, the labeled oligonucleotide can be
ligated to the other(s). In a preferred embodiment, a first
oligonucleotide that is offset by one to several nucleotides from
the branch point is selected (so that it reads into one of the
branch sequences), a second oligonucleotide reading from the first
and into the branch point sequence is also selected, and a set of
third oligonucleotides that correspond to all the possible branch
sequences with an overlap of the branch point sequence by one or a
few nucleotides (corresponding to the first oligonucleotide) is
selected. These oligonucleotides are hybridized to the target DNA,
and only the third oligonucleotide with the proper branch sequence
(that matches the branch sequence of the first oligonucleotide)
will produce a ligation product with the first and second
oligonucleotides.
EXAMPLE 30
Multiplexing Probes for Analyzing a Target Nucleic Acid
[0355] In this Example, sets of probes are labeled with different
labels so that each probe of a set can be differentiated from the
other probes in the set. Thus, the set of probes may be contacted
with target nucleic acid in a single hybridization reaction without
the loss of any probe information. In preferred embodiments, the
different labels are different radioisotopes, or different
flourescent labels, or different EMLs. These sets of probes may be
used in either Format I, Format II or Format III SBH.
[0356] In Format I SBH, the set of differently lableled probes are
hybridized to target nucleic acid which is fixed to a substrate
under conditions that allow differentiation between perfect matches
one base-pair mismatches. Specific probes which bind to the target
nucleic acid are identified by their different labels and perfect
matches are determined, at least in part, from this binding
information.
[0357] In Format II SBH, the target nucleic acids are labeled with
different probes and hybridized to arrays of probes. Specific
target nucleic acids which bind to the probes are identified by
their different labels and perfect matches are determined, at least
in part, form this binding information.
[0358] In Format III SBH, the set of differently labeled probes and
fixed probes are hybridized to a target nucleic acid under
conditions that allow perfect matches to be differentiated from one
base-pair mismatches. Labeled probes that are adjacent, on the
target, to a fixed probe are bound to the fixed probe, and these
products are detected and differentiated by their different
labels.
[0359] In a preferred embodiment, the different labels are EMLs,
which can be detected by electron capture mass spectrometry
(EC-MS). EMLs may be prepared from a variety of backbone molecules,
with certain aromatic backbones being particularly preferred, e.g.,
see Xu et al., J. Chromatog. 764:95-102 (1997). The EML is attached
to a probe in a reversible and stable manner, and after the rpobe
is hybridized to target nucleic acid, the EML is reomved from the
probe and identified by standard EC-MS (e.g., the EC-MS may be done
by a gas chromotograph-mass spectrometer).
EXAMPLE 31
Detection of Low Frequency Target Nucleic Acids
[0360] Format III SBH has sufficient discrimination power to
identify a sequence that is present in a sample at 1 part to 99
parts of a similar sequence that differs by a single nucleotide.
Thus, Format III can be used to identify a nucleic acid present at
a very low concentration in a sample of nucleic acids, e.g., a
sample derived from blood.
[0361] In one embodiment, the two sequences are for cystic fibrosis
and the sequences differ from each other by a deletion of three
nucleotides. Probes for the two sequences were as follows, probes
distinguishing the deletion from wild type were fixed to a
substrate, and a labeled contiguous probe was common to both. Using
these targets and probes, the deletion mutant could be detected
with Format III SBH when it was present at one part to ninety nine
parts of the wild-type.
EXAMPLE 32
Polaroid Apparatus and Method for Analyzing a Target Nucleic
Acid
[0362] An apparatus for analyzing a nucleic acid can be constructed
with two arrays of nucleic acids, and an optional material that
prevents the nucleic acids of the two arrays from mixing until such
mixing is desired. The arrays of the apparatus may be supported by
a variety of substrates, including but not limited to, nylon
membranes, nitrocellulose membranes, or other materials disclosed
above. In preferred embodiments, one of the substrate is a membrane
separated into sectors by hydrophobic strips, or a suitable support
material with wells which may contain a gel or sponge. In this
embodiment, probes are placed on a sector of the membrane, or in
the well, the gel, or sponge, and a solution (with or without
target nucleic acids) is added to the membrane or well so that the
probes are solubilized. The solution with the solubilized probes is
then allowed to contact the second array of nucleic acids. The
nucleic acids may be, but are not limited to, oligonucleotide
probes, or target nucleic acids, and the probes or target nucleic
acids may be labeled. The nucleic acids may be labeled with any
labels conventionally used in the art, including but not limited to
radioisotopes, flourscent labels or electrophore mass labels.
[0363] The material which prevents mixing of the nucleic acids may
be disposed between the two arrays in such a way that when the
material is removed the nucleic acids of the two arrays mix
together. This material may be in the form of a sheet, membrane, or
other barrier, and this material may be comprised of any material
that prevents the mixing of the nucleic acids.
[0364] This apparatus may be used in Format I SBH as follows: a
first array of the apparatus has target nucleic acids that are
fixed to the substrate, and a second array of the apparatus has
nucleic acid probes that are labeled and can be removed to
interrogate the target nucleic acid of the first array. The two
arrays are optionally separated by a sheet of material that
prevents the probes from contacting the target nucleic acid, and
when this sheet is removed the probes can interrogate the target.
After appropiate incubation and (optionally) washing steps the
array of targets may be "read" to determine which probes formed
perfect matches with the target. This reading may be automated or
can be done manually (e.g., by eye with an autoradiogram). In
Format II SBH, the procedure followed would be similar to that
described above except that the target is labeled and the probes
are fixed.
[0365] Alternatively, the apparatus may be used in Format III SBH
as follows: two arrays of nucleic acid probes are formed, the
nucleic acid probes of either or both arrays may be labeled, and
one of the arrays may be fixed to its substrate. The two arrays are
separated by a sheet of material that prevents the probes from
mixing. A Format II reaction is initiated by adding target nucleic
acid and removing the sheet allowing the probes to mix with each
other and the target. Probes which bind to adjacent sites on the
target are bound together (e.g., by base-stacking interactions or
by covalently joining the backbones), and the results are read to
determine which probes bound to the target at adjacent sites. When
one set of probes is fixed to the substrate, the fixed array can be
read to determine which probes from the other array are bound
together with the fixed probes. As with the above method, this
reading may be automated (e.g., with an ELISA reader) or can be
done manually (e.g., by eye with an autoradiogram).
[0366] The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention, and compositions and methods which
are functionally equivalent are within the scope of the invention.
Indeed, numerous modifications and variations in the practice of
the invention are expected to occur to those skilled in the art
upon consideration of the present preferred embodiments.
Consequently, the only limitations which should be placed upon the
scope of the invention are those which appear in the appended
claims.
[0367] All references cited within the body of the instant
specification are hereby incorporated by reference in their
entirety.
* * * * *