U.S. patent application number 10/152297 was filed with the patent office on 2003-04-24 for detection of nucleic acid hybrids.
This patent application is currently assigned to Promega Corporation. Invention is credited to Andrews, Christine Ann, Gu, Trent, Hartnett, James Robert, Kephart, Daniel, Leippe, Donna, Lewis, Martin K., Mandrekar, Michelle, Olson, Ryan J., Rhodes, Richard Byron, Shultz, John William, Welch, Roy, Wood, Keith V..
Application Number | 20030077621 10/152297 |
Document ID | / |
Family ID | 27400551 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030077621 |
Kind Code |
A1 |
Shultz, John William ; et
al. |
April 24, 2003 |
Detection of nucleic acid hybrids
Abstract
Processes are disclosed using the depolymerization of a nucleic
acid hybrid to qualitatively and quantitatively analyze for the
presence of a predetermined endogenous nucleic acid. Applications
of those processes include the detection of single nucleotide
polymorphisms, identification of single base changes, spieciation,
determination of viral load, genotyping, medical marker
diagnostics, and the like.
Inventors: |
Shultz, John William;
(Verona, WI) ; Lewis, Martin K.; (Madison, WI)
; Leippe, Donna; (Middleton, WI) ; Mandrekar,
Michelle; (Oregon, WI) ; Kephart, Daniel;
(Cottage Grove, WI) ; Rhodes, Richard Byron;
(Madison, WI) ; Andrews, Christine Ann; (Cottage
Grove, WI) ; Hartnett, James Robert; (Madison,
WI) ; Gu, Trent; (Madison, WI) ; Olson, Ryan
J.; (Middleton, WI) ; Wood, Keith V.;
(Madison, WI) ; Welch, Roy; (Palo Alto,
CA) |
Correspondence
Address: |
WELSH & KATZ, LTD
120 S RIVERSIDE PLAZA
22ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
Promega Corporation
Madison
WI
53711
|
Family ID: |
27400551 |
Appl. No.: |
10/152297 |
Filed: |
May 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10152297 |
May 20, 2002 |
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09383316 |
Aug 25, 1999 |
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6391551 |
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09383316 |
Aug 25, 1999 |
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09358972 |
Jul 21, 1999 |
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6235480 |
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09358972 |
Jul 21, 1999 |
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09252436 |
Feb 18, 1999 |
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6159693 |
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09252436 |
Feb 18, 1999 |
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09042287 |
Mar 13, 1998 |
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6335162 |
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Current U.S.
Class: |
435/6.11 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6823 20130101;
C12Q 1/6827 20130101; C12Q 1/66 20130101; C12Q 1/04 20130101; C12Q
1/6823 20130101; C12Q 2535/131 20130101; C12Q 2521/319 20130101;
C12Q 1/6827 20130101; C12Q 2535/131 20130101; C12Q 2521/319
20130101; C12Q 1/6823 20130101; C12Q 2565/301 20130101; C12Q
2565/1015 20130101; C12Q 2537/101 20130101; C12Q 1/6823 20130101;
C12Q 2565/627 20130101; C12Q 2535/131 20130101; C12Q 2521/319
20130101; C12Q 1/6827 20130101; C12Q 2565/627 20130101; C12Q
2535/131 20130101; C12Q 2521/319 20130101; C12Q 1/6827 20130101;
C12Q 2565/301 20130101; C12Q 2565/1015 20130101; C12Q 2537/101
20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 11, 1999 |
US |
PCT/US99/05304 |
Claims
What is claimed is:
1. A method for determining the presence or absence of a
predetermined endogenous nucleic acid target sequence in a nucleic
acid sample that comprises the steps of: (A) providing a treated
sample that may contain said predetermined nucleic acid target
sequence hybridized with a nucleic acid probe that includes an
identifier nucleotide in the 3'-terminal region; (B) admixing the
treated sample with a depolymerizing amount of an enzyme whose
activity is to release one or more nucleotides from the 3'-terminus
of a hybridized nucleic acid probe to form a treated reaction
mixture; (C) maintaining the treated reaction mixture for a time
period sufficient to permit the enzyme to depolymerize hybridized
nucleic acid and release identifier nucleotides therefrom; and (D)
analyzing for the presence of released identifier nucleotides to
obtain an analytical output, the analytical output indicating the
presence or absence of said endogenous nucleic acid target
sequence.
2. The method according to claim 1 wherein said identifier
nucleotide is a nucleoside triphosphate.
3. The method according to claim 1 wherein said analytical output
is obtained by luminescence spectroscopy.
4. The method according to claim 1 wherein said analytical output
is obtained by fluorescence spectroscopy.
5. The method according to claim 4 wherein said released identifier
nucleotide includes a fluorescent label.
6. The method according to claim 5 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
7. The method according to claim 1 wherein said analytical output
is obtained by mass spectrometry.
8. The method according to claim 7 wherein said released identifier
nucleotide includes a fluorescent label.
9. The method according to claim 7 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
10. The method according to claim 1 wherein said analytical output
is obtained by absorbance spectroscopy.
11. The method according to claim 1 including the further steps of
forming said treated sample by (a) admixing a sample to be assayed
with one or more nucleic acid probes to form a hybridization
composition, wherein the 3'-terminal region of said nucleic acid
probes (i) hybridize with partial or total complementarity to said
endogenous nucleic acid target sequence when that sequence is
present in the sample and (ii) include an identifier nucleotide;
(b) maintaining said hybridization composition for a time period
sufficient to form a treated sample that may contain said one
predetermined nucleic acid target sequence hybridized with a
nucleic acid probe.
12. The method according to claim 1 wherein said nucleic acid
sample is obtained from a biological sample.
13. A method for determining the presence or absence of at least
one predetermined endogenous nucleic acid target sequence in a
nucleic acid sample that comprises the steps of: (A) admixing a
sample to be assayed with one or more nucleic acid probes to form a
hybridization composition, wherein the 3'-terminal region of said
nucleic acid probes (i) hybridizes with partial or total
complementarity to at least one said predetermined endogenous
nucleic acid target sequence when that sequence is present in the
sample and (ii) includes an identifier nucleotide; (B) maintaining
said hybridization composition for a time period sufficient to form
a treated sample that may contain said predetermined endogenous
nucleic acid target sequence hybridized with a nucleic acid probe;
(C) admixing the treated sample with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a hybridized nucleic acid probe to form a
treated reaction mixture; (D) maintaining the treated reaction
mixture for a time period sufficient to permit the enzyme to
depolymerize hybridized nucleic acid and release identifier
nucleotides therefrom; and (E) analyzing for the presence of
released identifier nucleotides to obtain an analytical output, the
analytical output indicating the presence or absence of at least
one said endogenous nucleic acid target sequence.
14. The method according to claim 13 wherein said identifier
nucleotide is a nucleoside triphosphate.
15. The method according to claim 13 wherein said analytical output
is obtained by luminescence spectroscopy.
16. The method according to claim 13 wherein said analytical output
is obtained by fluorescence spectroscopy.
17. The method according to claim 16 wherein said released
identifier nucleotide includes a fluorescent label.
18. The method according to claim 17 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
19. The method according to claim 13 wherein said analytical output
is obtained by mass spectrometry.
20. The method according to claim 19 wherein said released
identifier nucleotide includes a fluorescent label.
21. The method according to claim 20 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
22. The method according to claim 13 wherein said analytical output
is obtained by absorbance spectroscopy.
23. The method according to claim 13 wherein said sample contains a
plurality of predetermined endogenous nucleic acid target sequences
and is admixed with a plurality of said nucleic acid probes.
24. The method according to claim 23 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with
partial complementarity to one endogenous target nucleic acid
sequence is greater than the analytical output when all of the
nucleic acid probes hybridize with total complementarity to their
respective nucleic acid target sequences.
25. The method according to claim 23 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with
partial complementarity to one endogenous target nucleic acid
sequence is less than the analytical output when all of the nucleic
acid probes hybridize with total complementarity to their
respective nucleic acid target sequences.
26. The method according to claim 23 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with total
complementarity to one endogenous nucleic acid target sequence is
greater than the analytical output when all of the nucleic acid
probes hybridize with partial complementarity to their respective
nucleic acid target sequences.
27. The method according to claim 23 wherein the analytical output
obtained when one of said nucleic acid probes hybridize with total
complementarity to one endogenous target nucleic acid sequence is
less than the analytical output when all of the nucleic acid probes
hybridize with partial complementarity to their respective nucleic
acid target sequences.
28. The method according to claim 13 wherein said enzyme whose
activity is to release nucleotides is a template-dependent
polymerase that, in the presence of pyrophosphate ions,
depolymerizes hybridized nucleic acids whose bases in the
3'-terminal region are matched with total complementarity.
29. The method according to claim 13 wherein said enzyme whose
activity is to release nucleotides exhibits a
3'.fwdarw.5'-exonuclease activity, depolymerizing hybridized
nucleic acids having one or more mismatched bases in the
3'-terminal region of the hybridized probe.
30. A method for determining the presence or absence of a specific
base in an endogenous nucleic acid target sequence in a sample to
be assayed that comprises the steps of: (A) admixing a sample to be
assayed with one or more nucleic acid probes to form an endogenous
hybridization composition, wherein the 3'-terminal region of at
least one of said nucleic acid probes (i) is substantially
complementary to said endogenous nucleic acid target sequence and
comprises at least one predetermined nucleotide at an interrogation
position, and (ii) includes an identifier nucleotide, and wherein
said nucleic acid target sequence comprises at least one specific
base whose presence or absence is to be determined (B) maintaining
said hybridization composition for a time period sufficient to form
a treated sample, wherein said interrogation position of the probe
is a nucleotide that is aligned with said specific base to be
identified in said endogenous target sequence, when present, so
that base pairing can occur; (C) admixing the treated sample with
an enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a hybridized nucleic acid probe to depolymerize
the hybrid and form a treated reaction mixture; (D) maintaining the
treated reaction mixture for a time period sufficient to release an
identifier nucleotide therefrom; and (E) analyzing for the presence
or absence of released identifier nucleotide to obtain an
analytical output that indicates the presence or absence of said
specific base to be identified.
31. The method according to claim 30 wherein the identifier
nucleotide is at the interrogation position.
32. The method according to claim 30 wherein said analytical output
is obtained by fluorescence spectroscopy.
33. The method according to claim 32 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
34. The method according to claim 30 wherein said analytical output
is obtained by mass spectrometry.
35. The method according to claim 32 wherein said released
identifier nucleotide includes a fluorescent label.
36. The method according to claim 34 wherein said released
identifier nucleotide includes a fluorescent label.
37. The method according to claim 30, wherein said nucleic acid
target sequence is selected from the group consisting of
deoxyribonucleic acid and ribonucleic acid.
38. The method according to claim 37, further comprising a first
probe, a second probe, a third probe and a fourth probe.
39. The method according to claim 38, wherein said interrogation
position of said first probe comprises a nucleic acid residue that
is a deoxyadenosine or adenosine residue, said interrogation
position of said second probe comprises a nucleic acid residue that
is a deoxythymidine or uridine residue, said interrogation position
of said third probe comprises a nucleic acid residue that is a
deoxyguanosine or guanosine residue, and said fourth nucleic acid
probe comprises a nucleic acid residue that is a deoxycytosine or
cytosine residue.
40. The method according to claim 30 wherein said sample to be
assayed comprises a plurality of endogenous nucleic acid target
sequences in which the presence or absence of a plurality of
specific bases is interrogated.
41. The method according to claim 40 wherein the analytical output
obtained when one of said exogenous nucleic acid probes hybridizes
with partial complementarity to one target nucleic acid sequence is
greater than the analytical output when all of the nucleic acid
probes hybridize with total complementarity to their respective
nucleic acid target sequences.
42. The method according to claim 40 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with
partial complementarity to one target endogenous nucleic acid
sequence is less than the analytical output when all of the nucleic
acid probes hybridize with total complementarity to their
respective nucleic acid target sequences.
43. The method according to claim 40 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with total
complementarity to one endogenous nucleic acid target sequence is
greater than the analytical output when all of the nucleic acid
probes hybridize with partial complementarity to their respective
nucleic acid target sequences.
44. The method according to claim 40 wherein the analytical output
obtained when one of said nucleic acid probes hybridize with total
complementarity to one target nucleic acid sequence is less than
the analytical output when all of the nucleic acid probes hybridize
with partial complementarity to their respective nucleic acid
target sequences.
45. The method according to claim 30 wherein said enzyme whose
activity is to release nucleotides is a template-dependent
polymerase that, in the presence of pyrophosphate ions,
depolymerizes hybridized nucleic acids whose bases in the
3'terminal region of the probe are matched with total
complementarity.
46. The method according to claim 30 wherein said enzyme whose
activity is to release nucleotides exhibits a
3'.fwdarw.5'-exonuclease activity, depolymerizing hybridized
nucleic acids having one or more mismatched bases at the
3'-terminus of the hybridized probe.
47. A method for determining the presence or absence of a first
endogenous nucleic acid target in a nucleic acid sample containing
that target or a substantially identical second target that
comprises the steps of: (A) admixing said sample to be assayed with
one or more nucleic acid probes to form a hybridization
composition, wherein said first and second endogenous nucleic acid
targets comprise a region of sequence identity except for at least
a single nucleotide at a predetermined position that differs
between the endogenous targets, and wherein said nucleic acid probe
(i) is substantially complementary to said nucleic acid target
region of sequence identity and comprises at least one nucleotide
at an interrogation position, said interrogation position of the
probe being aligned with said predetermined position of a target
when a target and probe are hybridized and (ii) includes an
identifier nucleotide in the 3'-terminal region; (B) maintaining
said hybridization composition for a time period sufficient to form
a treated sample wherein the nucleotide at said interrogation
position of said probe is aligned with the nucleotide at said
predetermined position of said target in said region of identity;
(C) admixing the treated sample with a depolymerizing amount an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a hybridized nucleic acid probe to form a
treated reaction mixture; (D) maintaining the treated reaction
mixture for a time period sufficient to release identifier
nucleotide and depolymerize said hybridized nucleic acid probe; and
(E) analyzing for the presence of released identifier nucleotide to
obtain an analytical output, said analytical output indicating the
presence or absence of said nucleotide at said predetermined region
and thereby the presence or absence of a first or second nucleic
acid target.
48. The method according to claim 47 wherein said analytical output
is obtained by fluorescence spectroscopy.
49. The method according to claim 48 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
50. The method according to claim 47 wherein said analytical output
is obtained by mass spectrometry.
51. The method according to claim 47 wherein said released
identifier nucleotide includes a fluorescent label.
52. The method according to claim 47 wherein said identifier
nucleotide is a nucleoside triphosphate.
53. The method according to claim 52 wherein said analytical output
is obtained by luminescence spectroscopy.
54. The method according to claim 53 wherein said analytical output
is obtained by absorbance spectroscopy.
55. The method according to claim 47 wherein said nucleic acid
target sequence is selected from the group consisting of
deoxyribonucleic acid and ribonucleic acid.
56. The method according to claim 47 further comprising a first
probe and a second probe.
57. The method according to claim 56 wherein said sample to be
assayed comprises a plurality first nucleic acid targets and second
substantially identical nucleic acid targets.
58. The method according to claim 57 wherein said first probe
comprises a nucleotide at said interrogation position that is
complementary to a first target nucleic acid at said predetermined
position, and said second probe comprises a nucleotide at the
interrogation position that is complementary to a second target
nucleic acid at said predetermined position.
59. The method according to claim 57 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with
partial complementarity to one target nucleic acid sequence is
greater than the analytical output when all of the nucleic acid
probes hybridize with total complementarity to their respective
nucleic acid target sequences.
60. The method according to claim 57 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with
partial complementarity to one target nucleic acid sequence is less
than the analytical output when all of the nucleic acid probes
hybridize with total complementarity to their respective nucleic
acid target sequences.
61. The method according to claim 57 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with total
complementarity to one nucleic acid target sequence is greater than
the analytical output when all of the nucleic acid probes hybridize
with partial complementarity to their respective nucleic acid
target sequences.
62. The method according to claim 57 wherein the analytical output
obtained when one of said nucleic acid probes hybridizes with total
complementarity to one target nucleic acid sequence is less than
the analytical output when all of the nucleic acid probes hybridize
with partial complementarity to their respective nucleic acid
target sequences.
63. The method according to claim 47 wherein said enzyme whose
activity is to release nucleotides is a template-dependent
polymerase that, in the presence of pyrophosphate ions,
depolymerizes hybridized nucleic acids whose bases in the
3'-terminal region are matched with total complementarity.
64. The method according to claim 47 wherein said enzyme whose
activity is to release nucleotides exhibits a
3'.fwdarw.5'-exonuclease activity, depolymerizing hybridized
nucleic acids having one or more mismatched bases in the
3'-terminal region of the hybridized probe.
65. A method for determining the number of known sequence repeats
present in a nucleic acid target sequence in a nucleic acid sample
that comprises the steps of: (A) providing a plurality of separate
treated samples, each treated sample containing a nucleic acid
target sequence hybridized with a nucleic acid probe wherein (a)
the nucleic acid target sequence contains (i) a plurality of known
sequence repeats and (ii) a non-repeated region downstream of the
repeats, and (b) the nucleic acid probe is one of a plurality of
different probes wherein said probes differ in the number of
complementary sequence repeats contained therein, each nucleic acid
probe containing (i) a plurality of sequence repeats complementary
to the known sequence repeat of alleles of the target nucleic acid,
(ii) an identifier nucleotide in the 3'-terminal region of the
probe and (iii) a 5'-terminal locker sequence that is complementary
to the non-repeated region of the target and comprises 1 to about
20 nucleotides. (B) admixing each treated sample with a
depolymerizing amount of an enzyme whose activity is to release one
or more nucleotides from the 3'-terminus of a hybridized nucleic
acid probe to form a treated depolymerization reaction mixture; (C)
maintaining the treated depolymerization reaction mixtures for a
time period sufficient to permit the enzyme to depolymerize
hybridized nucleic acid probe and release identifier nucleotide
therefrom; and (D) analyzing the samples for the presence of
released identifier nucleotide to obtain an analytical output
indicative of the number of sequence repeats present in said
nucleic acid target sequence.
66. The method according to claim 65 wherein said nucleic acid
sample comprises two nucleic acid target sequences representing
alleles and is homozygous with respect to the number of sequence
repeats in the two alleles.
67. The method according to claim 65 wherein said nucleic acid
sample comprises two nucleic acid target sequences representing
alleles and is heterozygous with respect to the number of sequence
repeats in the two alleles.
68. The method according to claim 65 wherein said identifier
nucleotide is a nucleotide that is part of the repeated
sequence.
69. The method according to claim 65 wherein said identifier
nucleotide of the probe sequence is complementary to a
non-repeating sequence located 3' to the repeated sequences of the
target nucleic acid.
70. The method according to claim 69 wherein said identifier
nucleotide is present in a sequence containing 1 to about 20
nucleic acids that is complementary to a non-repeating sequence
located 3' to the repeated sequences of the target nucleic
acid.
71. The method according to claim 65 wherein a repeated known
sequence present in a nucleic acid target sequence has a length of
2 to about 24 bases per repeat.
72. A one-pot method for determining the presence or absence of at
least one predetermined endogenous nucleic acid target sequence in
a nucleic acid sample that comprises the steps of: (A) admixing a
treated sample that may contain said predetermined endogenous
nucleic acid target sequence hybridized to a nucleic acid probe
whose 3'-terminal region is completely complementary to said
predetermined nucleic acid target sequence and includes an
identifier nucleotide with (i) a depolymerizing amount of an enzyme
whose activity in the presence of pyrophosphate is to release
identifier nucleotide as a nucleoside triphosphate from the
hybridized nucleic acid probe, (ii) adenosine 5' diphosphate, (iii)
pyrophosphate and (iv) NDPK to form a treated reaction mixture; (b)
maintaining the treated reaction mixture at a temperature of about
25 to about 80 degrees C. for a time period sufficient to permit
the enzyme to depolymerize hybridized nucleic acid probe, release
an identifier nucleotide in the 3'-terminal region as a nucleoside
triphosphate and to convert said nucleoside triphosphate and said
adenosine 5' diphosphate to adenosine 5' triphosphate; and (d)
analyzing for the presence of adenosine 5' triphosphate to obtain
an analytical output, the analytical output indicating the presence
or absence of at least one said nucleic acid target sequence.
73. The method according to claim 72 wherein said analytical output
is obtained by luminescence spectroscopy.
74. The method according to claim 72 including the further steps of
forming said treated sample by (a) admixing a sample to be assayed
with one or more nucleic acid probes to form a hybridization
composition, wherein the 3'-terminal region of said nucleic acid
probe (i) hybridizes with partial or total complementarity to a
nucleic acid target sequence when that sequence is present in the
sample and (ii) includes an identifier nucleotide; (b) maintaining
said hybridization composition for a time period sufficient to form
a treated sample that may contain said one predetermined nucleic
acid target sequence hybridized with a nucleic acid probe.
75. The method according to claim 72 wherein said depolymerizing
enzyme maintains activity at 60-90.degree. C.
76. The method according to claim 72 wherein said depolymerizing
enzyme is selected from the group consisting of the Tne triple
mutant DNA polymerase, Bst DNA polymerase, Ath DNA polymerase, Taq
DNA polymerase and Tvu DNA polymerase.
77. The method according to claim 72 wherein said NDPK is that
encoded by Pyrococcus furiosis.
78. A method for determining whether the nucleic acid target
sequence in a nucleic acid sample is an allele from a homozygous or
heterozygous locus that comprises the steps of: (A) providing a
plurality of separate treated samples, each sample containing (a) a
nucleic acid target sequence hybridized with (b) a nucleic acid
probe, said nucleic acid target sequence being that of a first
allele, a second allele or a mixture of said first and second
alleles from a locus of interest of said nucleic acid target, said
alleles differing in sequence at an interrogation position, said
nucleic acid probe containing an identifier nucleotide in the
3-terminal region that is aligned at an interrogation nucleotide
position of the target sequence when said probe and target are
hybridized; (B) admixing each treated sample with a depolymerizing
amount of an enzyme whose activity is to release one or more
nucleotides from the 3'-terminus of a hybridized nucleic acid probe
to form a treated reaction mixture; (C) maintaining the treated
reaction mixtures for a time period sufficient to permit the enzyme
to depolymerize hybridized nucleic acid probe and an release
identifier nucleotide; and (D) analyzing the samples for the
presence of released identifier nucleotide to obtain an analytical
output, the analytical output indicating whether the nucleic acid
target sequence in a nucleic acid sample is an allele from a
homozygous or a heterozygous locus.
79. The method of claim 78 wherein said analyzing comprises
analyzing the samples for the quantity of released identifier
nucleotide to obtain an analytical output, the analytical output
indicating whether the nucleic acid target sequence in the nucleic
acid sample is homozygous or heterozygous when compared to the
analytical output of an appropriate control.
80. The method according to claim 78 wherein said analytical output
indicates which allele is present when the nucleic acid target
sequence in a nucleic acid sample is homozygous at the locus of
interest.
81. The method according to claim 78 wherein said enzyme whose
activity is to release nucleotides is a template-dependent
polymerase that, in the presence of pyrophosphate ions,
depolymerizes hybridized nucleic acids whose bases in the
3'-terminal region are completely complementary to bases of said
nucleic acid target.
82. The method according to claim 79 wherein said analytical output
is obtained by luminescence spectroscopy.
83. A method for determining the loss of heterozygosity of a locus
of an allele that comprises the steps of: (A) providing a plurality
of separate treated samples, each sample containing (a) a nucleic
acid target sequence hybridized with (b) a nucleic acid probe, said
nucleic acid target sequence being that of a first allele or a
mixture of said first allele and a second allele of said nucleic
acid target, said alleles differing in sequence at an interrogation
position, said nucleic acid probe containing a 3'-terminal region
that hybridizes to a region of said nucleic acid target sequence
containing said interrogation nucleotide position when said probe
and target are hybridized and an identifier nucleotide; (B)
admixing each treated sample with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a hybridized nucleic acid probe to form a
treated reaction mixture; (C) maintaining the treated reaction
mixtures for a time period sufficient to depolymerize hybridized
nucleic acid probe and an release identifier nucleotide; and (D)
analyzing the samples for the quantity of released identifier
nucleotide to obtain an analytical output, the analytical output
indicating whether the nucleic acid target sequence in a nucleic
acid sample has lost heterozygosity at the locus of the allele.
84. The method of claim 83 wherein the quantity of said released
identifier nucleotide for said first allele is substantially less
that the quantity of said released identifier nucleotide for said
first allele of a known heterozygous control, and the quantity of
said released identifier nucleotide for said second allele is
substantially similar to the quantity of said released identifier
nucleotide for said second allele of a known heterozygous control,
indicating a loss of heterozygosity at the locus of said first
allele.
85. The method of claim 83 wherein the quantity of said released
identifier nucleotide for said second allele is substantially less
that the quantity of said released identifier nucleotide for said
second allele of a known heterozygous control, and the quantity of
said released identifier nucleotide for said first allele is
substantially similar to the quantity of said released identifier
nucleotide for said first allele of a known heterozygous control,
indicating a loss of heterozygosity at the locus of said second
allele.
86. The method according to claim 83 wherein said analytical output
is obtained by luminescence spectroscopy.
87. The method according to claim 83 wherein said analytical output
is obtained by absorbance spectrometry.
88. The method according to claim 83 wherein said analytical output
is obtained by fluorescence spectroscopy.
89. The method according to claim 88 wherein said released
identifier nucleotide includes a fluorescent label.
90. The method according to claim 89 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
91. The method according to claim 83 wherein said analytical output
is obtained by mass spectrometry.
92. The method according to claim 91 wherein said released
identifier nucleotide includes a fluorescent label.
93. The method according to claim 92 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
94. The method according to claim 83 wherein said enzyme whose
activity is to release nucleotides is a template-dependent
polymerase that, in the presence of pyrophosphate ions,
depolymerizes hybridized nucleic acids whose bases in the
3'-terminal region are completely complementary to bases of said
nucleic acid target.
95. The method according to claim 94 wherein the quantity of said
released identifier nucleotide for said first allele is
substantially less than the quantity of said released identifier
nucleotide for said first and second alleles, indicating a loss of
heterozygosity at the locus of said first allele.
96. The method according to claim 94 wherein the quantity of said
released identifier nucleotide for said second allele is
substantially less than the quantity of said released identifier
nucleotide for said first and second alleles, indicating a loss of
heterozygosity at the locus of said second allele.
97. A method for determining the presence of trisomy of an allele
that comprises the steps of: (A) providing a plurality of separate
treated samples, each sample containing (a) a nucleic acid target
sequence hybridized with (b) a nucleic acid probe, said nucleic
acid target sequence being that of a first allele, a second allele
or a mixture of said first and second alleles of said nucleic acid
target, said alleles differing in sequence at an interrogation
position, said nucleic acid probe containing a 3'-terminal region
that hybridizes to a region of said nucleic acid target sequence
containing said interrogation nucleotide position when said probe
and target are hybridized and an identifier nucleotide; (B)
admixing each treated sample with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a hybridized nucleic acid probe to form a
treated reaction mixture; (C) maintaining the treated reaction
mixtures for a time period sufficient to depolymerize hybridized
nucleic acid probe and an release identifier nucleotide; and (D)
analyzing the samples for released identifier nucleotide to obtain
an analytical output, the quantity of said analytical output
relative to an analytical output of a control sample indicating
whether a trisomy is present in the nucleic acid target
sequence.
98. The method of claim 97 wherein the ratio of the quantity of
said released identifier nucleotide for said first and second
allele is about 3 to zero, compared to the ratio of the quantity of
said released identifier nucleotide for said first and second
allele of a known heterozygous control of about 1 to 1, indicating
a trisomy at the locus of said first allele.
99. The method of claim 97 wherein the ratio of the quantity of
said released identifier nucleotide for said first and second
allele is about zero to 3, compared to the ratio of the quantity of
said released identifier nucleotide for said first and second
allele of a known heterozygous control of about 1 to 1, indicating
a trisomy at the locus of said second allele.
100. The method of claim 97 wherein the ratio of the quantity of
said released identifier nucleotide for said first and second
allele is about 2 to 1, compared to the ratio of the quantity of
said released identifier nucleotide for said first and second
allele of a known heterozygous control of about 1 to 1, indicating
a trisomy having two copies of the locus of said first allele and
one copy of the locus of said second allele.
101. The method of claim 97 wherein the ratio of the quantity of
said released identifier nucleotide for said first and second
allele is about 1 to 2, compared to the ratio of the quantity of
said released identifier nucleotide for said first and second
allele of a known heterozygous control of about 1 to 1, indicating
a trisomy having one copy of the locus of said first allele and two
copies of the locus of said second allele.
102. The method according to claim 97 wherein said analytical
output is obtained by luminescence spectroscopy.
103. The method according to claim 97 wherein said analytical
output is obtained by absorbance spectrometry.
104. The method according to claim 97 wherein said analytical
output is obtained by fluorescence spectroscopy.
105. The method according to claim 104 wherein said released
identifier nucleotide includes a fluorescent label.
106. The method according to claim 105 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
107. The method according to claim 97 wherein said analytical
output is obtained by mass spectrometry.
108. The method according to claim 107 wherein said released
identifier nucleotide includes a fluorescent label.
109. The method according to claim 108 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
110. The method according to claim 97 wherein said enzyme whose
activity is to release nucleotides is a template-dependent
polymerase that, in the presence of pyrophosphate ions,
depolymerizes hybridized nucleic acids whose bases in the
3'-terminal region are completely complementary to bases of said
nucleic acid target.
111. The method according to claim 97 wherein the quantity of said
released identifier nucleotide for said first allele is
substantially greater than the quantity of said released identifier
nucleotide of a homozygous control, indicating that said nucleic
acid target sequence has a trisomy.
112. The method according to claim 97 wherein the quantity of said
released identifier nucleotide for said second allele is
substantially greater than the quantity of said released identifier
nucleotide of a homozygous control, indicating that said nucleic
acid target sequence has a trisomy.
113. A kit for determining the presence or absence of a
predetermined endogenous nucleic acid target sequence in a nucleic
acid sample comprising: (A) an enzyme whose activity is to release
one or more nucleotides from the 3' terminus of a hybridized
nucleic acid probe; and (B) at least one nucleic acid probe, said
nucleic acid probe being complementary to said endogenous nucleic
acid target sequence.
114. A kit for determining the presence or absence of at least one
endogenous predetermined nucleic acid target sequence in a nucleic
acid sample comprising: (A) an enzyme whose activity in the
presence of pyrophosphate is to release identifier nucleotide as a
nucleoside triphosphate from hybridized nucleic acid probe; (B)
adenosine 5' diphosphate; (C) pyrophosphate; (D) a nucleoside
diphosphate kinase; and (E) at least one nucleic acid probe, said
nucleic acid probe being complementary to said predetermined
endogenous nucleic acid target sequence.
115. A kit for determining the presence or absence of a
predetermined endogenous nucleic acid target sequence in a nucleic
acid sample comprising: (A) an enzyme whose activity is to release
one or more nucleotides from the 3' terminus of a hybridized
nucleic acid probe; and (B) instructions for use.
116. A method for determining the presence or absence of a nucleic
acid target sequence containing an interrogation position in a
nucleic acid sample that comprises the steps of: (A) providing a
treated sample that contains a nucleic acid sample that may include
said nucleic acid target sequence hybridized with a nucleic acid
probe that is comprised of three sections, (i) a first section that
contains the probe 3'-terminal about 10 to about 30 nucleotides
that are complementary to the nucleic acid target sequence at
positions beginning about 1 to about 30 nucleic acids downstream of
said interrogation position of the target sequence, (ii) a
5'-terminal region of about 10 to about 200 nucleic acids in length
and having the identical sequence of said nucleic acid target
sequence, and (iii) an optional third section that contains zero to
about 50 nucleic acids that are not complementary to said nucleic
acid sample, and ; (B) extending said nucleic acid probe in a 3'
direction to form a second probe hybridized to the nucleic acid
sample as a second hybrid; (D) denaturing said second hybrid to
separate said second probe from said nucleic acid target sequence;
(E) renaturing said aqueous composition to form hairpin structures
from said second probe; (F) admixing the hairpin
structure-containing composition with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a nucleic acid hybrid to form a treated reaction
mixture; (G) maintaining the treated reaction mixture for a time
period sufficient to permit the enzyme to depolymerize hybridized
nucleic acid and release one or more nucleotides from the
3'-terminus therefrom; and (H) analyzing for the presence of
released identifier nucleotide to obtain an analytical output, the
analytical output indicating the presence or absence of said
nucleic acid target sequence.
117. A method for determining the presence or absence of a nucleic
acid target sequence, or a specific base within the target
sequence, in a nucleic acid sample, that comprises the steps of:
(A) providing a treated sample that contains a nucleic acid sample
that may include a nucleic acid target sequence hybridized with a
first nucleic acid probe as a first hybrid, said first probe being
comprised of at least two sections, a first section containing the
probe 3'-terminal about 10 to about 30 nucleotides that are
complementary to the target nucleic acid sequence at a position
beginning about 5 to about 30 nucleotides downstream of the target
interrogation position, a second section of the first probe
containing about 5 to about 30 nucleotides that are a repeat of the
target sequence from the interrogation position to about 10 to
about 30 nucleotides downstream of the interrogation position that
does not hybridize to said first section of the probe, and an
optional third section of the probe located between the first and
second sections of the probe that is zero to about 50 nucleotides
in length and comprises a sequence that does not hybridize to
either the first or second section of the probe; (B) extending the
first hybrid in the treated sample at the 3'-end of the first
probe, thereby extending the first probe past the interrogation
position and forming an extended first hybrid that includes an
interrogation position; (C) denaturing an aqueous composition of
the extended first hybrid to separate the two nucleic acid strands
and form an aqueous composition containing a separated target
nucleic acid and a separated extended first probe; (D) annealing
the extended first probe to a second probe that is about 10 to
about 30 nucleotides in length and is complementary to the extended
first probe at a position beginning about 5 to about 2000
nucleotides downstream of the interrogation position in the
extended first probe, thereby forming a second hybrid; (E)
extending the second hybrid at the 3'-end of the second probe until
that extension reaches the 5'-end of the extended first probe,
thereby forming a second extended hybrid containing a second
extended probe whose 3'-region includes an identifier nucleotide;
(F) denaturing an aqueous composition of the extended second hybrid
to separate the two nucleic acid strands and form an aqueous
composition containing separated extended first and second probes;
(G) cooling the aqueous composition to form a hairpin structure
from the separated extended second probe to form a hairpin
structure-containing composition; (H) admixing the hairpin
structure-containing composition with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a nucleic acid hybrid to form a treated reaction
mixture; (I) maintaining the reaction mixture for a time period
sufficient to release 3'-terminal region identifier nucleotides;
and (J) analyzing for the presence of released identifier
nucleotide to obtain an analytical output, the analytical output
indicating the presence or absence of said predetermined nucleic
acid target sequence or a specific base within the target
sequence.
118. The method according to claim 117 wherein said analytical
output is obtained by luminescence spectroscopy.
119. The method according to claim 117 wherein said analytical
output is obtained by fluorescence spectroscopy.
120. The method according to claim 119 wherein said released
identifier nucleotide includes a fluorescent label.
121. The method according to claim 120 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
122. The method according to claim 117 wherein said analytical
output is obtained by mass spectrometry.
123. The method according to claim 122 wherein said released
identifier nucleotide includes a fluorescent label.
124. The method according to claim 122 wherein said identifier
nucleotide is fluorescently labeled after release from said
hybrid.
125. The method according to claim 117 wherein said analytical
output is obtained by absorbance spectroscopy.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/358,972, filed on Jul. 21, 1999, which is a continuation-in-part
of U.S. Ser. No. 09/252,436, filed on Feb. 18, 1999, which is a
continuation-in-part of U.S. Ser. No. 09/042,287, filed Mar. 13,
1998, all of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to nucleic acid detection. More
specifically, the invention relates to the detection of targeted,
predetermined endogenous nucleic acid sequences in nucleic acid
target hybrids, and the various applications of their
detection.
BACKGROUND OF THE INVENTION
[0003] Methods to detect nucleic acids and to detect specific
nucleic acids of interest provide a foundation upon which the large
and rapidly growing field of molecular biology is built. There is
constant need for alternative methods and products. The reasons for
selecting one method over another are varied, and include a desire
to avoid radioactive materials, the lack of a license to use a
technique, the cost or availability of reagents or equipment, the
desire to minimize the time spent or the number of steps, the
accuracy or sensitivity for a certain application, the ease of
analysis, or the ability to automate the process.
[0004] The detection of nucleic acids or specific nucleic acids is
often a portion of a process rather than an end in itself. There
are many applications of the detection of nucleic acids in the art,
and new applications are always being developed. The ability to
detect and quantify nucleic acids is useful in detecting
microorganisms, viruses and biological molecules, and thus affects
many fields, including human and veterinary medicine, food
processing and environmental testing. Additionally, the detection
and/or quantification of specific biomolecules from biological
samples (e.g. tissue, sputum, urine, blood, semen, saliva) has
applications in forensic science, such as the identification and
exclusion of criminal suspects and paternity testing as well as
medical diagnostics.
[0005] Some general methods to detect nucleic acids are not
dependent upon a priori knowledge of the nucleic acid sequence. A
nucleic acid detection method that is not sequence specific, but is
RNA specific is described in U.S. Pat. No. 4,735,897, where RNA is
depolymerized using a polynucleotide phosphorylase (PNP) in the
presence of phosphate or using a ribonuclease. PNP stops
depolymerizing when a double-stranded RNA segment is encountered,
sometimes as the form of secondary structure of single-stranded
RNA, as is common in ribosomal RNA, transfer RNA, viral RNA, and
the message portion of mRNA. PNP depolymerization of the
polyadenylated tail of MRNA in the presence of inorganic phosphate
forms ADP. Alternatively, depolymerization using a ribonuclease
forms AMP. The formed AMP is converted to ADP with myokinase, and
ADP is converted into ATP by pyruvate kinase or creatine
phosphokinase. Either the ATP or the byproduct from the
organophosphate co-reactant (pyruvate or creatine) is detected as
an indirect method of detecting mRNA.
[0006] In U.S. Pat. No. 4,735,897, ATP is detected by a luciferase
detection system. In the presence of ATP and oxygen, luciferase
catalyzes the oxidation of luciferin, producing light that can then
be quantified using a luminometer. Additional products of the
reaction are AMP, pyrophosphate and oxyluciferin.
[0007] Duplex DNA can be detected using intercalating dyes such as
ethidium bromide. Such dyes are also used to detect hybrid
formation.
[0008] Hybridization methods to detect nucleic acids are dependent
upon knowledge of the nucleic acid sequence. Many known nucleic
acid detection techniques depend upon specific nucleic acid
hybridization in which an oligonucleotide probe is hybridized or
annealed to nucleic acid in the sample or on a blot, and the
hybridized probes are detected.
[0009] A traditional type of process for the detection of
hybridized nucleic acid uses labeled nucleic acid probes to
hybridize to a nucleic acid sample. For example, in a Southern blot
technique, a nucleic acid sample is separated in an agarose gel
based on size and affixed to a membrane, denatured, and exposed to
the labeled nucleic acid probe under hybridizing conditions. If the
labeled nucleic acid probe forms a hybrid with the nucleic acid on
the blot, the label is bound to the membrane. Probes used in
Southern blots have been labeled with radioactivity, fluorescent
dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase and
acridinium esters.
[0010] Another type of process for the detection of hybridized
nucleic acid takes advantage of the polymerase chain reaction
(PCR). The PCR process is well known in the art (U.S. Pat. Nos.
4,683,195, 4,683,202, and 4,800,159). To briefly summarize PCR,
nucleic acid primers, complementary to opposite strands of a
nucleic acid amplification target sequence, are permitted to anneal
to the denatured sample. A DNA polymerase (typically heat stable)
extends the DNA duplex from the hybridized primer. The process is
repeated to amplify the nucleic acid target. If the nucleic acid
primers do not hybridize to the sample, then there is no
corresponding amplified PCR product. In this case, the PCR primer
acts as a hybridization probe. PCR-based methods are of limited use
for the detection of nucleic acid of unknown sequence.
[0011] In a PCR method, the amplified nucleic acid product may be
detected in a number of ways, e.g. incorporation of a labeled
nucleotide into the amplified strand by using labeled primers.
Primers used in PCR have been labeled with radioactivity,
fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline
phosphatase, acridinium esters, biotin and jack bean urease. PCR
products made with unlabeled primers may be detected in other ways,
such as electrophoretic gel separation followed by dye-based
visualization.
[0012] Fluorescence techniques are also known for the detection of
nucleic acid hybrids. U.S. Pat. No. 5,691,146 describes the use of
fluorescent hybridization probes that are fluorescence-quenched
unless they are hybridized to the target nucleic acid sequence.
U.S. Pat. No. 5,723,591 describes fluorescent hybridization probes
that are fluorescence-quenched until hybridized to the target
nucleic acid sequence, or until the probe is digested. Such
techniques provide information about hybridization, and are of
varying degrees of usefulness for the determination of single base
variances in sequences. Some fluorescence techniques involve
digestion of a nucleic acid hybrid in a 5'.fwdarw.3' direction to
release a fluorescent signal from proximity to a fluorescence
quencher, for example, TaqMan.RTM. (Perkin Elmer; U.S. Pat. Nos.
5,691,146 and 5,876,930).
[0013] Enzymes having template-specific polymerase activity for
which some 3'.fwdarw.5' depolymerization activity has been reported
include E. coli DNA Polymerase (Deutscher and Kornberg, J. Biol.
Chem., 244(11):3019-28 (1969)), T7 DNA Polymerase (Wong et al.,
Biochemistry 30:526-37 (1991); Tabor and Richardson, J. Biol. Chem.
265:8322-28 (1990)), E. coli RNA polymerase (Rozovskaya et al.,
Biochem. J. 224:645-50 (1994)), AMV and RLV reverse transcriptases
(Srivastava and Modak, J. Biol. Chem. 255:2000-4 (1980)), and HIV
reverse transcriptase (Zinnen et al., J. Biol. Chem. 269:24195-202
(1994)). A template-dependent polymerase for which 3' to 5'
exonuclease activity has been reported on a mismatched end of a DNA
hybrid is phage 29 DNA polymerase (de Vega, M. et al. EMBO J.,
15:1182-1192, 1996).
[0014] A variety of methodologies currently exist for detection of
single nucleotide polymorphisms (SNPs) that are present in genomic
DNA. SNPs are DNA point mutations or insertions/deletions that are
present at measurable frequencies in the population. SNPs are the
most common variations in the genome. SNPs occur at defined
positions within genomes and can be used for gene mapping, defining
population structure, and performing functional studies. SNPs are
useful as markers because many known genetic diseases are caused by
point mutations and insertions/deletions.
[0015] In rare cases where an SNP alters a fortuitous restriction
enzyme recognition sequence, differential sensitivity of the
amplified DNA to cleavage can be used for SNP detection. This
technique requires that an appropriate restriction enzyme site be
present or introduced in the appropriate sequence context for
differential recognition by the restriction endonuclease. After
amplification, the products are cleaved by the appropriate
restriction endonuclease and products are analyzed by gel
electrophoresis and subsequent staining. The throughput of analysis
by this technique is limited because samples require processing,
gel analysis, and significant interpretation of data before SNPs
can be accurately determined.
[0016] Single strand conformational polymorphism (SSCP) is a second
technique that can detect SNPs present in an amplified DNA segment
(Hayashi, K. Genetic Analysis: Techniques and Applications 9:73-79,
1992). In this method, the double stranded amplified product is
denatured and then both strands are allowed to reanneal during
electrophoresis in non-denaturing polyacrylamide gels. The
separated strands assume a specific folded conformation based on
intramolecular base pairing. The electrophoretic properties of each
strand are dependent on the folded conformation. The presence of
single nucleotide changes in the sequence can cause a detectable
change in the conformation and electrophoretic migration of an
amplified sample relative to wild type samples, allowing SNPs to be
identified. In addition to the limited throughput possible by
gel-based techniques, the design and interpretation of SSCP based
experiments can be difficult. Multiplex analysis of several samples
in the same SSCP reaction is extremely challenging. The sensitivity
required in mutation detection and analysis has led most
investigators to use radioactively labeled PCR products for this
technique.
[0017] In the amplification refractory mutation system (ARMS, also
known as allele specific PCR or ASPCR), two amplification reactions
are used to determine if a SNP is present in a DNA sample (Newton
et al. Nucl Acids Res 17:2503, 1989; Wu et al. PNAS 86:2757, 1989).
Both amplification reactions contain a common primer for the target
of interest. The first reaction contains a second primer specific
for the wild type product which will give rise to a PCR product if
the wild type gene is present in the sample. The second PCR
reaction contains a primer that has a single nucleotide change at
or near the 3' end that represents the base change that is present
in the mutated form of the DNA. The second primer, in conjunction
with the common primer, will only function in PCR if genomic DNA
that contains the mutated form of genomic DNA is present. This
technique requires duplicate amplification reactions to be
performed and analyzed by gel electrophoresis to ascertain if a
mutated form of a gene is present. In addition, the data must be
manually interpreted.
[0018] Single base extension is a technique that allows the
detection of SNPs by hybridizing a single strand DNA probe to a
captured DNA target (Nikiforov, T. et al. Nucl Acids Res
22:4167-4175). Once hybridized, the single strand probe is extended
by a single base with labeled dideoxynucleotides. The labeled,
extended products are then detected using calorimetric or
fluorescent methodologies.
[0019] A variety of technologies related to real-time (or kinetic)
PCR have been adapted to perform SNP detection. Many of these
systems are platform based, and require specialized equipment,
complicated primer design, and expensive supporting materials for
SNP detection. In contrast, the process of this invention has been
designed as a modular technology that can use a variety of
instruments that are suited to the throughput needs of the
end-user. In addition, the coupling of luciferase detection
sensitivity with standard oligonucleotide chemistry and
well-established enzymology provides a flexible and open system
architecture. Alternative analytical detection methods, such as
mass spectroscopy, HPLC, and fluorescence detection methods can
also be used in the process of this invention, providing additional
assay flexibility.
[0020] SNP detection using real-time amplification relies on the
ability to detect amplified segments of nucleic acid as they are
during the amplification reaction. Three basic real-time SNP
detection methodologies exist: (i) increased fluorescence of double
strand DNA specific dye binding, (ii) decreased quenching of
fluorescence during amplification, and (iii) increased fluorescence
energy transfer during amplification (Wittwer, C. et al.
Biotechniques 22:130-138, 1997). All of these techniques are
non-gel based and each strategy will be briefly discussed.
[0021] A variety of dyes are known to exhibit increased
fluorescence in response to binding double stranded DNA. This
property is utilized in conjunction with the amplification
refractory mutation system described above to detect the presence
of SNP. Production of wild type or mutation containing PCR products
are continuously monitored by the increased fluorescence of dyes
such as ethidium bromide or SYBER Green as they bind to the
accumulating PCR product. Note that dye binding is not selective
for the sequence of the PCR product, and high non-specific
background can give rise to false signals with this technique.
[0022] A second SNP detection technology for real time PCR, known
generally as exonuclease primers (TaqMan.RTM. probes), utilizes the
5' exonuclease activity of thermostable polymerases such as Taq to
cleave dual-labeled probes present in the amplification reaction
(Wittwer, C. et al. Biotechniques 22:130-138, 1997; Holland, P et
al PNAS 88:7276-7280, 1991). While complementary to the PCR
product, the probes used in this assay are distinct from the PCR
primer and are dually-labeled with both a molecule capable of
fluorescence and a molecule capable of quenching fluorescence. When
the probes are intact, intramolecular quenching of the fluorescent
signal within the DNA probe leads to little signal. When the
fluorescent molecule is liberated by the exonuclease activity of
Taq during amplification, the quenching is greatly reduced leading
to increased fluorescent signal.
[0023] An additional form of real-time PCR also capitalizes on the
intramolecular quenching of a fluorescent molecule by use of a
tethered quenching moiety. The molecular beacon technology utilizes
hairpin-shaped molecules with an internally-quenched fluorophore
whose fluorescence is restored by binding to a DNA target of
interest (Kramer, R. et al. Nat. Biotechnol. 14:303-308, 1996).
Increased binding of the molecular beacon probe to the accumulating
PCR product can be used to specifically detect SNPs present in
genomic DNA.
[0024] A final general fluorescent detection strategy used for
detection of SNPs in real time utilizes synthetic DNA segments
known as hybridization probes in conjunction with a process known
as fluorescence resonance energy transfer (FRET) (Wittwer, C. et
al. Biotechniques 22:130-138, 1997; Bernard, P. et al. Am. J.
Pathol. 153:1055-1061, 1998). This technique relies on the
independent binding of labeled DNA probes on the target sequence.
The close approximation of the two probes on the target sequence
increases resonance energy transfer from one probe to the other,
leading to a unique fluorescence signal. Mismatches caused by SNPs
that disrupt the binding of either of the probes can be used to
detect mutant sequences present in a DNA sample.
[0025] A number of gene-level defects have been implicated in the
etiology of human disease. Researchers have used several techniques
to detect these genetic mutations for prevention or diagnosis of
these disease states. Van Essen et al. [J. Med. Genet. 34:805-12
(1997)] report that 65-70% of Duchenne and Becker muscular
dystrophy patients exhibit rearrangements in the dystrophin gene,
as detected by Southern blotting or multiplex PCR. Microlesions in
these two forms of muscular dystrophy are typically detected using
single strand conformational analysis, heteroduplex analysis, and
the protein truncation test.
[0026] Calvano et al. [Clin. Genet. 52:17-22 (1997)] report the use
of PCR fragments used as fluorescent probes for the detection of
female carriers of Duchenne and Becker muscular dystrophy.
Jongpiputvanich et al. [J. Med. Assoc. Thai. 79(Supp. 1):S15-21
(1996)] report the use of multiplex PCR and microsatellite or STR
analysis for diagnosis and carrier detection in a Duchenne muscular
dystrophy family. Pastore et al. [Mol. Cell. Probes 10:129-37
(1996)] developed a quantitative PCR analysis method using
radiolabeled PCR products for the detection of macrodeletion
carriers of Duchenne and Becker muscular dystrophy. Katayama et al.
[Fetal Diagn. Ther. 9:379-84 (1994)] studied the efficacy of PCR
for prenatal diagnosis of Duchenne muscular dystrophy. These
workers used PCR-restriction fragment length polymorphism analysis,
multiplex PCR, and dinucleotide repeat polymorphism analysis to
diagnose affected male fetuses and detect carrier female fetuses in
the first trimester.
[0027] A polymorphism in the human gap junctional protein connexin
37 was studied as a prognostic marker for atherosclerosis. Boerma
et al. Intern. Med. 246:211-218 (1999). A restriction fragment
length polymorphism in the proline variant of the connexin 37 gene
was used to show that this allele was over-represented in patients
with atherosclerotic plaques. Shohet et al. [Arterioscler. Thromb.
Vasc. Biol. 19:1975-78 (1999)] report the frequency of the -514T
allele of hepatic lipase in white men with coronary artery disease.
In this population, postheparin plasma hepatic lipase activity was
15 to 20% lower in heterozygotes and 30% lower in homozygotes
compared to controls. A novel missense mutation in the presenilin-1
gene was detected in a family with presenile familial Alzheimer's
disease (FAD). Sugiyama et al. Mutat. 14:90 (1999). These workers
report that over 50 such missense mutations in the presenilin-1
gene have been reported in families with FAD. Sensitive, reliable
assays for these and other gene-level defects have several
potential diagnostic and preventative applications in human and
animal health care.
[0028] In summary, there is a need for alternative methods for the
detection of nucleic acid hybrids. There is a great demand for such
methods to determine the presence or absence of nucleic acid
sequences that differ slightly from sequences that might otherwise
be present. There is a great demand for methods to determine the
presence or absence of sequences unique to a particular species in
a sample. There is also a great demand for methods that are more
highly sensitive than the known methods, highly reproducible and
automatable.
[0029] It would be beneficial if another method were available for
detecting the presence of a sought-after, predetermined target
nucleotide sequence or allelic variant. It would also be beneficial
if such a method were operable using a sample size of the microgram
to picogram scale. It would further be beneficial if such a
detection method were capable of providing multiple analyses in a
single assay (multiplex assays). The disclosure that follows
provides such methods.
BRIEF SUMMARY OF THE INVENTION
[0030] A method of this invention is used to determine the presence
or absence of a predetermined (known) endogenous nucleic acid
target sequence in a nucleic acid sample. Such a method utilizes an
enzyme that can depolymerize the 3'-terminus of an oligonucleotide
probe hybridized to a nucleic acid target sequence to release one
or more identifier nucleotides whose presence can then be
determined.
[0031] One embodiment of the invention contemplates a method for
determining the presence or absence of a predetermined endogenous
nucleic acid target sequence in a nucleic acid sample. Thus, the
presence or absence of at least one predetermined endogenous
nucleic acid target sequence is sought to be determined. More than
one such predetermined endogenous target sequence can also be
present in the sample being assayed, and the presence or absence of
more than one predetermined endogenous nucleic acid target sequence
can be determined. The embodiment comprises the following
steps.
[0032] A treated sample is provided that may contain a
predetermined endogenous nucleic acid target sequence hybridized
with a nucleic acid probe that includes an identifier nucleotide in
the 3'-terminal region. The treated sample is admixed with a
depolymerizing amount of an enzyme whose activity is to release one
or more nucleotides from the 3'-terminus of a hybridized nucleic
acid probe to form a treated reaction mixture. The treated reaction
mixture is maintained under depolymerizing conditions for a time
period sufficient to permit the enzyme to depolymerize hybridized
nucleic acid and release identifier nucleotides therefrom.
[0033] An analytical output is obtained by analyzing for the
presence or absence of released identifier nucleotides. The
analytical output indicates the presence or absence of the
nucleotide at the predetermined region, and, thereby, the presence
or absence of a first nucleic acid target. The analytical output is
obtained by various techniques as discussed herein.
[0034] It is contemplated that an analytical output of the methods
of the invention can be obtained in a variety of ways. The
analytical output can be ascertained by luminescence spectroscopy.
In some preferred embodiments, analysis for released 3'-terminal
region indicator nucleotides comprises the detection of ATP, either
by a luciferase detection system (luminescence spectroscopy) or an
NADH detection system (absorbance spectroscopy). In particularly
preferred embodiments where greater sensitivity is desired, ATP
molecules are formed by a phosphate transferring step, for example
using an enzyme such as NDPK in the presence of ADP, from the
nucleoside triphosphates produced by the depolymerizing step. In
some embodiments the ATP is amplified to form a plurality of ATP
molecules. In the ATP detection embodiments, typically the enzyme
(NDPK) for converting nucleotides and added ADP into ATP is present
in the depolymerization reaction with the depolymerizing enzyme,
and when they are present together, they are denoted as a "one pot"
method.
[0035] In an alternative embodiment, the analytical output is
obtained by fluorescence spectroscopy. Use of a wide variety of
fluorescence detection methods is contemplated. In one exemplary
contemplated method, an identifier nucleotide includes a
fluorescent label. An identifier nucleotide can be fluorescently
labeled prior to, or after, release of the identifier nucleotide.
It is also contemplated that other than a released identifier
nucleotide contains a fluorescent tag. In such an embodiment, the
release of nucleotides in a process of the invention is ascertained
by a determination of a difference in the length of the
polynucleotide probe, for example by capillary electrophoresis
imaged by a fluorescent tag at the 5' terminus of the probe or in a
region other than the 3' terminal region.
[0036] In an alternative embodiment the analytical output is
obtained by mass spectrometry. It is preferred here that an
identifier nucleotide be a nucleotide analog or a labeled
nucleotide and have a molecular mass that is different from the
mass of a usual form of that nucleotide, although a difference in
mass is not required. It is also noted that with a fluorescently
labeled identifier nucleotide, the analytical output can also be
obtained by mass spectrometry. It is also contemplated that the
analysis of released nucleotide be conducted by ascertaining the
difference in mass of the probe after a depolymerization step of a
process of the invention.
[0037] In another alternative embodiment, the analytical output is
obtained by absorbance spectroscopy. Such analysis monitors the
absorbance of light in the ultraviolet and visible regions of the
spectrum to determine the presence of absorbing species. In one
aspect of such a process, released nucleotides are separated from
hybridized nucleic acid and other polynucleotides by chromatography
(e.g. HPLC or GC) or electrophoresis (e.g. PAGE or capillary
electrophoresis). Either the released identifier nucleotide or the
remainder of the probe can be analyzed for to ascertain the release
of the identifier nucleotide in a process of the invention. In
another aspect of such a process a label may be incorporated in the
analyzed nucleic acid.
[0038] In a contemplated embodiment, a sample to be assayed is
admixed with one or more nucleic acid probes under hybridizing
conditions to form a hybridization composition. The 3'-terminal
region of the nucleic acid probe hybridizes with partial or total
complementarity to the nucleic acid target sequence when that
sequence is present in the sample. The 3'-terminal region of the
nucleic acid probe includes an identifier nucleotide. The
hybridization composition is maintained under hybridizing
conditions for a time period sufficient to form a treated sample
that may contain said predetermined nucleic acid target sequence
hybridized with a nucleic acid probe. The treated sample is admixed
with a depolymerizing amount of an enzyme whose activity is to
release one or more nucleotides from the 3'-terminus of a
hybridized nucleic acid probe to form a treated reaction mixture.
The treated reaction mixture is maintained under depolymerizing
conditions for a time period sufficient to permit the enzyme to
depolymerize hybridized nucleic acid and release identifier
nucleotides therefrom. The presence of released identifier
nucleotides is analyzed to obtain an analytical output, the
analytical output indicating the presence or absence of the nucleic
acid target sequence. The analytical output may be obtained by
various techniques as discussed above.
[0039] One method of the invention contemplates interrogating the
presence or absence of a specific base in a nucleic acid target
sequence in a sample to be assayed, and comprises the following
steps.
[0040] A hybridization composition is formed by admixing a sample
to be assayed with one or more nucleic acid probes under
hybridizing conditions. The sample to be assayed may contain a
nucleic acid target sequence to be interrogated. The nucleic acid
target comprises at least one base whose presence or absence is to
be identified. The hybridization composition includes at least one
nucleic acid probe that is substantially complementary to the
nucleic acid target sequence and comprises at least one
predetermined nucleotide at an interrogation position, and an
identifier nucleotide in the 3'-terminal region.
[0041] A treated sample is formed by maintaining the hybridization
composition under hybridizing conditions for a time period
sufficient for base pairing to occur when a probe nucleotide at an
interrogation position is aligned with a base to be identified in
the target sequence. A treated reaction mixture is formed by
admixing the treated sample with an enzyme whose activity is to
release one or more identifier nucleotides from the 3'-terminus of
a hybridized nucleic acid probe to depolymerize the hybrid. The
treated reaction mixture is maintained under depolymerizing
conditions for a time period sufficient to permit the enzyme to
depolymerize the hybridized nucleic acid and release an identifier
nucleotide.
[0042] An analytical output is obtained by analyzing for the
presence or absence of released identifier nucleotides. The
analytical output indicates the presence or absence of the specific
base or bases to be identified. The analytical output is obtained
by various techniques, as discussed herein. Preferably, an
identifier nucleotide is at the interrogation position.
[0043] In one aspect of a method of the invention, the nucleic acid
target sequence is selected from the group consisting of
deoxyribonucleic acid and ribonucleic acid.
[0044] A method that identifies the particular base present at an
interrogation position, optionally comprises a first probe, a
second probe, a third probe, and a fourth probe. An interrogation
position of the first probe comprises a nucleic acid residue that
is a deoxyadenosine or adenosine residue. An interrogation position
of the second probe comprises a nucleic acid residue that is a
deoxythymidine or uridine residue. An interrogation position of the
third probe comprises a nucleic acid residue that is a
deoxyguanosine or guanosine residue. An interrogation position of
the fourth nucleic acid probe comprises a nucleic acid residue that
is a deoxycytosine or cytosine residue.
[0045] In another aspect of the invention, the sample containing a
plurality of target nucleic acid sequences is admixed with a
plurality of the nucleic acid probes. Several analytical outputs
can be obtained from such multiplexed assays. In a first
embodiment, the analytical output obtained when at least one
nucleic acid probes hybridizes with partial complementarity to one
target nucleic acid sequence is greater than the analytical output
when all of the nucleic acid probes hybridize with total
complementarity to their respective nucleic acid target sequences.
In a second embodiment, the analytical output obtained when at
least one nucleic acid probe hybridizes with partial
complementarity to one target nucleic acid sequence is less than
the analytical output when all of the nucleic acid probes hybridize
with total complementarity to their respective nucleic acid target
sequences. In a third embodiment, the analytical output obtained
when at least one nucleic acid probe hybridizes with total
complementarity to one nucleic acid target sequence is greater than
the analytical output when all of the nucleic acid probes hybridize
with partial complementarity to their respective nucleic acid
target sequences. In a fourth embodiment, the analytical output
obtained when at least one nucleic acid probe hybridizes with total
complementarity to one target nucleic acid sequence is less than
the analytical output when all of the nucleic acid probes hybridize
with partial complementarity to their respective nucleic acid
target sequences. The depolymerizing enzymes are as described
herein.
[0046] Yet another embodiment of the invention contemplates a
method for determining the presence or absence of a first
endogenous nucleic acid target in a nucleic acid sample that may
contain that target or may contain a substantially identical second
target. For example, the second target may have a base
substitution, deletion or addition relative to the first nucleic
acid target. This embodiment comprises the following steps.
[0047] A sample to be assayed is admixed with one or more nucleic
acid probes under hybridizing conditions to form a hybridization
composition. The first and second nucleic acid targets each
comprise a region of sequence identity except for at least a single
nucleotide at a predetermined position that differs between the
targets. The nucleic acid probe is substantially complementary to
the nucleic acid target region of sequence identity and comprises
at least one nucleotide at an interrogation position. An
interrogation position of the probe is aligned with the
predetermined position of a target when a target and probe are
hybridized. The probe also includes an identifier nucleotide in the
3'-terminal region.
[0048] The hybridization composition is maintained under
hybridizing conditions for a time period sufficient to form a
treated sample wherein the nucleotide at the interrogation position
of the probe is aligned with the nucleotide at the predetermined
position in the region of identity of the target.
[0049] A treated reaction mixture is formed by admixing the treated
sample with a depolymerizing amount of an enzyme whose activity is
to release one or more nucleotides from the 3'-terminus of a
hybridized nucleic acid probe. The reaction mixture is maintained
under depolymerization conditions for a time period sufficient to
permit the enzyme to depolymerize the hybridized nucleic acid and
release the identifier nucleotide.
[0050] An analytical output is obtained by analyzing for the
presence or absence of released identifier nucleotides. The
analytical output indicates the presence or absence of the
nucleotide at the predetermined region, and; thereby, the presence
or absence of a first nucleic acid target.
[0051] One aspect of the above method is comprised of a first probe
and a second probe. The first probe comprises a nucleotide at an
interrogation position that is complementary to a first nucleic
acid target at a predetermined position. The second probe comprises
a nucleotide at an interrogation position that is complementary to
a second nucleic acid target at a predetermined position.
[0052] In one aspect of a process of the invention, the
depolymerizing enzyme, whose activity is to release nucleotides, is
a template-dependent polymerase, whose activity is to depolymerize
hybridized nucleic acid whose 3'-terminal nucleotide is matched, in
the 3'.fwdarw.5' direction in the presence of pyrophosphate ions to
release one or more nucleotides. Thus, the enzyme's activity is to
depolymerize hybridized nucleic acid to release nucleotides under
depolymerizing conditions. Preferably, this enzyme depolymerizes
hybridized nucleic acids whose bases in the 3'-terminal region of
the probe are matched with total complementarity to the
corresponding bases of the nucleic acid target. The enzyme will
continue to release properly paired bases from the 3'-terminus and
will stop when the enzyme arrives at a base that is mismatched.
[0053] In an alternative aspect of the process (method), the
depolymerizing enzyme, whose activity is to release nucleotides,
exhibits a 3'.fwdarw.5' exonuclease activity in which hybridized
nucleic acids having one or more mismatched bases at the
3'-terminus of the hybridized probe are depolymerized. Thus, the
enzyme's activity is to depolymerize hybridized nucleic acid to
release nucleotides under depolymerizing conditions. In this
embodiment, the hybrid may be separated from the free probe prior
to enzyme treatment. In some embodiments, an excess of target may
be used so that the concentration of free probe in the enzyme
reaction is extremely low.
[0054] In still another alternative aspect of a process of the
invention, the depolymerizing enzyme exhibits a 3' to 5'
exonuclease activity on a double-stranded DNA substrate having one
or more matched bases at the 3' terminus of the hybrid. The
enzyme's activity is to depolymerize hybridized nucleic acid to
release nucleotides containing a 5' phosphate under depolymerizing
conditions.
[0055] A further embodiment of the invention, such as is used for
Single Tandem Repeat (STR) detection, contemplates a method for
determining the number of known sequence repeats that are present
in an endogenous nucleic acid target sequence in a nucleic acid
sample. A method for determining the number of known sequence
repeats comprises the following steps. A plurality of separate
treated samples is provided. Each treated sample contains a nucleic
acid target sequence hybridized with a nucleic acid probe. The
nucleic acid target sequence contains a plurality of known sequence
repeats and a downstream non-repeated region. Each nucleic acid
probe contains a different number of complementary repeats of the
known sequence, an identifier nucleotide in the 3'-terminal region
and a 5'-terminal locker sequence. The 5'-terminal locker sequence
is complementary to the downstream non-repeated region of the
target and comprises 1 to about 20 nucleotides, preferably 5 to 20
nucleotides, most preferably 10 to 20 nucleotides. The various
probes represent complements to possible alleles of the target
nucleic acid. A treated depolymerization reaction mixture is formed
by admixing each treated sample with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a hybridized nucleic acid probe. The treated
depolymerization reaction mixture is maintained under
depolymerizing conditions for a time period sufficient to permit
the enzyme to depolymerize the hybridized nucleic acid probe and
release an identifier nucleotide. The samples are analyzed for the
presence or absence of released identifier nucleotide to obtain an
analytical output. The analytical output from the sample whose
probe contained the same number of sequence repeats as present in
the target nucleic acid is indicative of and determines the number
of sequence repeats present in the nucleic acid target.
[0056] In one aspect of the method, the nucleic acid sample
contains two nucleic acid targets representing alleles at a locus,
and is homozygous with respect to the number of known sequence
repeats of the two alleles. In an alternative method of the
invention, the nucleic acid sample is heterozygous with respect to
the two alleles at the locus. In another method of the invention,
an identifier nucleotide is a nucleotide that is part of the region
containing a repeated sequence. In an alternative method of the
invention, an identifier nucleotide of the probe sequence is part
of the region containing a non-repeating sequence that is
complementary to that located in the target nucleic acid 5' to the
repeated known sequence. In this latter aspect of the method, the
identifier nucleotide is present in a sequence containing 1 to
about 20 nucleic acids that is complementary to a non-repeating
sequence of the target nucleic acid located in the probe 3' to the
known sequence repeats. The repeated known sequence present in a
nucleic acid target sequence typically has a length of 2 to about
24 bases per repeat.
[0057] A further embodiment of the invention contemplates a method
using thermostable DNA polymerase as a depolymerizing enzyme for
determining the presence or absence of at least one predetermined
endogenous nucleic acid target sequence in a nucleic acid sample,
and comprises the following steps.
[0058] A treated sample is provided that may contain a
predetermined endogenous nucleic acid target sequence hybridized to
a nucleic acid probe whose 3'-terminal region is complementary to
the predetermined nucleic acid target sequence and includes an
identifier nucleotide in the 3'-terminal region. A treated
depolymerization reaction mixture is formed by admixing a treated
sample with a depolymerizing amount of a enzyme whose activity is
to release an identifier nucleotide from the 3'-terminus of a
hybridized nucleic acid probe. In a preferred one-pot embodiment,
the depolymerizing enzyme is thermostable and more preferably, the
treated reaction mixture also contains (i) adenosine 5'
diphosphate, (ii) pyrophosphate, and (iii) a thermostable
nucleoside diphosphate kinase (NDPK).
[0059] The treated sample is maintained under depolymerizing
conditions at a temperature of about 4.degree. C. to about
90.degree. C., more preferably at a temperature of about 20.degree.
C. to about 90.degree. C., and most preferably at a temperature of
about 25.degree. C. to about 80.degree. C., for a time period
sufficient to permit the depolymerizing enzyme to depolymerize the
hybridized nucleic acid probe and release an identifier nucleotide
as a nucleoside triphosphate. In preferred one-pot reactions, the
time period is also sufficient to permit NDPK enzyme to transfer a
phosphate from the released nucleoside triphosphate to added ADP,
thereby forming ATP. The presence or absence of a nucleic acid
target sequence is determined from the analytical output obtained
using ATP. In a preferred method of the invention, analytical
output is obtained by luminescence spectrometry.
[0060] In another aspect of the thermostable enzyme one-pot method
for determining the presence or absence of a predetermined
endogenous nucleic acid target sequence in a nucleic acid sample,
the treated sample is formed by the following further steps. A
hybridization composition is formed by admixing the sample to be
assayed with one or more nucleic acid probes under hybridizing
conditions. The 3'-terminal region of the nucleic acid probe (i)
hybridizes with partial or total complementarity to a nucleic acid
target sequence when that sequence is present in the sample, and
(ii) includes an identifier nucleotide. A treated sample is formed
by maintaining the hybridization composition under hybridizing
conditions for a time period sufficient for the predetermined
endogenous nucleic acid target sequence to hybridize with the
nucleic acid probe.
[0061] Preferably, the depolymerizing enzyme is from a group of
thermophilic DNA polymerases comprising Tne triple mutant DNA
polymerase, Tne DNA polymerase, Taq DNA polymerase, Ath DNA
polymerase, Tvu DNA polymerase, Bst DNA polymerase, and Tth DNA
polymerase. The Tne triple mutant DNA polymerase is a preferred
thermophilic enzyme and is discussed in greater detail hereinafter.
In another aspect of the method, the NDPK is that encoded for by
the thermophilic bacteria Pyrococcus furiosis (Pfu).
[0062] A still further method of the invention contemplates
determining whether the presence or absence of a nucleic acid
target sequence in a nucleic acid sample results from a locus that
is homozygous or heterozygous for the two alleles at the locus.
This method is comprised of the following steps. A plurality of
separate treated samples is provided. Each sample may contain a
nucleic acid target sequence hybridized with a nucleic acid probe.
The nucleic acid target sequence consists of either a first allele,
a second allele, or a mixture of first and second alleles of the
nucleic acid target. The alleles differ in sequence at an
interrogation position. The nucleic acid probe contains an
identifier nucleotide in the 3'-terminal region that is aligned at
an interrogation nucleotide position of the target sequence when
the probe and target are hybridized.
[0063] A treated reaction mixture is formed by admixing each
treated sample with a depolymerizing amount of an enzyme whose
activity is to release one or more nucleotides from the 3'-terminus
of a hybridized nucleic acid probe. The treated reaction mixture is
maintained under depolymerizing conditions for a time period
sufficient to permit the enzyme to depolymerize the hybridized
nucleic acid probe and release an identifier nucleotide. The
samples are analyzed for the presence or absence of released
identifier nucleotides to obtain an analytical output. The
analytical output is quantifiable and thus determines whether the
sample is homozygous or heterozygous when compared to the
analytical output of appropriate controls.
[0064] A multiplexed version of this embodiment is also
contemplated, wherein probes for two or more alleles are provided
in one reaction--each probe is distinguishable, but preferably each
probe has the same length. Then, after hybridization,
depolymerization, and analysis according to the invention, the
relative analytical output for the various distinguishable
identifier nucleotides or remaining probes will show whether the
sample is homozygous or heterozygous and for which alleles. Another
multiplexed version of this embodiment is contemplated, wherein
probes for alleles at a plurality of loci are provided. Preferably,
the different loci have substantially different target sequences.
Probes for the various alleles at each locus are preferably of the
same length. Each of the probes should be distinguishable either by
analysis of the released identifier nucleotide or by analysis of
the remaining probe after depolymerization.
[0065] Another embodiment of the invention contemplates a method
for determining the loss of heterozygosity (LOH) of a locus of an
allele that comprises the following steps.
[0066] A plurality of separate treated samples is provided, each
sample containing a nucleic acid target sequence hybridized with a
nucleic acid probe. The nucleic acid target sequence is that of a
first allele or a mixture of the first allele and a second allele
of the nucleic acid target, wherein the alleles differ in sequence.
The nucleic acid probe contains a 3'-terminal region that
hybridizes to a target sequence when the probe and target are
hybridized.
[0067] Each treated sample is admixed with a depolymerizing amount
of an enzyme whose activity is to release one or more nucleotides
from the 3'-terminus of a hybridized nucleic acid probe to form a
treated reaction mixture. The treated reaction mixture is
maintained under depolymerizing conditions for a time period
sufficient to depolymerize hybridized nucleic acid probe and
release identifier nucleotides. The samples are then analyzed for
the quantity of released identifier nucleotides to obtain an
analytical output, the analytical output indicating whether the
nucleic acid target sequence in a nucleic acid sample has lost
heterozygosity at the locus of the allele.
[0068] In preferred LOH embodiments, the analytical output is
obtained by luminescence spectroscopy, absorbance spectrometry,
mass spectrometry or fluorescence spectroscopy. In another
preferred embodiment, the released identifier nucleotide includes a
fluorescent label. The identifier nucleotide is optionally
fluorescently labeled after release from the hybrid.
[0069] It is contemplated that in the above analytical methods,
either the released identifier nucleotide or the remainder of the
probe can be evaluated to determine whether identifier nucleotide
had been released, as described herein.
[0070] In another preferred LOH embodiment, the enzyme whose
activity is to release nucleotides is a template-dependent
polymerase that, in the presence of pyrophosphate ions,
depolymerizes hybridized nucleic acids whose bases in the
3'-terminal region are completely complementary to bases of the
nucleic acid target. The depolymerization proceeds from the 3'
terminal nucleotide of the probe and stops when it reaches a base
that is not complementary to the corresponding target base.
[0071] In one aspect of the LOH embodiment, the quantity of the
released identifier nucleotides for the first allele is
substantially less than the quantity of the released identifier
nucleotide for the first allele of a known heterozygous control
sample, and the quantity of the released identifier nucleotides for
the second allele is substantially similar to that of the released
identifier nucleotide for the second allele of a known heterozygous
control sample, indicating a loss of heterozygosity at the locus of
the first allele.
[0072] In another aspect of the LOH embodiment, the quantity of the
released identifier nucleotides for the second allele is
substantially less than the quantity of the released identifier
nucleotides for the second allele of a known heterozygous control
sample, and the quantity of the released identifier nucleotides for
the first allele is substantially similar to that of the released
identifier nucleotide for the first allele of a known heterozygous
control sample, indicating a loss of heterozygosity at the locus of
the second allele. The known heterozygous control has analytical
output for its treated sample indicating alleles one and two are
present in the sample at about a 1:1 ratio. A sample with loss of
heterozygosity has an analytical output for the treated samples
indicating alleles one and two are present in the sample at a 1:0
or 0:1 ratio respectively when compared to the analytical output of
a known heterozygous control sample.
[0073] A still further preferred embodiment of the invention
contemplates a method for determining the presence of trisomy of an
allele that comprises the following steps.
[0074] A plurality of separate treated samples is provided, wherein
each sample contains a nucleic acid target sequence hybridized with
a nucleic acid probe. The nucleic acid target sequence is that of a
first allele, a second allele or a mixture of the first and second
alleles of the nucleic acid target. The alleles differ in sequence
at an interrogation position. The nucleic acid probe contains a
3'-terminal region that hybridizes to a region of the nucleic acid
target sequence containing the interrogation nucleotide position
when the probe and target are hybridized. The nucleic acid probe
also contains an identifier nucleotide.
[0075] Each treated sample is admixed with a depolymerizing amount
of an enzyme whose activity, under depolymerizing conditions, is to
release one or more nucleotides from the 3'-terminus of a
hybridized nucleic acid probe to form a treated reaction mixture.
The treated reaction mixture is maintained for a time period
sufficient to depolymerize hybridized nucleic acid probe and
release identifier nucleotides. The samples are analyzed for
released identifier nucleotides to obtain an analytical output, the
magnitude of the analytical output relative to an analytical output
of an appropriate control sample indicating whether a trisomy is
present in the nucleic acid target sequence.
[0076] For trisomy analysis, preferably the analytical output is
obtained by luminescence spectroscopy, absorbance spectrometry,
fluorescence spectroscopy, or mass spectrometry. In one preferred
embodiment, the released identifier nucleotide includes a
fluorescent label. The identifier nucleotide is optionally
fluorescently labeled after release from the hybrid.
[0077] In a preferred embodiment for trisomy analysis, the enzyme
whose activity is to release nucleotides is a template-dependent
polymerase, that, in the presence of pyrophosphate ions,
depolymerizes hybridized nucleic acids whose bases in the 3'
terminal region are completely complementary to bases of said
nucleic acid target.
[0078] In one embodiment, the quantity of released identifier
nucleotides for the first allele is substantially greater than the
quantity of the released identifier nucleotides of a control sample
homozygous for the first allele, indicating that the nucleic acid
target sequence has a trisomy. Preferably, the quantity of released
identifier nucleotides is expressed as a ratio. For example, a
normal heterozygote has about a 1:1 ratio of the analytical output
for the two alleles. If the trisomy is homozygous for either
allele, the ratio is about three times the value for that allele in
a normal heterozygote that has none of the other allele. If the
trisomy is heterozygous, then the ratio is about 2:1 of one allele
to the other when compared to the analytical output of a control
heterozygote.
[0079] A still further embodiment of the invention contemplates
determining the presence or absence of a nucleic acid target
sequence in a nucleic acid sample with a probe that is hybridized
to the target and then modified to be able to form a hairpin
structure. This embodiment comprises the following steps.
[0080] A treated sample is provided that contains a nucleic acid
sample that may include a nucleic acid target sequence having an
interrogation position hybridized with a nucleic acid probe. The
probe is comprised of at least two sections. The first section
contains the probe 3'-terminal about 10 to about 30 nucleotides.
These nucleotides are complementary to the target strand sequence
at positions beginning about 1 to about 30 nucleotides downstream
of the interrogation position. The second section of the probe is
located at the 5'-terminal region of the probe and contains about
10 to about 20 nucleotides of the target sequence. This sequence
spans the region in the target from the nucleotide at or just
upstream (5') of the interrogation position, to the nucleotide just
upstream to where the 3'-terminal nucleotide of the probe anneals
to the target. An optional third section of the probe, from zero to
about 50, and preferably about zero to about 20 nucleotides in
length and comprising a sequence that does not hybridize with
either the first or second section, is located between the first
and second sections of the probe.
[0081] The probe of the treated sample is extended in a
template-dependent manner, as by admixture with dNTPs and a
template-dependent polymerase, at least through the interrogation
position, thereby forming an extended probe/target hybrid. In a
preferred embodiment, the length of the probe extension is limited
by omission from the extension reaction of a DNTP complementary to
a nucleotide of the target sequence that is present upstream of the
interrogation position and absent between the nucleotide
complementary to the 3'-end of the interrogation position.
[0082] The extended probe/target hybrid is separated from any
unreacted dNTPs. The extended probe/target hybrid is denatured to
separate the strands. The extended probe strand is permitted to
form a hairpin structure.
[0083] A treated reaction mixture is formed by admixing the hairpin
structure-containing composition with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of an extended probe hairpin structure. The
reaction mixture is maintained under depolymerizing conditions for
a time period sufficient for the depolymerizing enzyme to release
3'-terminus nucleotides, and then analyzed for the presence of
released identifier nucleotides. The analytical output indicates
the presence or absence of the nucleic acid target sequence.
[0084] A still further embodiment of the invention, termed
REAPERTM, also utilizes hairpin structures. This method
contemplates determining the presence or absence of a nucleic acid
target sequence, or a specific base within the target sequence, in
a nucleic acid sample, and comprises the following steps. A treated
sample is provided that contains a nucleic acid sample that may
include a nucleic acid target sequence hybridized with a first
nucleic acid probe strand.
[0085] The hybrid is termed the first hybrid. The first probe is
comprised of at least two sections. The first section contains the
probe 3'-terminal about 10 to about 30 nucleotides that are
complementary to the target nucleic acid sequence at a position
beginning about 5 to about 30 nucleotides downstream of the target
interrogation position. The second section of the first probe
contains about 5 to about 30 nucleotides that are a repeat of the
target sequence from the interrogation position to about 10 to
about 30 nucleotides downstream of the interrogation position, and
does not hybridize to the first section of the probe. An optional
third section of the probe, located between the first and second
sections of the probe, is zero to about 50, preferably up to about
20, nucleotides in length and comprises a sequence that does not
hybridize to either the first or second section.
[0086] The first hybrid in the treated sample is extended at the
3'-end of the first probe, thereby extending the first probe past
the interrogation position and forming an extended first hybrid
whose sequence includes an interrogation position. The extended
first hybrid is comprised of the original target nucleic acid and
extended first probe. The extended first hybrid is then denatured
in an aqueous composition to separate the two nucleic acid strands
of the hybridized duplex and form an aqueous solution containing a
separated target nucleic acid and a separated extended first
probe.
[0087] A second probe, that is about 10 to about 2000, preferably
about 10 to about 200, most preferably about 10 to about 30
nucleotides in length and is complementary to the extended first
probe at a position beginning about 5 to about 2000, preferably
about 5 to about 200, nucleotides downstream of the interrogation
position in extended first probe, is annealed to the extended first
probe, thereby forming the second hybrid. The second hybrid is
extended at the 3'-end of the second probe until that extension
reaches the 5'-end of the extended first probe, thereby forming a
second extended hybrid whose 3'-region includes an identifier
nucleotide. In preferred embodiments the extending polymerase for
both extensions does not add a nucleotide to the 3' end that does
not have a corresponding complementary nucleotide in the
template.
[0088] An aqueous composition of the extended second hybrid is
denatured to separate the two nucleic acid strands. The aqueous
composition so formed is cooled to form a "hairpin structure" from
the separated extended second probe when the target sequence is
present in the original nucleic acid sample.
[0089] A treated reaction mixture is formed by admixing the hairpin
structure-containing composition with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a nucleic acid hybrid. The reaction mixture is
maintained under depolymerizing conditions for a time period
sufficient to release 3'-terminal region identifier nucleotides,
and then analyzed for the presence of released identifier
nucleotides. The analytical output indicates the presence or
absence of the nucleic acid target sequence.
[0090] The present invention has many benefits and advantages,
several of which are listed below.
[0091] One benefit of the invention is that, in some embodiments,
nucleic acid hybrids can be detected with very high levels of
sensitivity without the need for radiochemicals or
electrophoresis.
[0092] An advantage of the invention is that the presence or
absence of one or more target nucleic acid(s) can be detected
reliably, reproducibly, and with great sensitivity.
[0093] A further benefit of the invention is that quantitative
information can be obtained about the amount of a target nucleic
acid sequence in a sample.
[0094] A further advantage of the invention is that very slight
differences in nucleic acid sequence are detectable, including
single nucleotide polymorphisms (SNPs).
[0095] Yet another benefit of the invention is that the presence or
absence of a number of target nucleic acid sequences can be
determined in the same assay.
[0096] Yet another advantage of the invention is that the presence
or absence of a target nucleic acid can be determined with a small
number of reagents and manipulations.
[0097] Another benefit of the invention is that the processes lend
themselves to automation.
[0098] Still another benefit of the invention is its flexibility of
use in many different types of applications and assays including,
but not limited to, detection of mutations, translocations, and
SNPs in nucleic acid (including those associated with genetic
disease), determination of viral load, species identification,
sample contamination, and analysis of forensic samples.
[0099] Still further benefits and advantages of the invention will
become apparent from the specification and claims that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0100] In the drawings forming a portion of this disclosure,
[0101] FIG. 1. illustrates the Reaper.TM. assay as illustrated in
Example 89. FIG. 1A illustrates the first hybrid formed by the
annealing of nucleic acid target SEQ ID NO:111 (111) to first probe
SEQ ID NO: 112 (112). An arrow points to an interrogation position
in 111.
[0102] FIG. 1B illustrates the first extended hybrid formed by the
annealing of ill to the extended 112. Extended 112 is first
extended probe SEQ ID NO: 113 (113).
[0103] FIG. 1C illustrates the second hybrid formed by annealing of
113 from the denatured nucleic acid molecule shown in FIG. 1B to
the second probe denoted SEQ ID NO: 114 (114). An arrow points to
the interrogation position in 113.
[0104] FIG. 1D illustrates the extended second hybrid formed by the
annealing of 113 and the extended 114 strand denoted SEQ ID NO: 115
(115).
[0105] FIG. 1E illustrates the 115 strand denatured from FIG. 1D
and forming a hairpin structure. An arrow points to the
interrogation position at the 3'-terminus of the hybrid.
Definitions
[0106] To facilitate understanding of the invention, a number of
terms are defined below.
[0107] "Nucleoside", as used herein, refers to a compound
consisting of a purine [guanine (G) or adenine (A)] or pyrimidine
[thymine (T), uridine (U) or cytidine (C)] base covalently linked
to a pentose, whereas "nucleotide" refers to a nucleoside
phosphorylated at one of its pentose hydroxyl groups. "XTP", "XDP"
and "XMP" are generic designations for ribonucleotides and
deoxyribonucleotides, wherein the "TP" stands for triphosphate,
"DP" stands for diphosphate, and "MP" stands for monophosphate, in
conformity with standard usage in the art. Subgeneric designations
for ribonucleotides are "NMP", "NDP" or "NTP", and subgeneric
designations for deoxyribonucleotides are "dNMP", "dNDP" or "dNTP".
Also included as "nucleoside", as used herein, are materials that
are commonly used as substitutes for the nucleosides above such as
modified forms of these bases (e.g. methyl guanine) or synthetic
materials well known in such uses in the art, such as inosine.
[0108] A "nucleic acid," as used herein, is a covalently linked
sequence of nucleotides in which the 3' position of the pentose of
one nucleotide is joined by a phosphodiester group to the 5'
position of the pentose of the next, and in which the nucleotide
residues (bases) are linked in specific sequence; i.e., a linear
order of nucleotides. A "polynucleotide," as used herein, is a
nucleic acid containing a sequence that is greater than about 100
nucleotides in length. An "oligonucleotide," as used herein, is a
short polynucleotide or a portion of a polynucleotide. An
oligonucleotide typically contains a sequence of about two to about
one hundred bases. The word "oligo" is sometimes used in place of
the word "oligonucleotide" .
[0109] A base "position" as used herein refers to the location of a
given base or nucleotide residue within a nucleic acid.
[0110] A "nucleic acid of interest," as used herein, is any
particular nucleic acid one desires to study in a sample.
[0111] The term "isolated" when used in relation to a nucleic acid
or protein, refers to a nucleic acid sequence or protein that is
identified and separated from at least one contaminant (nucleic
acid or protein, respectively) with which it is ordinarily
associated in its natural source. Isolated nucleic acid or protein
is present in a form or setting that is different from that in
which it is found in nature. In contrast, non-isolated nucleic
acids or proteins are found in the state they exist in nature.
[0112] As used herein, the term "purified" or "to purify" means the
result of any process which removes some contaminants from the
component of interest, such as a protein or nucleic acid. The
percent of a purified component is thereby increased in the
sample.
[0113] The term "wild-type," as used herein, refers to a gene or
gene product that has the characteristics of that gene or gene
product that is most frequently observed in a population and is
thus arbitrarily designated the "normal" or "wild-type" form of the
gene. In contrast, the term "modified" or "mutant" as used herein,
refers to a gene or gene product that displays modifications in
sequence and/or functional properties (i.e., altered
characteristics) when compared to the wild-type gene or gene
product.
[0114] Nucleic acids are known to contain different types of
mutations. As used herein, a "point" mutation refers to an
alteration in the sequence of a nucleotide at a single base
position. A "lesion", as used herein, refers to site within a
nucleic acid where one or more bases are mutated by deletion or
insertion, so that the nucleic acid sequence differs from the
wild-type sequence.
[0115] A "single nucleotide polymorphism" or SNP, as used herein,
is a variation from the most frequently occurring base at a
particular nucleic acid position.
[0116] Homologous genes or alleles from different species are also
known to vary in sequence. Regions of homologous genes or alleles
from different species can be essentially identical in sequence.
Such regions are referred to herein as "regions of identity." It is
contemplated herein that a "region of substantial identity" can
contain some "mismatches," where bases at the same position in the
region of identity are different. This base position is referred to
herein as "mismatch position." DNA molecules are said to have a
"5'-terminus" (5' end) and a "3'-terminus" (3' end) because nucleic
acid phosphodiester linkages occur to the 5' carbon and 3' carbon
of the pentose ring of the substituent mononucleotides. The end of
a polynucleotide at which a new linkage would be to a 5' carbon is
its 5' terminal nucleotide. The end of a polynucleotide at which a
new linkage would be to a 3' carbon is its 3' terminal nucleotide.
A terminal nucleotide, as used herein, is the nucleotide at the end
position of the 3'- or 5'-terminus. As used herein, a nucleic acid
sequence, even if internal to a larger oligonucleotide or
polynucleotide, also can be said to have 5'- and 3'-ends. For
example, a gene sequence located within a larger chromosome
sequence can still be said to have a 5'- and 3'-end.
[0117] As used herein, the 3'-terminal region of the nucleic acid
probe refers to the region of the probe including nucleotides
within about 10 residues from the 3'-terminal position.
[0118] In either a linear or circular DNA molecule, discrete
elements are referred to as being "upstream" or "5'" relative to an
element if they are bonded or would be bonded to the 5'-end of that
element. Similarly, discrete elements are "downstream" or "3'"
relative to an element if they are or would be bonded to the 3'-end
of that element. Transcription proceeds in a 5' to 3' manner along
the DNA strand. This means that RNA is made by the sequential
addition of ribonucleotide-5'-triphosphates to the 3'-terminus of
the growing chain (with the elimination of pyrophosphate).
[0119] As used herein, the term "target nucleic acid" or "nucleic
acid target" refers to a particular nucleic acid sequence of
interest. Thus, the "target" can exist in the presence of other
nucleic acid molecules or within a larger nucleic acid
molecule.
[0120] As used herein, the term "nucleic acid probe" refers to an
oligonucleotide or polynucleotide that is capable of hybridizing to
another nucleic acid of interest. A nucleic acid probe may occur
naturally as in a purified restriction digest or be produced
synthetically, recombinantly or by PCR amplification. As used
herein, the term "nucleic acid probe" refers to the oligonucleotide
or polynucleotide used in a method of the present invention. That
same oligonucleotide could also be used, for example, in a PCR
method as a primer for polymerization, but as used herein, that
oligonucleotide would then be referred to as a "primer". Herein,
oligonucleotides or polynucleotides may contain a phosphorothioate
bond.
[0121] As used herein, the terms "complementary" or
"complementarity" are used in reference to nucleic acids (i.e., a
sequence of nucleotides) related by the well-known base-pairing
rules that A pairs with T and C pairs with G. For example, the
sequence 5'-A-G-T-3', is complementary to the sequence 3'-T-C-A-5'.
Complementarity can be "partial," in which only some of the nucleic
acid bases are matched according to the base pairing rules. On the
other hand, there may be "complete" or "total" complementarity
between the nucleic acid strands when all of the bases are matched
according to base pairing rules. The degree of complementarity
between nucleic acid strands has significant effects on the
efficiency and strength of hybridization between nucleic acid
strands as known well in the art. This is of particular importance
in detection methods that depend upon binding between nucleic
acids, such as those of the invention. The term "substantially
complementary" refers to any probe that can hybridize to either or
both strands of the target nucleic acid sequence under conditions
of low stringency as described below or, preferably, in polymerase
reaction buffer (Promega, M195A) heated to 95.degree. C. and then
cooled to room temperature. As used herein, when the nucleic acid
probe is referred to as partially or totally complementary to the
target nucleic acid, that refers to the 3'-terminal region of the
probe (i.e. within about 10 nucleotides of the 3'-terminal
nucleotide position).
[0122] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acid strands.
Hybridization and the strength of hybridization (i.e., the strength
of the association between nucleic acid strands) is impacted by
many factors well known in the art including the degree of
complementarity between the nucleic acids, stringency of the
conditions involved affected by such conditions as the
concentration of salts, the T.sub.m (melting temperature) of the
formed hybrid, the presence of other components (e.g., the presence
or absence of polyethylene glycol), the molarity of the hybridizing
strands and the G:C content of the nucleic acid strands.
[0123] As used herein, the term "stringency" is used in reference
to the conditions of temperature, ionic strength, and the presence
of other compounds, under which nucleic acid hybridizations are
conducted. With "high stringency" conditions, nucleic acid base
pairing will occur only between nucleic acid fragments that have a
high frequency of complementary base sequences. Thus, conditions of
"weak" or "low" stringency are often required when it is desired
that nucleic acids which are not completely complementary to one
another be hybridized or annealed together. The art knows well that
numerous equivalent conditions can be employed to comprise low
stringency conditions.
[0124] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature". The melting temperature is the
temperature at which 50% of a population of double-stranded nucleic
acid molecules becomes dissociated into single strands. The
equation for calculating the Tm of nucleic acids is well-known in
the art. The Tm of a hybrid nucleic acid is often estimated using a
formula adopted from hybridization assays in 1 M salt, and commonly
used for calculating T.sub.m for PCR primers: [(number of
A+T).times.20.degree. C.+(number of G+C).times.4.degree. C.]. C. R.
Newton et al. PCR, 2.sup.nd Ed., Springer-Verlag (New York: 1997),
p. 24. This formula was found to be inaccurate for primers longer
that 20 nucleotides. Id. Other more sophisticated computations
exist in the art which take structural as well as sequence
characteristics into account for the calculation of T.sub.m. A
calculated T.sub.m is merely an estimate; the optimum temperature
is commonly determined empirically.
[0125] The term "homology," as used herein, refers to a degree of
complementarity. There can be partial homology or complete homology
(i.e., identity). A partially complementary sequence that at least
partially inhibits a completely complementary sequence from
hybridizing to a target nucleic acid is referred to using the
functional term "substantially homologous."
[0126] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous," as used herein, refers to a probe that can hybridize
to a strand of the double-stranded nucleic acid sequence under
conditions of low stringency.
[0127] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous," as used herein,
refers to a probe that can hybridize to (i.e., is the complement
of) the single-stranded nucleic acid template sequence under
conditions of low stringency.
[0128] The term "interrogation position," as used herein, refers to
the location of a given base of interest within a nucleic acid
probe. For example, in the analysis of SNPs, the "interrogation
position" in the probe is in the position that would be
complementary to the single nucleotide of the target that may be
altered from wild type. The analytical output from a method of the
invention provides information about a nucleic acid residue of the
target nucleic acid that is complementary to an interrogation
position of the probe. An interrogation position is within about
ten bases of the actual 3'-terminal nucleotide of the nucleic acid
probe, although not necessarily at the 3'-terminal nucleotide
position. The interrogation position of the target nucleic acid
sequence is opposite the interrogation position of the probe, when
the target and probe nucleic acids are hybridized.
[0129] The term "identifier nucleotide," as used herein, refers to
a nucleotide whose presence is to be detected in a process of the
invention to identify that a depolymerization reaction has
occurred. The particular application of a method of the invention
affects which residues are considered an identifier nucleotide. For
a method using ATP detection (e.g. luciferase/luciferin or NADH)
wherein, during analysis, all nucleotides released in the
depolymerization are "converted" to ATP with an enzyme such as
NDPK, all nucleotides released are identifier nucleotides.
Similarly, for a method using absorbance detection that does not
distinguish between nucleotides, all released nucleotides are
identifier nucleotides. For a mass spectrometric detection wherein
all the released nucleotides are analyzed, all released nucleotides
can be identifier nucleotides; alternatively a particular
nucleotide (e.g. a nucleotide analog having a distinctive mass) can
be detected. For fluorescence detection, a fluorescently-labeled
nucleotide is an identifier nucleotide. The nucleotide may be
labeled prior to or after release from the nucleic acid. For
radiographic detection, a radioactively-labeled nucleotide is an
identifier nucleotide. In some cases, the release of identifier
nucleotide is deduced by analyzing the remainder of the probe after
a depolymerization step of the invention. Such analysis is
generally by a determination of the size or mass of the remaining
probe and can be by any of the described analytical methods (e.g. a
fluorescent tag on the 5'-terminus of the probe to monitor its
molecular weight following capillary electrophoresis).
[0130] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to a class of enzymes, each of which
cut double-stranded DNA. Some restriction endonucleases cut double
strand DNA at or near a specific nucleotide sequence, such as the
enzyme commonly referred to as BamH I that recognizes the double
strand sequence 5'GGATCC 3'. However, other representatives of such
enzymes cut DNA in a non-specific manner such as the DNA
endonuclease DNase I.
[0131] The term "sample," as used herein, is used in its broadest
sense. A sample suspected of containing a nucleic acid can comprise
a cell, chromosomes isolated from a cell (e.g., a spread of
metaphase chromosomes), genomic DNA, RNA, cDNA and the like.
[0132] The term "detection," as used herein, refers to
quantitatively or qualitatively identifying a nucleotide or nucleic
acid within a sample.
[0133] The term "depolymerization," as used herein, refers to the
removal of a nucleotide from the 3' end of a nucleic acid.
[0134] The term "allele," as used herein, refers to an alternative
form of a gene and the term "locus," as used herein, refers to a
particular place on a nucleic acid molecule.
DETAILED DESCRIPTION OF THE INVENTION
[0135] A method of this invention is used to determine the presence
or absence of at least one predetermined (known) endogenous nucleic
acid target sequence in a nucleic acid sample. A nucleic acid
target is "predetermined" in that its sequence must be known to
design a probe that hybridizes with that target. However, it should
be noted that a nucleic acid target sequence, as used with respect
to a process of this invention, may merely act as a reporter to
signal the presence of a different nucleic acid whose presence is
desired to be determined. That other nucleic acid of interest does
not have to have a predetermined sequence. Furthermore, in many
embodiments, a process of the invention is useful in determining
the identity of a base within a target where only enough of the
sequence is known to design a probe that hybridizes to that target
with partial complementarity at the 3'-terminal region of the
probe.
[0136] Such a method utilizes an enzyme that can depolymerize the
3'-terminus of an oligonucleotide probe hybridized to the nucleic
acid target sequence to release one or more identifier nucleotides
whose presence or absence can then be determined as an analytical
output that indicates the presence or absence of the target
sequence.
[0137] A nucleic acid target sequence is predetermined (or known)
in that a nucleic acid probe is provided to be partially or totally
complementary to that nucleic acid target sequence. A nucleic acid
target sequence is a portion of nucleic acid sample with which the
probe hybridizes if that target sequence is present in the
sample.
[0138] A nucleic acid target is "endogenous" when it is a natural
part of the sample being assayed. For example, analysis of a human
body sample to ascertain its genetic makeup (e.g. trisomy or
Duchennes muscular dystrophy analysis) will have an endogenous
nucleic acid target. On the other hand, analysis of a human body
sample to search for the presence of a viral pathogen has an
exogenous target. However, analysis of a microbiological sample to
determine its genetic makeup (e.g. speciation) has an endogenous
target.
[0139] A first step of the method is admixing a sample to be
assayed with one or more nucleic acid probes. The admixing of the
first step is typically carried out under low stringency
hybridizing conditions to form a hybridization composition. In such
a hybridization composition, the 3'-terminal region of the nucleic
acid probe(s) (i) hybridizes with partial or total complementarity
to a nucleic acid target sequence that may be present in the
sample; and (ii) includes an identifier nucleotide in the
3'-terminal region.
[0140] Preferably, the nucleic acid probe is designed to not
hybridize with itself to form a hairpin structure in such a way as
to interfere with hybridization of the 3'-terminal region of the
probe to the target nucleic acid. Parameters guiding probe design
are well known in the art.
[0141] The hybridization composition is maintained under
hybridizing conditions for a time period sufficient to form a
treated sample that may contain at least one predetermined nucleic
acid target sequence hybridized with a nucleic acid probe.
[0142] In the event that the sample to be assayed does not contain
a target sequence to which the probe hybridizes, no hybridization
takes place. When a method of the present invention is used to
determine whether a particular target sequence is present or absent
in a sample to be assayed, the resulting treated sample may not
contain a substrate for the enzymes of the present invention. As a
result, a 3' terminal region identifier nucleotide is not released
and the analytical output is at or near background levels.
[0143] The treated sample is admixed with a depolymerizing amount
of an enzyme whose activity is to release one or more identifier
nucleotides from the 3'-terminal region of the probe that is
hybridized to the nucleic acid target to form a depolymerization
reaction mixture. The choice of enzyme used in the process
determines if a match or mismatch at the 3'-terminal nucleotide
results in release of that 3'-terminal nucleotide. Further
information regarding specific enzyme reaction conditions is
discussed in detail hereinafter.
[0144] The depolymerization reaction mixture is maintained under
depolymerizing conditions for a time period sufficient to permit
the enzyme to depolymerize hybridized nucleic acid and release
identifier nucleotides therefrom to form a treated reaction
mixture.
[0145] The presence or absence of released identifier nucleotides
is then determined to obtain an analytical output. The analytical
output indicates the presence or absence of at least the one
nucleic acid target sequence.
[0146] Processes of the invention can also be concerned with the
degree of hybridization of the target to the 3'-terminal region of
the probe. Examples hereinafter show that the distinction between a
matched and mismatched base becomes less notable as a single
mismatch is at a position further upstream from the 3'-terminal
region position. There is very little discrimination between a
match and mismatch when a single mismatch is ten to twelve residues
from the 3'-terminal nucleotide position, whereas great
discrimination is observed when a single mismatch is at the
3'-terminus. Therefore, when the degree of complementarity (partial
or total complementarity) of a nucleic acid probe hybridized to a
target nucleic acid sequence is referred to herein in regard to an
identifier nucleotide, this is to be understood to be referring to
within the 3'-terminal region, up to about ten residues of the
3'-terminal position.
[0147] The sufficiency of the time period for hybridization can be
empirically ascertained for a control sample for various
hybridizing conditions and nucleic acid probe/target combinations.
Exemplary maintenance times and conditions are provided in the
specific examples hereinafter and typically reflect low stringency
hybridization conditions. In practice, once a suitable set of
hybridization conditions and maintenance time periods are known for
a given set of probes, an assay using those conditions provides the
correct result if the nucleic acid target sequence is present.
Typical maintenance times are about 5 to about 60 minutes.
[0148] The conditions and considerations with respect to
hybridization of PCR primers to template nucleic acid in PCR are
applicable to the hybridization of nucleic acid probes to target
sequences in a process of the invention. Such hybridization
conditions are well known in the art, and are a matter of routine
experimentation depending on factors including the sequence of the
nucleic acid probe and the target nucleic acid [sequence identity
(homology), length and G+C content] molar amounts of nucleic acid
present, buffer, salt content and duplex Tm among other
variables.
[0149] Processes of the invention are sensitive and hybridization
conditions of low stringency (e.g. temperature of 0-4.degree. C.)
are sufficient, but moderate stringency conditions (i.e.
temperatures of 40-60.degree. C.) also permit hybridization and
provide acceptable results. This is true for all processes of the
invention.
[0150] In one contemplated embodiment of the invention, the enzyme
whose activity is to depolymerize hybridized nucleic acid to
release nucleotides from the probe 3'-terminal end is a
template-dependent polymerase. In such an embodiment, the reverse
of a polymerase reaction is used to depolymerize a nucleic acid
probe, and the identifier nucleotide is released when the
3'-terminal nucleotide of the nucleic acid probe hybridizes with
total complementarity to its nucleic acid target sequence. A signal
confirms the presence of a nucleic acid target sequence that has
the sequence sufficiently complementary to the nucleic acid probe
to be detected by the process of the invention.
[0151] In an embodiment that uses a 3'.fwdarw.5' exonuclease
activity of a polymerase, such as Klenow or T4 DNA polymerase (but
not limited to those two enzymes), to depolymerize a nucleic acid
probe, an identifier nucleotide is released when the 3'-terminal
residue of the nucleic acid probe is mismatched and therefore there
is only partial complementarity of the 3'-terminus of the nucleic
acid probe to its nucleic acid target sequence. In this embodiment,
to minimize background, the hybrid is typically purified from the
un-annealed nucleic acid prior to the enzyme reaction, which
releases identifier nucleotides. A signal confirms the presence of
a nucleic acid target sequence that is not totally complementary to
the nucleic acid probe.
[0152] In an embodiment that uses a 3'.fwdarw.5' exonuclease
activity of Exonuclease III to depolymerize a nucleic acid probe,
an identifier nucleotide is released when the 3'-terminal residue
of the nucleic acid probe is matched to the target nucleic acid in
the hybrid. A signal confirms the presence of a nucleic acid target
that is complementary to the probe at the released identifier
nucleotide.
[0153] It is thus seen that hybridization and depolymerization can
lead to the release of an indicator nucleotide or to little or no
release of such a nucleotide, depending upon whether the
probe:target hybrid is matched or mismatched at the 3'-terminal
region. This is also dependent on the type of enzyme used and the
type of end, matched or mismatched, that the enzyme requires for
depolymerization activity.
[0154] The magnitude of a contemplated analytical output under
defined conditions is dependent upon the amount of released
nucleotides. Where an identifier nucleotide is released, an
analytical output can be provided that has a value greater than
background. Where an identifier nucleotide is not released either
because the target sequence was not present in the original sample
or because the probe and depolymerizing enzyme chosen do not
provide release of a 3'-terminal nucleotide when the target is
present, or if the match/mismatch state of the 3'-terminal
nucleotide did not match that required for the enzyme used to
release a 3'-terminal nucleotide, the analytical output is
substantially at a background level.
[0155] In an example dealing with background levels, the net
relative light values for analytical output are typically
calculated as follows. First the results from matching samples are
averaged, then the net light production from the matching and
mismatching samples is determined and the net light production from
the matching reaction is divided by that seen in the mismatch
reaction. The net light production was determined by subtracting
the estimated light contribution from the probes and template
present in the reactions from the total light produced. The light
production from the template reaction was considered to be the
total of that contributed from the template specifically and that
contributed by contaminating ATP from various reaction components.
The net increase from the probes alone was calculated by
subtracting the average "No Target DNA" values from the probe
values since this subtracts the contributions from contaminating
ATP from the probe values. Thus, the formula used to determine the
net light production from the reactions was:
Net Light=Total light-[(target alone)+(probe alone-No DNA)]
[0156] For example, in the case of an assay to determine which of
two alleles are present, the net light values are used to determine
the signal ratio by dividing the signal from the first allele probe
by the signal from the second allele probe.
[0157] It has been observed that very different detection ratios
can be calculated from two sets of probes but that the signal
ratios from the different target genotypes are easily
distinguishable from each other. In addition, a mismatching allele
probe gave a relatively low light signal in the absence of nucleic
acid target using low concentrations of Klenow exo-. If such
manipulations were not used, the light signal from the probe alone
would be a large contribution to the total signal of the samples
containing the probe, making sensitive allele discrimination more
difficult.
[0158] Depolymerization reactions and enzymes useful in such
reactions are discussed below.
[0159] Depolymerization
[0160] Nucleic acid polymerases generally catalyze the elongation
of nucleic acid chains. The reaction is driven by the cleavage of a
pyrophosphate released as each nucleotide is added. Each
nucleoside-5'--triphosphate has three phosphate groups linked to
carbon five of the ribose or deoxyribose sugar. The addition of a
nucleotide to a growing nucleic acid results in formation of an
internucleoside phosphodiester bond. This bond is characterized in
having a 3' linkage to carbon 3 of ribose or deoxyribose and a 5'
linkage to carbon 5 of ribose or deoxyribose. Each nucleotide is
added through formation of a new 3'.fwdarw.5' linkage, so the
nucleic acid strand grows in a 5' to 3' direction.
[0161] Depolymerization in its strictest sense means the reverse of
polymerization so that in the present context, an internucleotide
phosphodiester bond is broken between the two 3'-terminal bases in
the presence of pyrophosphate and a polymerase enzyme to form a
nucleic acid that is one nucleotide shorter and a nucleoside
triphosphate. A somewhat more encompassing definition is
contemplated here. In accordance with that definition, the
3'-terminal nucleotide is removed from a nucleic acid in a reaction
catalyzed by an enzyme, but the nucleotide formed can be a
monophosphate and pyrophosphate is not always required.
[0162] The former reactions are referred to herein as
pyrophosphorolysis reactions whereas the latter reactions are
referred to as exonuclease reactions. These two types of
depolymerization are discussed below.
[0163] It is to be understood that the depolymerization reaction of
interest in the invention is that depolymerization occurring in the
3'-terminal region of the nucleic acid probe. This depolymerization
reaction releases identifier nucleotide, as discussed herein.
[0164] A. Pyrophosphorolysis
[0165] In some embodiments of the present invention, a method
comprises depolymerizing the nucleic acid (NA) at a 3'-terminal
nucleotide by enzymatically cleaving the terminal internucleoside
phosphodiester bond in the presence of pyrophosphate, or an
analogue thereof, to form an XTP as illustrated by the following
reaction on double-stranded DNA having a 5' overhang:
1 5'. . . TpApCpGpGpCpT-3'OH 3'. . . ApTpGpCpCpGpApCpTp-5' .dwnarw.
enzyme + PPi 5'. . . TpApCpGp-3'OH 3'. . . ApTpGpCpCpGpApCpTp-5' +
dGTP + dCTP + dTTP
[0166] Several polymerases are known to catalyze the reverse of the
polymerization process. This reverse reaction is called
"pyrophosphorolysis." The pyrophosphorolysis activity of DNA
polymerase was demonstrated by Deutscher and Kornberg, J. Biol.
Chem., 244:3019-28 (1969). Other template-dependent nucleic acid
polymerases capable of pyrophosphorolysis include, but are not
limited to, DNA polymerase .alpha., DNA polymerase .beta., T4 DNA
polymerase, Taq polymerase, Tne polymerase, Tne triple mutant
polymerase, Tth polymerase, Tvu polymerase, Ath polymerase, Bst
polymerase, E. coli DNA polymerase I, Klenow fragment, Klenow exo
minus (exo-), AMV reverse transcriptase, RNA polymerase and MMLV
reverse transcriptase. However, not all polymerases are known to
possess pyrophosphorolysis activity. For example, poly(A)
polymerase has been reported to not catalyze pyrophosphorylation.
(See Sippel, Eur. J. Biochem. 37:31-40 (1973)).
[0167] A mechanism of pyrophosphorolysis has been suggested for RNA
polymerase. Although understanding of the mechanism is not
necessary to use the present invention, it is believed that the
partial transfer of a Mg.sup.2+ ion from the attacking
pyrophosphate to the phosphate of the internucleoside
phosphodiester bond of the RNA can increase the nucleophilic
reactivity of the pyrophosphate and the electrophilicity of the
diester as described in Rozovskaya et al., Biochem. J., 224:645-50
(1984). The internucleoside phosphodiester bond is enzymatically
cleaved by the addition of pyrophosphate to the nucleoside 5'
phosphate and a new phosphodiester bond is formed between the
pyrophosphate and the nucleoside monophosphate.
[0168] The pyrophosphorolysis reaction can be summarized as
follows:
Reaction 1: NA.sub.n+PP.sub.i-NA.sub.n-1+XTP
[0169] wherein NA is a nucleic acid, n is the number of nucleotide
bases, PP.sub.i is pyrophosphate and XTP is either a DNTP molecule
or NTP molecule. The reaction can then be repeated so as to produce
at least two XTP molecules. It should be noted that the reaction
can be repeated on the same nucleic acid molecule or on a plurality
of different nucleic acid molecules.
[0170] In a preferred embodiment in the case of the reverse of
polymerase activity (pyrophosphorolysis), a preferred substrate is
a DNA probe hybridized to a nucleic acid target sequence with total
complementarity at its 3'-terminus, including an identifier residue
at the 3'-terminal region. In an example of this preferred
embodiment, when the nucleic acid probe is hybridized to a nucleic
acid target sequence such that there is one base mismatch at the
3'-terminal nucleotide of the nucleic acid probe, the nucleic acid
probe is inefficiently depolymerized through the reverse
polymerization reaction. Thus, such a substrate is not an ideal
substrate for depolymerization.
[0171] The non-ideality of the substrate for depolymerization via a
reverse of the polymerization reaction is recognized with a single
base mismatch as far in as about 10 residues from the 3'-terminus
of the nucleic acid probe. With a single base mismatch 12 residues
from the 3'-terminus of the probe, the depolymerization reaction
can occur to approximately the same extent as when there is no
mismatch and the nucleic acid probe is totally complementary to the
nucleic acid target sequence.
[0172] It is thus contemplated that the reactivity of the
depolymerization reaction is a continuum that is related to the
efficiency of the substrate. A partially complementary hybrid is a
less efficient depolymerization substrate than a totally
complementary hybrid for the reverse of a polymerization reaction.
It is contemplated that this differential reactivity be used to
enhance the discrimination between matches and mismatches at
certain positions (e.g. an interrogation position). When a
substrate hybrid is totally complementary, it will give a fairly
high analytical output. A mismatch can be intentionally introduced
to destabilize the substrate hybrid. Such a destabilization can
increase the difference in analytical output between bases
substituted at an interrogation position that is different from the
destabilizing base position.
[0173] Several chemical compounds are known in the art to be
substitutable for pyrophosphate in pyrophosphorolysis reactions.
Rozovskaya, et al., Biochem. J., 224:645-650 (1984). Exemplary
compounds and their released nucleotide product are shown in the
table below, along with the nucleotide product (where the
ribonucleoside or deoxyribonucleoside is denoted "Nuc") of
pyrophosphorolysis. 1
[0174] Preferred reaction mixtures for depolymerization by
pyrophosphorolysis, including suitable buffers for each nucleic
acid polymerase analyzed, are described in greater detail in the
Examples. Typically, under these conditions, sufficient NTP or dNTP
is released to accurately detect or assay extremely low amounts of
nucleic acids (e.g., about 5-1000 picograms). ATP can be produced
by conversion from XTP by an enzyme such as NDPK (in the presence
of ADP) prior to analysis or the ATP can be further amplified prior
to analysis.
[0175] Even though the preferred reaction conditions for
polymerization and depolymerization by pyrophosphorolysis are
similar, the rates of these reactions can vary greatly. For
example, AMV and RLV reverse transcriptases catalyze
pyrophosphorolysis under optimal conditions at a rate of about
fifty- to one hundred-fold less than polymerization as demonstrated
in Srivastavan and Modak, J. Biol. Chem., 255(5):2000-04 (1980).
Thus, the high efficiency of the pyrophosphorolysis reaction was
unexpected, and appears to be associated with extremely low levels
of DNA substrate, in contrast to previous DNA pyrophosphorolysis
studies conducted using much greater amounts of DNA.
[0176] Although not wishing to be bound by theory, a possible
explanation for this effect might also be that the molar
concentrations of free deoxyribonucleoside triphosphates produced
at very low DNA levels would be predicted to be very low. Indeed,
these levels are expected to be far below the Michaelis constant
(K.sub.m) of the enzyme. Thus, reincorporation of released dNTPs
would be expected to be vanishingly small.
[0177] The pyrophosphorolysis activity of different nucleic acid
polymerases also varies. For example, T4 polymerase and Tne DNA
polymerase possess very high pyrophosphorolysis activity as
measured by a luciferase assay for ATP produced by
pyrophosphorolysis. Pyrophosphorolysis using T4 polymerase resulted
in about a 10 fold increase in light production as compared to
MMLV-RT and a 4 fold increase in light production as compared to
Taq polymerase.
[0178] During the development of the invention disclosed in the
parent application, it was discovered that the detection of some
types of nucleic acids at low picogram levels is generally enhanced
by fragmenting or partially digesting the nucleic acid. Preferably,
fragmentation is accomplished by sonication or restriction enzyme
digestion of the nucleic acid in order to provide a plurality of
smaller nucleic acid fragments. Although an understanding of the
mechanism is not necessary in order to practice the present
invention, this step probably enhances detection because the
pyrophosphorolysis reaction only proceeds from the nucleic acid
ends. By providing a greater number of nucleic acid ends, more
reactions are allowed to occur at any one time.
[0179] It should be noted that DNA ends can be present within a
molecule as well as at the end of a linear DNA fragment. For
example, polymerases can catalyze pyrophosphorolysis from a gap or
a nick in a DNA segment. The type of enzyme and substrate used for
pyrophosphorolysis reactions determine whether fragmentation is
necessary.
[0180] The type of DNA end resulting from restriction enzyme
digestion also affects the pyrophosphorolysis activity of different
nucleic acid polymerases. For example, Klenow exo-, MMLV-RT and Tag
polymerase catalyze pyrophosphorolysis of DNA fragments with
5'-overhangs and with blunt-ends, but have little or no
pyrophosphorolysis activity with 3'-overhangs. In contrast, T4 DNA
polymerase catalyzes both 3'- and 5'-end overhang and blunt-end
mediated pyrophosphorolysis. Thus, T4 DNA polymerase is a preferred
enzyme for pyrophosphorolysis of a hybrid with a 3'-overhang. When
other nucleic acid polymerases are utilized for pyrophosphorolysis
of restriction enzyme treated DNA, it is contemplated that care is
taken to match the end specificity of the polymerase with the type
of end created by the restriction endonuclease. Such care is well
within the skill of those in the art.
[0181] Tabor and Richardson, J. Biol. Chem. 265 (14):8322-28 (1990)
reported unwanted pyrophosphorolysis mediated by T7 DNA
polymerase-catalyzed DNA sequencing by the chain termination
method. Those authors note that, even at the most sensitive sites,
the rate of unwanted pyrophosphorolysis is at least 100,000 times
slower than the rate of polymerization.
[0182] By definition, DNA sequencing is directed to ascertaining an
unknown DNA sequence, rather than the detection of a known DNA
sequence. In DNA sequencing by the chain termination method,
oligonucleotide primers are extended by T7 DNA polymerase supplied
with exogenous dNTPs and dideoxy NTPs. When a dideoxy NTP is
incorporated into an elongating primer, no further polymerization
can take place. These dideoxy-terminated fragments are then
resolved on a DNA sequencing gel. However, in certain instances
unwanted pyrophosphorolysis removes a 3'-terminal dideoxynucleotide
from the elongated primer, which allows T7 DNA polymerase to
catalyze additional polymerization. This additional polymerization
leads to the degradation (loss) of specific
dideoxynucleotide-terminated fragments on DNA sequencing gels. In
other words, the resulting DNA sequencing gel will exhibit "holes"
or gaps where the DNA sequence cannot be determined.
[0183] Tabor and Richardson, above, noted that when dNTPs are
present in high concentrations, these pyrophosphorolysis sites
occur once in several thousand nucleotides. Those authors have
identified a canonical sequence, 5' dIdAdN.sub.1ddN.sub.2 3', which
is especially sensitive to pyrophosphorolysis when dITP is
substituted for dGTP. This unwanted T7 DNA polymerase-mediated
pyrophosphorolysis reaction can be avoided by the addition of
pyrophosphatase, which eliminates PP.sub.i from the DNA sequencing
reaction mixture. Pyrophosphatase, it is reported, eliminates the
gaps in a DNA sequencing gel, permitting the accurate determination
of a DNA sequence using T7 DNA polymerase-mediated dideoxy
sequencing.
[0184] The present invention, in contrast, seeks to exploit DNA
polymerase-mediated pyrophosphorolysis, by optimizing conditions
for this reverse reaction to take place. The present invention is
directed to the detection of a known sequence in a target nucleic
acid, rather than ascertaining an unknown nucleic acid sequence
using the polymerization activity of T7 DNA polymerase.
[0185] The pyrophosphorolysis reported by Tabor and Richardson
cannot detect the presence of a specific nucleic acid sequence. In
fact, DNA sequencing by the dideoxy method relies upon the
incorporation of dideoxy nucleotides into an elongating primer. The
T7 DNA polymerase-mediated pyrophosphorolysis reported by those
authors is equally random, although there is reported a preference
for the above-mentioned canonical sequence. According to Tabor and
Richardson, in the absence of pyrophosphatase, one would only note
gaps in a DNA sequencing gel, and those gaps would not provide any
information as to the DNA sequence at those gaps. There is
accordingly no method disclosed for identifying the release of 3'
dideoxy nucleotides by the reported T7 DNA polymerase-mediated
pyrophosphorolysis.
[0186] Further, it is contemplated that the type of polymerase used
in the pyrophosphorolysis reaction is matched to the correct
nucleic acid substrate in order to produce the best results. In
general, DNA polymerases and reverse transcriptases are preferred
for depolymerizing DNA, whereas RNA polymerases are preferred for
depolymerizing RNA. Reverse transcriptases or DNA polymerases with
reverse transcriptase activity are preferred for depolymerizing
RNA-DNA hybrids.
[0187] In the parent application, it was surprisingly determined
that poly(A) polymerase can catalyze pyrophosphorolysis, even
though no such reaction had been previously reported. Indeed,
poly(A) polymerase has been widely reported to not catalyze
pyrophosphorolysis. (See e.g., Sippel, Eur. J. Biochem., 37:31-40
(1973) and Sano and Feix, Eur. J. Biochem., 71:577-83 (1976)). In
these preferred embodiments of the invention disclosed in the
parent application, the manganese chloride present in the
previously reported buffers is omitted, the concentration of sodium
chloride is decreased, and the pH value is lowered from about 8.0
to about 7.5. Furthermore, the poly(A) polymerase
pyrophosphorolysis reaction buffer contains about 50 mM Tris-Cl pH
7.5, 10 mM MgCl.sub.2, 50 mM NaCl, and 2 mM NaPP.sub.i (sodium
pyrophosphate).
[0188] It is important to note that the depolymerization reaction
is the reverse of the polymerization reaction. Therefore, as
increasing amounts of free nucleoside triphosphates are produced by
depolymerization, a state of equilibrium can theoretically be
attained in which polymerization and depolymerization reactions are
balanced. Alternatively, where small amounts of nucleic acid are
detected, the reaction can go essentially to completion without
reaching equilibrium, (i.e., the nucleic acid target is
depolymerized into its constituent subunit nucleotides by greater
than 50%). This factor is important in quantitative assays because
the total amount of nucleotides released is proportional to the
amount of signal generated in the detection assay.
[0189] When used for qualitative detection of nucleic acid, as long
as a threshold level of nucleotides is produced, it is not
necessary that the reaction reach equilibrium or go essentially to
completion. In preferred embodiments, the mixture of nucleoside
triphosphate molecules produced by depolymerization is preferably
converted to ATP as described below. For either quantitative or
qualitative detection, a detectable threshold ATP concentration of
approximately 1.times.10.sup.-12 molar in 100 .mu.l of sample is
preferably provided for detection of light in a typical luciferase
assay.
[0190] In some preferred embodiments, oligonucleotide probes are
typically utilized at about 100 ng to about 1 .mu.g per 20 .mu.L
depolymerization reaction. That amount provides a probe to target
weight ratio of about 200:1 to about 1,000:1.
[0191] In a preferred embodiment of the present invention, nucleic
acid polymerase and pyrophosphate (PP.sub.i) or an analogue
thereof, are added to a hybridized sample containing from less than
about 100 .mu.g of target nucleic acid, to less than about 10 pg of
nucleic acid. Typical target nucleic acids are present at about 1
to about 5 ng in the sample to be assayed, with a target nucleic
acid length of about 30 to about 1000 bp being preferred.
[0192] Next, the hybridized nucleic acid is degraded
(depolymerized) by pyrophosphorolysis, releasing free NTPs or
dNTPs. Enzymes useful in the pyrophosphorolysis reaction include,
but are not limited to, those noted previously such as the
following polymerases: AMV reverse transcriptase, MMLV reverse
transcriptase, DNA polymerase alpha and beta, Tag polymerase, Tne
polymerase, Ath polymerase, Tvu polymerase, Tne triple mutant
polymerase, T4 DNA polymerase, E. coli DNA polymerase I, Klenow
fragment, Klenow exo minus, Tth polymerase, and poly(A)
polymerase.
[0193] Most preferably, Klenow exo minus (Klenow exo-) or Tne
triple mutant polymerase is utilized for DNA pyrophosphorolysis
reactions because of their efficient utilization of 5' overhanging
DNA ends.
[0194] When using enzymes that utilize 5' overhang substrates, it
is preferred that the 3' end of the target nucleic acid extends
beyond the 5' end of the nucleic acid probe. In this way, the only
5' overhang substrate is that where the 5' end of the target
nucleic acid overhangs the 3' terminal region of the nucleic acid
probe. An alternative method of limiting depolymerization to the
nucleic acid probe is chemical modification of the ends of other
nucleic acids in the sample, such as, for example, making a
phosphorothioate linkage at the 3'-terminus of the target nucleic
acid.
[0195] A depolymerizing enzyme is preferably present in an amount
sufficient to depolymerize a hybridized target:probe. That amount
can vary with the enzyme used, the depolymerization temperature,
the buffer, and the like, as are well-known in the art. For a
typical reaction carried out in a 20 .mu.L volume, about 0.25 to
about 1 unit (U) of an enzyme such as Klenow exo- is used. About 1
to about 5 U of the thermostable enzymes are used for
depolymerization at elevated temperatures.
[0196] Luciferase, which is part of the preferred ATP detection
system, is inhibited by PP.sub.i. In preferred embodiments, care is
taken to avoid transferring a highly inhibiting amount of PP.sub.i
to the ATP detection reaction. Preferably, the amount of PP.sub.i
carried over to the ATP detection reaction results in a
concentration of PP.sub.i in the luciferase detection reaction of
less than about 100 .mu.M, although less than about 10 .mu.M is
desirable. Therefore, the amount of PP.sub.i utilized in the
pyrophosphorolysis reaction is determined by the size of the
aliquot that is taken for use in the luciferase detection system.
It is contemplated that the aliquot size can vary depending upon
the test system used, but the amount of PP.sub.i transferred or
carried over to the luciferase detection reaction corresponds to
the PP.sub.i concentration parameters described above, so that the
concentration of PP.sub.i is at least below about 100 .mu.M, and
preferably below about 10 .mu.M.
[0197] In one preferred embodiment of the invention, the enzyme
whose activity is to depolymerize is a template-dependent
polymerase. The depolymerization reaction is a reverse of the
polymerization reaction. In a contemplated embodiment, the
polymerization reaction is reversed in the presence of
pyrophosphate in a reaction referred to as pyrophosphorolysis.
[0198] In some preferred embodiments, the reaction conditions are
preferably adjusted to further favor depolymerization of a nucleic
acid probe that is hybridized with its target nucleic acid sequence
by providing a higher concentration of nucleic acid probe than its
target nucleic acid sequence.
[0199] One strategy to favor the depolymerization of a probe:target
hybrid is that the probe be in excess over the nucleic acid target
in the hybridization step after denaturing of duplex target nucleic
acid.
[0200] Another strategy to favor the depolymerization of a
probe:target hybrid is to isolate only the strand of nucleic acid
target to which the probe is complementary. There are several
techniques that can be used to achieve this end.
[0201] In one technique, phosphorothioate linkages are utilized at
the 5'-terminus of a target nucleic acid amplifying primer
sequence, e.g., at the 1 to about 10 5'-most residues. Upon PCR
amplification of the target, the phosphorothioate linkages of the
primer become incorporated into the amplified target nucleic acid
as part of one of a pair of complementary strands. Treatment of the
double-stranded resulting molecule with T7 polymerase exonuclease 6
removes the non-phosphorothioate-containing strand. This technique
is illustrated in detail in the Examples hereinafter.
[0202] In another technique, strand isolation can be accomplished
by amplifying the target nucleic acid using PCR primers
incorporated into the extended nucleic acid strand (with which a
nucleic acid probe useful herein is designed to hybridize) that are
not labeled, whereas primers for the complementary strand are
labeled, such as with biotin. Then, the amplified nucleic acid is
denatured and added to streptavidin linked to a solid support. A
useful material is Streptavidin MagneSphere.RTM. paramagnetic
particles (Promega, Z548A), where a magnet can be used to separate
the desired target nucleic acid strand from its biotinylated
complementary strand.
[0203] B. Exonuclease Digestion
[0204] In other embodiments of the present invention, a method
comprises depolymerizing the nucleic acid at a 3'-terminal
nucleotide by enzymatically cleaving the terminal internucleoside
phosphodiester bond to form an XMP as illustrated by the following
reaction on double-stranded DNA having a 5'-overhang:
2 5'. . . GpCpTpApApGpT-3'OH 3'. . . CpGpApTpTpCpApCpTp-5' .dwnarw.
enzyme 5'. . . GpCpTpA-3'OH 3'. . . CpGpApTpTpCpApCpTp-5' + dAMP +
dGMP + dTMP
[0205] For example, such a hydrolysis reaction can be catalyzed by
Klenow or Exonuclease III in the presence or absence of NTPs.
[0206] In some embodiments (e.g., quantitative assays for nucleic
acids), the depolymerizing step is repeated essentially to
completion or equilibrium to obtain at least two nucleotide
molecules from a strand of minimally three nucleotides in order to
increase detection sensitivity. In alternative embodiments, (e.g.,
qualitative detection of DNA), the depolymerizing step need not be
repeated if there are sufficient nucleic acid molecules present to
generate a signal.
[0207] In another embodiment of the present invention, terminally
mismatched hybridized nucleic acid probes are first depolymerized
into NMP or dNMP by exonuclease digestion according to the
following reaction:
Reaction 2: NA.sub.n+H.sub.2O.fwdarw.NA.sub.n-1+XMP
[0208] wherein NA.sub.n is a nucleic acid, XMP is either a DNMP or
NMP, and n is the number of nucleotides in the nucleic acid.
[0209] This depolymerization reaction is shown more specifically
below in the following reaction on double-stranded DNA having a
5'-overhang and mismatched bases at the 3'-terminus:
3 5'. . . CpTpApApGPC-3'OH 3'. . . GpApTpTpCpApCpTp-5' .dwnarw.
enzyme 5'. . . CpTpApApG-3'OH 3'. . . GpApTpTpCpApCpTp-5' +
dCMP
[0210] For example, such a depolymerization reaction can be
catalyzed by bacteriophage T4 polymerase in the absence of NTPs. In
preferred embodiments, the released nucleotides, XMPs, are produced
by nuclease digestion.
[0211] Nuclease digestion can be accomplished by a variety of
nucleases that release a nucleotide with a 5' phosphate, including
S1 nuclease, nuclease BAL 31, mung bean nuclease, exonuclease III
and ribonuclease H. Nuclease digestion conditions and buffers are
known in the art. Nucleases and buffers for their use are available
from commercial sources.
[0212] In the biosynthesis of purine and pyrimidine
mononucleotides, phosphoribosyl-1-pyrophosphate (PRPP) is the
obligatory ribose-5'-phosphate donor. PRPP itself is formed in a
reaction catalyzed by PRPP synthetase through the transfer of
pyrophosphate from ATP to ribose-5'-phosphate. This reaction is
known to be reversible as described in Sabina et al., Science,
223:1193-95 (1984).
[0213] In some embodiments of the present invention, the NMP or
dNMP produced by nuclease digestion is preferably converted
directly to NTP or DNTP by the enzyme PRPP synthetase in the
following reaction:
Reaction 3: XMP+PRPP.fwdarw.XTP+ribose-5'-PO.sub.4
[0214] wherein XMP is either AMP or DAMP, and XTP is either ATP or
DATP. Preferably, this reaction produces a threshold ATP
concentration of approximately 1.times.10.sup.-12 M in 100 .mu.L of
sample.
[0215] In this reaction, the pyrophosphate group of PRPP is
enzymatically transferred to XMP molecules, forming XTP molecules.
Examples of suitable reaction conditions and buffers are set forth
elsewhere herein.
[0216] Utilization of the PRPP reaction in the nucleic acid
detection system of the present invention has advantages over
previously reported methods. For example, only one step is
necessary to convert an AMP or DAMP to ATP or DATP, thereby
simplifying the detection system. In addition, contamination of the
detection reaction with exogenous ATP, ADP, or AMP is less likely
using methods of the present invention, as compared to previously
reported methods.
[0217] In an embodiment wherein the depolymerizing enzyme exhibits
3'.fwdarw.5' exonuclease activity, the substrate is a
double-stranded or single-stranded nucleic acid having a
3'-hydroxyl terminus. Enzymes having 3'.fwdarw.5' exonuclease
activity that are useful in a process of the invention include E.
coli DNA polymerase I, Klenow fragment and bacteriophage T4 DNA
polymerase. E. coli DNA polymerase I holoenzyme is not preferred in
a process of the invention because it is preferable to avoid the
5'.fwdarw.3' exonuclease activity that degrades probe:target
hybrids regardless of the degree of hybridization at the
3'-terminus. Bacteriophage .lambda. exonuclease has only
5'.fwdarw.3' exonuclease activity, so it is not a contemplated
enzyme. Similarly, Taq DNA polymerase has a very low level of
3'.fwdarw.5' exonuclease activity. Exonuclease III (Exo III) has 3'
exonuclease activity on blunt-ended substrates or those having
5'-overhangs or nicks with 3'-hydroxyl groups, and is thus useful
in a process of the invention for depolymerizing hybrids with
matched 3' terminal nucleotides. However, Exo III is not limited to
hybrids having only partially complementary 3'-termini, it requires
a double stranded end, i.e. a matched terminal nucleotide.
[0218] In an embodiment of the invention where the enzyme's
activity is a 3'.fwdarw.5' exonuclease activity, the hybridized
nucleic acid probe is depolymerized from its 3'-terminal
nucleotide. In a preferred embodiment in the case of a 3'.fwdarw.5'
exonuclease activity of a polymerase, the preferred substrate is a
nucleic acid probe hybridized to a nucleic acid target sequence
with partial complementarity at its 3'-terminal region, most
preferably with a mismatch at its 3'-terminal residue that is an
identifier nucleotide.
[0219] A contemplated method is particularly useful in a multiplex
assay environment in which a plurality of probes is utilized to
determine whether one or more of a plurality of predetermined
endogenous nucleic acid sequences is present or absent in a sample.
A particularly useful area for such multiplex assays is in
screening assays where the usual analytical output indicates that
the sought-after gene target is absent.
[0220] In one illustrative embodiment, a nucleic acid sample is
screened for the presence of a plurality of predetermined mutant
genes. In this embodiment, the mutants usually are not present and
the analytical output is, for example, at about background levels
except where a mutation is present. In another embodiment, a
plurality of samples is examined for the presence or absence of
microbe-specific genes. Here, again, where a population of healthy
individuals, animals, or presumably sterile food is sampled, the
absence of the sought-after genes provides an analytical output
that is about background levels, and only in the rare instance does
a greater than the background output appear.
[0221] In a multiplexed embodiment of the above process, the sample
is admixed with a plurality of different nucleic acid probes,
preferably after amplification of the multiple nucleic acid targets
as needed. In this embodiment of the invention, the analytical
output for a certain result with one of the probes is
distinguishable from the analytical output from the opposite result
with all of the probes.
[0222] In preferred embodiments, the ATP produced via NDPK
conversion of released nucleotides in the presence of ADP is
detected by a luciferase detection system or an NADH detection
system. In still another embodiment of the present invention, the
pyrophosphate transferring step and the phosphate transferring step
are performed in a single pot reaction. In other preferred
embodiments, if increased sensitivity is required, the ATP
molecules can be amplified.
[0223] In a contemplated multiplex embodiment, information about
the presence or absence of a plurality of endogenous nucleic acid
target sequences is determined using a process of the invention on
a single nucleic acid sample, by admixing the sample with a
plurality of nucleic acid probes for the various endogenous nucleic
acid targets.
[0224] In a first multiplex embodiment of the invention, the
analytical output obtained when at least one of the nucleic acid
probes hybridizes with partial complementarity to its target
nucleic acid sequence is greater than the analytical output when
all of the nucleic acid probes hybridize with total complementarity
to their respective nucleic acid target sequences. Preferably, in
such an embodiment, the enzyme whose activity is to depolymerize
hybridized nucleic acid to release nucleotides exhibits a
3'.fwdarw.5'-exonuclease activity, depolymerizing hybridized
nucleic acids having one or more mismatched bases at the
3'-terminus of the hybridized probe.
[0225] In a second multiplex embodiment of the invention, the
analytical output obtained when at least one of said nucleic acid
probes hybridizes with partial complementarity to its target
nucleic acid sequence is less than the analytical output when all
of the nucleic acid probes hybridize with total complementarity to
their respective nucleic acid target sequences. Preferably, in such
an embodiment, the enzyme whose activity is to depolymerize
hybridized nucleic acid to release nucleotides is a
template-dependent polymerase.
[0226] In a third multiplex embodiment of the invention, the
analytical output obtained when at least one of said nucleic acid
probes hybridizes with total complementarity to its nucleic acid
target sequence is greater than the analytical output when all of
the nucleic acid probes hybridize with partial complementarity to
their respective nucleic acid target sequences. Preferably, in such
an embodiment, the enzyme whose activity is to depolymerize
hybridized nucleic acid to release nucleotides is a
template-dependent polymerase.
[0227] In a fourth multiplex embodiment of the invention, the
analytical output obtained when at least one of said nucleic acid
probes hybridizes with total complementarity to its target nucleic
acid sequence is less than the analytical output when all of the
nucleic acid probes hybridize with partial complementarity to their
respective nucleic acid target sequences. Preferably, in such an
embodiment, the enzyme whose activity is to depolymerize hybridized
nucleic acid to release nucleotides exhibits a
3'.fwdarw.5'-exonuclease activity, depolymerizing hybridized
nucleic acids having one or more mismatched bases at the
3'-terminus of the hybridized probe.
[0228] Analytical Output
[0229] The analytical output is obtained by detection of the
released identifier products, either the released nucleotides or
the remainder of the probe. Exemplary detection systems include the
light emitting luciferase detection system, the NADH light
adsorption detection system (NADH detection system), fluorescence
emissions and mass spectrometry. These detection systems are
discussed hereinbelow.
[0230] The fact that nucleotides were released (a qualitative
determination), or even the number of nucleotides released (a
quantitative determination) can be deduced through examination of
the probe after depolymerization. The determination of the size of
an oligonucleotide is well known in the art. For example gel
separation and chromatographic separations are well known. Gel
imaging techniques that take advantage of fluorescence and
absorbance spectroscopy as well as radiographic methods. Mass
spectrometry of oligonucleotides is also becoming more common.
[0231] A. Detection Of ATP
[0232] Luciferase detection systems are particularly useful for
detecting ATP. In the presence of ATP and oxygen, luciferase
catalyzes the oxidation of luciferin, producing light that can then
be quantified using a luminometer. Additional products of the
reaction are AMP, pyrophosphate and oxyluciferin.
[0233] In particularly preferred embodiments, ATP detection buffer
referred to as L/L reagent (Promega, FF2021) is utilized. In some
embodiments, Luciferase Assay Reagent (LAR) buffer (Promega, E152A)
is used instead of L/L reagent. Preferably, about 5 to 10 ng of
luciferase are used in the reaction. Although it is not intended
that the present invention be limited to a specific concentration
of luciferase, greater amounts of luciferase have a tendency to
increase non-specific background.
[0234] It is contemplated that in some embodiments, the dNTPs or
NTPs produced by pyrophosphorolysis or nuclease digestion are
converted to XTP, which can then be used directly as substrate for
luciferase, permitting detection of the nucleic acid. However, the
preferred substrate for luciferase is ATP, as demonstrated by Moyer
and Henderson, Anal. Biochem., 131:187-89 (1983). When DNA is the
initial substrate, NDPK is conveniently utilized to catalyze the
conversion of dNTPs to ATP by the following general reaction:
Reaction 4: dNTP*+ADP.fwdarw.dNDP+ATP*
[0235] wherein dNTP is a mixture of deoxyribonucleoside
triphosphates and dNDP is the corresponding deoxyribonucleoside
diphosphate. In Reaction 4, the terminal 5'-triphosphate (P*) of
the dNTP is transferred to ADP to form ATP.
[0236] Enzymes catalyzing this reaction are generally known as
nucleoside diphosphate kinases (NDPKs). NDPKs are ubiquitous,
relatively nonspecific enzymes. For a review of NDPK, see Parks and
Agarwal, in The Enzymes, Volume 8, P. Boyer Ed. (1973).
[0237] The conversion of NTPs or dNTPs to ATP by NDPK is preferably
accomplished by adding NDPK and a molar excess of ADP over the
amounts of NTPs or dNTPs expected to be produced by
pyrophosphorolysis or nuclease digestion, followed by
pyrophosphorylation by PRPP synthetase. The utilization of ADP
requires optimization of the amount of ADP added. Too much ADP
results in high background levels.
[0238] NDPK (EC 2.7.4.6) preparations from several biological
sources are commercially available from several suppliers. For
example yeast NDPK is available from Sigma Chemical Co., St. Louis,
Mo., whereas bovine NDPK is available from ICN Biochemicals, Inc.,
Costa Mesa, Calif. The particular NDPK selected for most uses
described herein is typically a matter of choice.
[0239] A further embodiment of the invention, such as is used for
Single Tandem Repeat (STR) detection, contemplates a method for
determining the number of known repeated sequences that are present
in a nucleic acid target sequence in a nucleic acid sample. A
method for determining the number of repeated known sequences
comprises the following steps. A plurality of separately treated
samples is provided. Each sample contains a nucleic acid target
sequence, containing a plurality of known repeated sequences and a
non-repeated region, hybridized with a nucleic acid probe. Each
nucleic acid probe contains a different number of complementary
known repeated sequences of alleles of the target nucleic acid, an
identifier nucleotide in the 3'-terminal region and a 5'-terminal
locker sequence that is complementary to the non-repeated region of
the target. A treated reaction mixture is formed by admixing each
treated sample with a depolymerizing amount of an enzyme whose
activity is to release one or more nucleotides from the 3'-terminus
of a hybridized nucleic acid probe. The treated reaction mixture is
maintained for a time period sufficient to permit the enzyme to
depolymerize the hybridized nucleic acid probe and release an
identifier nucleotide. The samples are analyzed for the presence or
absence of released identifier nucleotide to obtain an analytical
output. The analytical output from the sample whose probe contained
the same number of sequence repeats as present in the target
nucleic acid is indicative of and determines the number of sequence
repeats present in the nucleic acid target.
[0240] In one aspect of the method, the target nucleic acid is
homozygous with respect to the number of the repeated sequences at
the two alleles. In an alternative method of the invention, the
target nucleic acid is heterozygous for the repeated sequences. In
another method of the invention, an identifier nucleotide is a
nucleotide that is part of the region containing a repeated
sequence. In an alternative method of the invention, an identifier
nucleotide of the probe sequence is part of the region containing a
non-repeating sequence that is complementary to that located in the
target nucleic acid 5' to the repeated sequences. In this latter
aspect of the method, the identifier nucleotide is present in a
sequence containing 1 to about 20 nucleic acids that is
complementary to a non-repeating sequence of the target nucleic
acid located in the probe 3' to the repeated sequences. The
repeated known sequence present in a nucleic acid target sequence
typically has a length of 2 to about 24 bases per repeat. Di- and
tri-nucleotide repeats are well known in the art.
[0241] A contemplated thermostable NDPK, such as Pfu NDPK, is
advantageously utilized in a so-called one-step or one-pot method
of this invention. Here, a treated sample that may contain the
predetermined nucleic acid target sequence hybridized with a
nucleic acid probe that includes an identifier nucleotide in the
3'-terminal region is admixed with a depolymerizing amount of an
enzyme whose activity in the presence of pyrophosphate is to
release identifier nucleotides as nucleoside triphosphates from the
hybridized nucleic acid probe, adenosine 5' diphosphate (ADP),
pyrophosphate and NDPK to form a treated reaction mixture. The
treated reaction mixture so formed is maintained for a time period
sufficient to permit the enzyme to depolymerize the probe and to
permit NDPK to convert the XTP present into ATP (as shown in
reaction 4). The amount of ATP formed is determined by the
production of an analytical output, with that output providing the
indication of the presence or absence of the presence of the target
nucleic acid sequence.
[0242] Although yeast, bovine or another NDPK can be used in these
reactions, it is preferred to utilize a thermostable NDPK such as
the Pfu NDPK along with a thermostable depolymerizing enzyme such
as the Tne triple mutant DNA polymerase (discussed below), Bst DNA
polymerase, Ath DNA polymerase, Taq DNA polymerase and Tvu DNA
polymerase along with a reaction temperature of about 50.degree. C.
to about 90.degree. C. The use of these thermostable enzymes at an
above temperature can enhance the sensitivity of the method.
[0243] The Tne triple mutant DNA polymerase is described in detail
in WO 96/41014, whose disclosures are incorporated by reference,
and its 610 residue amino acid sequence is provided as SEQ ID NO:35
of that document. That enzyme is referred to in WO 96/41014 as Tne
M284 (D323A,D389A).
[0244] Briefly, that enzyme is a triple mutant of the polymerase
encoded by the thermophilic eubacterium Thermotoga neapolitana
(ATCC 49049). The amino-terminal 283 residues of the native
sequence are deleted and the aspartic acid residues at positions
323 and 389 of the native sequence are replaced by alanine residues
in this recombinant enzyme. This recombinant enzyme is thus a
deletion and replacement mutant of the native enzyme.
[0245] Deletion of the amino-terminal sequence removes the 5'
exonuclease activity of the native enzyme, whereas replacement of
the two aspartic acid residues removes a magnesium binding site
whose presence facilitates exonuclease activity, and this triple
mutant also exhibited no 3' exonuclease activity relative to the
recombinant native enzyme. This triple mutant enzyme exhibited a
half-life at 97.5.degree. C. of 66 minutes as compared to the full
length recombinant enzyme that exhibited a half-life of only 5
minutes at that temperature.
[0246] A reaction containing NDPK contains about 0.01 to 0.50 .mu.M
ADP, preferably about 0.05 .mu.M ADP. Various useful buffers and
other reaction components are set forth elsewhere. NDPK is itself
present in an amount sufficient to catalyze the desired conversion
of ADP to ATP. In a typical assay starting from a 20 .mu.L
depolymerization reaction, about 0.1 U of NDPK are used.
[0247] Where larger volumes of reactants are used, with the target
and probe concentrations being approximately proportionately
larger, the amount of NDPK or the other enzymes discussed herein
can be used in a similar larger proportion relative to the amount
discussed for the 20 .mu.L reaction. Indeed, a 20 .mu.L reaction
has been successfully scaled down about two fold and scaled
upwardly by a factor of about 20.
[0248] B. NADH Detection
[0249] In the NADH detection system, a combination of two enzymes,
phosphoglycerate kinase and glyceraldehyde phosphate dehydrogenase,
is used to catalyze the formation of NAD from NADH in the presence
of ATP. Because NADH is fluorescent whereas NAD is not, ATP is
measured as a loss in fluorescence intensity. Examples of NADH
based ATP assays are disclosed in U.S. Pat. Nos. 4,735,897,
4,595,655, 4,446,231 and 4,743,561, and UK Patent Application GB
2,055,200, all of which are herein incorporated by reference.
[0250] C. Mass Spectrometric Analysis
[0251] In one method of the invention, the presence of released
nucleotides is analyzed via mass spectrometry. In an embodiment of
a method using mass spectrometry, the treated reaction mixture is
ionized in a manner such that all components of the treated
reaction mixture in the molecular weight range of the released
identifier nucleotides are measured. Very small differences in
molecular weight can be detected using mass spectrographic methods
(different isotopes of the same atom are detectable), so any
variation from a natural nucleic acid, including a single atom
substitution (e.g. a fluorine in place of a hydrogen atom or a
replacement of a hydrogen by a deuterium atom) in the identifier
nucleotide gives rise to a detectable difference. Nucleic acid
analogs used in methods of the invention should not interfere with
either the hybridization of the nucleic acid probe or
depolymerization of the hybridized probe.
[0252] Additionally, mass spectrometry can discriminate between
individual nucleotides or nucleosides. For example, if the
3'-identifier nucleotide used in the instant invention was a G
nucleotide, mass spectrometry can be used to detect the release of
that G nucleotide in a method of the present invention. Similarly,
mass spectrometry can detect the release of an A, T or C
nucleotide, based on the differences in atomic weight of these
compounds. Thus, in a multiplexing embodiment of the present
invention, mass spectrometry can be used to resolve the presence of
one or more of these 3'-identifier nucleotides.
[0253] In a particularly useful aspect of this embodiment, a mass
spectral technique referred to as DIOS (desorption/ionization on
silicon) was recently reported by Wei et al., Nature, 399:243(1999)
that can accurately perform one or multiple assays on picogram or
attagram amounts using commercially available mass spectrographs
adapted with a specialized porous silicon sample well. The older,
well known, MALDI mass spectrographic assay techniques can also be
utilized.
[0254] In an embodiment of a multiplex method using mass
spectrometry, multiple different identifier nucleotides can be used
in the various nucleic acid probes. Using such a technique the
presence of the different identifier nucleotides is direct evidence
of the presence of the nucleic acid target sequences.
[0255] D. Fluorescence Spectroscopic Analysis
[0256] A wide variety of fluorescence detection methods can be used
herein. In one exemplary contemplated method, an identifier
nucleotide includes a fluorescent label. An identifier nucleotide
can be fluorescently labeled prior to, or after, release of the
identifier nucleotide. In one embodiment when the nucleotide is a
fluorescent label, the analytical output is obtained by
fluorescence spectroscopy. In an alternative embodiment when the
nucleotide is a fluorescent label, the analytical output is
obtained by mass spectrometry.
[0257] In a preferred embodiment of the invention, the fluorescent
label is part of a fluorescent analog of a nucleotide. Fluorescent
nucleotide analogs are widely known and commercially available from
several sources. An exemplary source is NEN.TM. Life Science
Products (Boston, Mass.), who offer dideoxy-, deoxy-, and
ribonucleotide analogs a labeled with fluorescein, coumarin,
tetramethylrhodamine, naphthofluorescein, pyrene, Texas Redo, and
Lissamine . Other suppliers include Amersham Pharmacia Biotech
(Uppsala, Sweden; Piscataway, N.J.) and MBI Fermentas, Inc.
(Amherst, N.Y.).
[0258] An advantage to using fluorescent labels and fluorescence
spectroscopy analysis is that there are multiple different labels.
Such different labels would be particularly useful in a multiplex
embodiment of the invention. Different fluorescent labels would be
used in different probes, so that the detection of a particular
fluorescently-labeled nucleotide analog as a released identifier
nucleotide could be used to deduce which nucleic acid targets are
present.
[0259] For example, fluorescein has a 488 nm excitation and 520 nm
emission wavelength, whereas rhodamine (in the form of tetramethyl
rhodamine) has 550 nm excitation and 575 nm emission wavelength. A
fluorescence detector provides an excitation source and an emission
detector. The emission wavelengths of 520 nm and 575 nm are easily
distinguishable using fluorescence spectroscopy.
[0260] On a per molecule basis, fluorescence spectroscopy is about
10-fold more sensitive than absorbance spectroscopy. A very wide
variety of fluorescence spectroscopy-based detectors are
commercially available for reading fluorescence values of single
tubes, flow cells and multi-well plates, among others. For example,
Labsystems Multiskan models of microplate readers are widely
available with a spectral range of 400 to 750 nm, and filters for
340, 405, 414, 450, 492, 540, 620, and 690 nm (e.g. Fisher
Scientific, Pittsburgh, Pa.).
[0261] It is contemplated that a released identifier nucleotide
could be labeled before or after depolymerization using
cross-linking chemistry well known in the art with commercially
available reagents. For example, fluorescein isothiocyanate and
rhodamine B isothiocyanate are both available from Aldrich Chemical
Company (Milwaukee, Wis.). References to fluorescein
isothiocyanate's use in labeling biological molecules include
Nature, 193:167 (1962), Methods Enzymol. 26:28 (1972), Anal.
Biochem., 57:227 (1974), Proc. Natl. Acad. Sci., U.S., 72:459
(1975).
[0262] It is contemplated that for many embodiments of the
invention, it is useful to separate released fluorescent identifier
nucleotides from those bound to an oligonucleotide, such as a
probe. Thus, the separation techniques well known in the art and
discussed above are useful with such an embodiment, including HPLC
fitted with a fluorescence detector. The enhanced sensitivity of
fluorescence relative to other spectroscopic techniques can be used
to increase the sensitivity of a detection or quantification
process of the invention.
[0263] E. Absorbance Spectroscopic Analysis
[0264] An absorbance spectrographic analysis step is contemplated
to provide an analytical output, thereby provide for the
determination of the presence or absence released identifier
nucleotide, and indicate the presence or absence of said nucleic
acid target sequence. This embodiment contemplates the
chromatographic separation of a reaction mixture that has been
treated with a depolymerizing amount of an enzyme whose activity is
to release one or more nucleotides from the 3'-terminus of a
hybridized nucleic acid.
[0265] In an illustrative embodiment, a multiplexed assay for the
presence of several different nucleic acid target sequences in a
sample is analyzed by absorbance spectroscopy. Several labeled
probes to various nucleic acid target sequences are added to a
nucleic acid sample. The labels on the probes may be various
nucleotide analogs, a different one for each probe. A
depolymerizing enzyme is added, such as Klenow exo-, releasing the
labeled nucleotides and other nucleotides from the 3'-termini of
probes hybridized to target sequences when the 3' terminal
nucleotide is matched.
[0266] The reaction solution is loaded onto a pre-equilibrated High
Pressure Liquid Chromatography (HPLC) column and eluted under
conditions that separate the nucleotide analogs from the natural
nucleotides. Useful media for chromatographic separation of
nucleotides, bases, and nucleosides include reverse phase media,
such as a reverse phase C18 column or ODS-80T.sub.M or ODS-120T
TSK-GEL by TosoHaas (Montgomeryville, Pa.), anion exchange media,
such as DEAE-25SW or SP-25W TSK-GEL by TosoHaas (Montgomeryville,
Pa.), or affinity media, such as Boronate-5PW TSK-GEL by TosoHaas
(Montgomeryville, Pa.). Example 20 illustrates an embodiment of the
present invention using HPLC.
[0267] The HPLC column is fitted with an absorbance detector to
monitor the column effluent. Hence, "absorbance spectroscopy" for
this type of analysis. Typical wavelengths for monitoring HPLC
detection of nucleotides are 250 nm, 260 nm and 280 nm. Such
separations of nucleotides and nucleotide analogs are well known in
the art. Revich et al., J. Chromatography, 317:283-300 (1984), and
Perrone & Brown, J. Chromatography, 317:301-310 (1984) provide
examples of the HPLC separation of dNTPs.
[0268] Identification of the separated nucleotide analogs can be
accomplished by comparison of the retention times (as monitored by
absorbance of effluent at various times) of standards of the
nucleotide analogs separated on the same HPLC column under the same
conditions. Alternatively, the identity of the nucleotide analogs
collected in separate fractions (as determined by continually
monitoring the absorbance of the column effluent) can be determined
by other standard analytical methods, such as nuclear magnetic
resonance or atomic analysis (H,C,N).
[0269] In this illustrative example using depolymerization with
Klenow exo-, the presence of a released identifier nucleotide from
a particular probe indicates the presence of the target sequence
that hybridize with that probe.
[0270] In an alternative embodiment, the released nucleotides from
a depolymerization reaction mixture are separated on a gas
chromatograph fitted with an absorbance detector to monitor column
effluent.
[0271] Coupled Reactions
[0272] In some embodiments, certain of the above reactions can be
performed as single pot reactions. A "single pot reaction" is a
reaction wherein at least two enzymes (i.e., E1 and E2) with
catalytic activity are present in the same reaction mix and act on
one or more substrate(s) (i.e., S1 and S2). In some embodiments,
the reactions catalyzed by the enzymes occur simultaneously where
E1 acts on S1 and E2 acts on S2. Alternatively, the reactions
catalyzed by E1 and E2 can occur in a step-wise or coupled manner
(e.g., where E1 acts on S1 to produce an intermediate S2.sub.i and
E2 then acts on S2.sub.i). Of course, in yet other embodiments,
such a coupled reaction can also be essentially simultaneous.
[0273] The ability to utilize combinations or mixtures of the
enzymes of the present invention in single pot reactions is
surprising, in light of the extremely low levels of nucleic acid
detection that are achieved using the present invention. This low
level detection is possible even though some enzymes are used under
suboptimal conditions. As previously described, it was found to be
necessary to optimize the concentration of PP.sub.i utilized in the
pyrophosphorolysis reactions to minimize inhibition of luciferase.
Therefore, aliquots from the NMP-, dNMP-, NTP-, DNTP- and
ATP-producing reactions can be directly added to L/L Reagent for
luciferase detection without any purification of the reaction
products. The luciferase reaction is not poisoned or otherwise
quenched by the components of the reactions. This desirable feature
permits automation and high throughput analysis with a minimal
amount of time and effort, and it also permits great flexibility in
the design of the overall detection schemes. However, it is not
intended that the present invention be limited to any particular
reaction condition, reagents, or embodiments.
[0274] In some preferred embodiments, the pyrophosphorolysis
reaction producing dNTP and the NDPK catalyzed reaction in which
the NTPs or dNTPs are converted to ATP are performed in a single
pot reaction in the nucleic acid polymerase buffer in these
embodiments. NDPK activity is sufficient to convert DNTP to ATP,
even though the polymerase buffer conditions are suboptimal for
NDPK activity.
[0275] The polymerase enzyme and NDPK can both be present initially
in the reaction, or the NDPK can be added directly to the reaction
after an incubation period sufficient for the production of NTP or
dNTP. Alternatively, a nucleic acid polymerase and NDPK can be
provided in the same vessel or mixture for use in the reactions
described above. The mixture preferably contains the nucleic acid
polymerase and NDPK in a concentration sufficient to catalyze the
production of ATP when in the presence of a nucleic acid,
pyrophosphate and ADP.
[0276] Preferably, the polymerase is provided in a concentration of
about 0.1 to 100 U/reaction (i.e., where "U" is units) most
preferably at about 0.5 U/reaction. Preferably, the NDPK is
provided in a concentration of 0.1 to 100 U/reaction, most
preferably at about 0.1 U/reaction. In further preferred
embodiments, the mixture is substantially free of contaminating
ATP.
[0277] Similarly, the PRPP synthetase and NDPK reactions can be
carried out in a single pot reaction in the PRPP synthetase buffer.
Again, in these embodiments, NDPK activity is sufficient even
though conditions for NDPK activity are suboptimal.
[0278] The nuclease-digested sample containing free NMPs and dNMPs
can be added to a reaction mix initially containing PRPP synthetase
and NDPK, or added to a PRPP synthetase reaction followed by
addition to a reaction mix containing NDPK. By way of example,
certain preferred buffers and reaction components can be found in
the Examples. However, it is not intended that the present
invention be limited to specific buffers or reaction
components.
[0279] PRPP synthetase and NDPK can be provided in the same vessel
or mixture for use in the reactions described above. The mixture
preferably contains the PRPP synthetase and NDPK in a concentration
sufficient to catalyze the production of ATP when in the presence
of PRPP and ADP. Preferably, the NDPK is provided in a
concentration of 0.1 to 100 U/reaction, most preferably at about
0.1 U/reaction. Preferably, the PRPP synthetase is provided in a
concentration of 0.001 to 10 U/reaction, most preferably at about
0.01 U/reaction. If amplification is desired, the PRPP synthetase
reaction is preferably heat inactivated, otherwise the PRPP
synthetase converts the added AMP to ATP.
[0280] The pyrophosphorolysis reaction and amplification reaction
can also be performed in a single pot reaction. In this single pot
reaction, poly(A) polymerase or any suitable template-dependent
polymerase can be used, including, but not limited to, AMV reverse
transcriptase, MMLV reverse transcriptase, DNA polymerase alpha or
beta, Taq polymerase, Tth polymerase, Tne polymerase, Tne triple
mutant polymerase, Tvu polymerase, Ath polymerase, E. coli DNA
polymerase I, T4 DNA polymerase, Klenow fragment, Klenow exo minus,
or poly(A) polymerase.
[0281] In some embodiments, a first enzyme for converting AMP to
ADP can be myokinase (e.g., adenylate kinase) or NMPK, and in other
embodiments, a second enzyme for converting ADP to ATP can be
pyruvate kinase or NDPK. In addition, in preferred embodiments, the
reaction is fed AMP. In particularly preferred embodiments,
apyrase-treated AMP is utilized to reduce background due to
contaminating ADP and ATP. Preferably 1 .mu.L of 1 U/.mu.L apyrase
is added to 19 .mu.L of 10 mM AMP, followed by incubation at room
temperature for 30 minutes and heat inactivation of the apyrase by
incubation at 70.degree. C. for 10 minutes.
[0282] High energy phosphate donors are also added to the reaction.
In preferred embodiments, when pyruvate kinase is utilized, PEP is
added. In other preferred embodiments, when NDPK is utilized, dCTP
is added. Preferably, the high energy phosphate donor is added
about 15 minutes after a pre-incubation with the polymerase,
although this is not necessary. These reactions can be
characterized as follows:
Reaction 5: NA.sub.n+PP.sub.i.fwdarw.NA.sub.n-1+XTP
XTP+AMP.fwdarw.ADP+XDP
ADP+D-P.fwdarw.ATP+D
[0283] wherein NA is a nucleic acid, XTP is a nucleoside
triphosphate (either a deoxynucleoside or ribonucleoside
triphosphate), XDP is a nucleoside diphosphate (either a
deoxynucleoside or ribonucleoside diphosphate), and D-P is a high
energy phosphate donor. It should be appreciated that this reaction
produces ATP, the preferred substrate for luciferase, from
dNTPs.
[0284] The amplification reaction proceeds as described in reaction
5 to produce a threshold ATP concentration of approximately
1.times.10.sup.-12 Molar in 100 .mu.L of sample. Preferably, the
polymerase is provided in a concentration of about 0.1 to 100
U/reaction, most preferably at about 0.5 U/reaction. Preferably,
the NDPK is provided in a concentration of 0.1 to 100 U/reaction,
most preferably at about 0.1 U/reaction. Preferably, the mixture is
substantially free of contaminating ATP.
[0285] Probe-Mediated Specific Nucleic Acid Detection
[0286] Depolymerization reactions can be used to interrogate the
identity of a specific base in a nucleic acid. For example, the
identity of single base point mutations, deletions, or insertions
in a nucleic acid can be determined as follows.
[0287] In one embodiment, a nucleic acid probe is synthesized that
is substantially complementary to a target nucleic acid containing
or suspected of containing a point mutation. It will be recognized
that various hybridization conditions can be used, so as to vary
the stringency at which hybridization occurs. Thus, depending upon
the system utilized, the complementarity of the probe can be
varied. Depending on the length of the probe, the GC content, and
the stringency of the hybridization conditions, the probe can have
as many as 10 base mismatches with the target nucleic acid, and
preferably less than 5 mismatches. Most preferably, the probe has
only one base mismatch with the target nucleic acid or is
completely complementary to the target nucleic acid.
[0288] The nucleic acid probe comprises single-stranded nucleic
acid (e.g., DNA or RNA). The probe can be of varying lengths,
preferably from about 10 to 100 bases, most preferably about 10 to
30 bases. In particularly preferred embodiments, the probe is
complementary to the target at all bases between an interrogation
position and 3' end of the nucleic acid probe.
[0289] In preferred embodiments, the probe is designed to have a
predetermined nucleotide at an interrogation position. When the
complementary probe base pairs or hybridizes to the target nucleic
acid, the base at an interrogation position aligns with the base in
the nucleic acid target whose identity is to be determined under
conditions such that base pairing can occur. It is contemplated
that an interrogation position can be varied within the probe. For
example, in some preferred embodiments, an interrogation position
is preferably within 10 bases of the 3' end of the nucleic acid
probe. In still other preferred embodiments, an interrogation
position is within 6 bases of the 3' end of the nucleic acid probe.
In particularly preferred embodiments, an interrogation position is
at the next to last or last base at the 3' end of the nucleic acid
probe.
[0290] In some preferred embodiments, four different probes of
equal length are synthesized, each having a different nucleotide at
an interrogation position. Accordingly, it is contemplated that in
some embodiments, a set of DNA probes includes a first probe with a
deoxyadenosine residue at an interrogation position, a second probe
with a deoxythymidine residue at an interrogation position, a third
probe with a deoxyguanosine residue at an interrogation position,
and a fourth probe with a deoxycytosine residue at an interrogation
position. Likewise, it is also contemplated that a set of RNA
probes includes a first probe with an adenosine residue at an
interrogation position, a second probe with a uridine residue at an
interrogation position, a third probe with a guanosine residue at
an interrogation position, and a fourth probe with a cytosine
residue at an interrogation position.
[0291] In the next step of some embodiments, the probe or probes
are hybridized to the target nucleic acid in separate reactions so
that a probe nucleic acid-target nucleic acid complex is formed. It
is contemplated that hybridization conditions can vary depending on
the length and base composition of the probe. In the probe-target
nucleic acid complex, the nucleotide at an interrogation position
is aligned with the specific base to be identified in the nucleic
acid. In embodiments in which a set of probes is utilized, a
different reaction is performed with each probe. In a multiplex
embodiment, the set of probes can be used simultaneously. Because
the probes differ at an interrogation position, only one of the
probes is complementary to the specific base in the target nucleic
acid that is aligned with an interrogation position.
[0292] In the next step of some embodiments, the nucleic acid
probe-target nucleic acid complexes are individually reacted under
conditions permitting depolymerization of the probe. The preferred
reaction conditions for depolymerization are described above and in
the following Examples. The nucleotides are then detected. In
preferred embodiments, the reaction mix also contains reagents
necessary to catalyze the conversion of XTP to ATP equivalents as
described in reaction 4 and in the following Examples. In some
preferred embodiments, the nucleotides and/or ATP produced by the
depolymerization reaction are then detected by either a luciferase
or NADH detection system. Complementarity of the base at an
interrogation position of the nucleic acid probe to the
corresponding base in the nucleic acid target is characterized by
detection of a signal generated from ATP following
depolymerization.
[0293] In particularly preferred embodiments, the identity of the
specific base is determined by comparing the amount of ATP produced
in each reaction. Depolymerization of the probe proceeds from its
3' end. When the base at an interrogation position is not
complementary to the specific base in the nucleic acid, very little
or no ATP is produced, and thus no signal results. In alternative
embodiments, this method can be practiced with from one to four
probes. It is contemplated that utilizing multiple probes, (e.g.,
each with a different base at an interrogation position), may prove
unnecessary if a positive signal is produced (e.g., with the first
probe tested).
[0294] In yet another preferred embodiment, the probe-mediated
specific nucleic acid detection method of the present invention can
be used to simply identify or detect a nucleic acid of interest.
For this method, a nucleic acid probe (e.g., DNA or RNA) is
utilized which is substantially complementary to the target nucleic
acid, which can be RNA or DNA. In a particularly preferred
embodiment, the nucleic acid probe is entirely complementary to the
target nucleic acid. The nucleic acid probe comprises
single-stranded nucleic acid (e.g., DNA or RNA). The probe can be
of varying lengths, preferably from about 10 to about 1000 bases,
most preferably about 10 to 100 bases. Detection is carried out as
described above. The nucleic acid probe-nucleic acid target complex
is exposed to conditions permitting depolymerization of the probe,
which results in the production of XTPs. Detection of the nucleic
acid of interest is characterized by a difference in the signal
generated by the XTPs produced. Preferably, the XTPs are converted
to ATP as described above and the ATP detected by a luciferase or
NADH detection system.
[0295] In another embodiment, the presence or absence of a lesion
in the target nucleic acid can be detected. A lesion may either be
an insertion mutation or a deletion mutation in the wild-type
target nucleic acid. The wild-type target nucleic acid contains a
region of complementarity, to which the nucleic acid probe can
hybridize. Thus, the region of complementarity in the wild-type
target nucleic acid is defined by the 5' and 3' ends of the nucleic
acid probe. When the region of complementarity contains a lesion,
the nucleic acid probe may still hybridize to the target nucleic
acid, but the hybridization is only partial. Depending on the size
and nature of the lesion, either the 5' or 3' end of the probe may
hybridize to the target nucleic acid, or a hybridization structure
characterized by the presence of a loop may be formed. In each of
these cases, depolymerization will be prevented. Preferably, the
nucleic acid probe is designed so that the lesion to be detected
begins about less than ten bases from 3' end of the probe,
preferably less than about 6 bases. The nucleic acid probe
comprises single-stranded nucleic acid (e.g., DNA or RNA). The
probe can be of varying lengths, preferably from about 10 to about
1000 bases, most preferably about 10 to 100 bases.
[0296] Detection of a nucleic acid containing a lesion is
characterized by the difference of a signal generated from the XTP
produced. Preferably, the XTPs are converted to ATP as described
above and the ATP detected by a luciferase or NADH detection
system.
[0297] It is contemplated that an increase in the signal
(analytical output) produced by the probe-mediated specific nucleic
acid detection methods of the invention can be realized by a novel
cycling method. In this embodiment of the invention, two probes are
designed that are complementary to each other and have a 3'
overhang at each end when they hybridize to one another. In
preferred embodiments, the probes are designed so that the 3'
overhang is a single base overhang. In alternative embodiments, the
probes also can hybridize to a target nucleic acid. In particularly
preferred embodiments, a polymerase that acts from the 3' end of
nucleic acids and does not recognize 3' overhangs is utilized for
the depolymerization reaction, such as Klenow exo-.
[0298] In preferred embodiments, the first step of the reaction
involves hybridization of an excess of one of the probes to the
target nucleic acid in the presence of the polymerase and under
conditions permitting depolymerization as described above. In some
embodiments, no 31 overhang exists, and the depolymerase reaction
proceeds from the 31 end of the probe. In some embodiments, the
reaction is terminated by separating the probe from the target
nucleic acid by heating the probe-target nucleic acid complexes. On
average, as few as one base is removed from probes that were bound
to the target nucleic acid, and fractions of shortened probes are
created.
[0299] In the second step, an excess of the second probe is added
to the reaction. Due to the law of mass action, the shortened
probes produced in the first step have a tendency to bind to the
newly added complementary probes, whereas the non-shortened probes
bind to the target nucleic acid. The shortened probes that bind to
the complementary probes produce a complex with no 3' overhang on
one end, and are depolymerized. This effectively doubles the amount
of substrate available for the depolymerization reaction. Steps one
and two can be repeated additional times until the desired level of
detection is achieved. In an alternative preferred embodiment, the
reactions can be coupled with NDPK as described above, to produce
ATP equivalents that are detectable by a luciferase-based or
NADH-based assay system.
[0300] The ability to interrogate the identity of a specific base
in a nucleic acid also permits discrimination between nucleic acids
from different species, or even from different alleles. The ability
to detect and discriminate between nucleic acids of related or
unrelated species also permits the identification of species
contained within a given nucleic acid-containing sample. For
example, the method can be used to determine which species of
several related bacteria are contained within a sample (e.g.,
clinical samples, environmental samples, or samples from non-human
animals).
[0301] In preferred embodiments of this method, nucleic acids with
substantially identical sequences from at least two species or
alleles are detected. The region of identity (target nucleic acid
sequence) contains at least a single nucleotide mismatch between
the species or alleles in at least one predetermined position and
also contains a 3' end and a 5' end or the identification of a
nucleic acid sequence unique to each species to be identified.
[0302] Next, in some embodiments, an RNA or DNA probe that is
substantially complementary to the region of identity is
synthesized. The probe can be of varying lengths, preferably from
about 10 to 1000 bases, most preferably about 10 to 100 bases. As
above, this complementary probe includes an interrogation
position.
[0303] An interrogation position can be varied within the probe.
For example, an interrogation position is preferably within 10
bases of the 3' end of the nucleic acid probe. More preferably, an
interrogation position is within 6 bases of the 3' end of the
nucleic acid probe. Most preferably, an interrogation position is
at the next to last or last base of the 3' end of the nucleic acid
probe.
[0304] The nucleic acid probes are designed so that the base at an
interrogation position is complementary to the nucleotide at the
predetermined position of one species or allele, but not another
due to the mismatch. Likewise, a second probe can be synthesized
that is complementary at an interrogation position to the
nucleotide at the predetermined position of a second species or
allele.
[0305] This same procedure can be employed to identify the presence
or absence of multiple species within a given sample. In these
embodiments, all that is required is the identification of
substantially identical sequences between species that contain base
mismatches or the identification of a nucleic acid sequence unique
to each species to be identified. Similarly, this procedure can be
used for quantitative analysis of the number of alleles at a loci
in a sample. By comparing the quantity of analytical output
relative to an appropriate internal or external control, the number
of alleles at a locus can be determined. These comparative
quantities can be expressed in terms of the ratio of one allele to
the other in one sample versus that same ratio measurement in a
control sample. In this process, events such as loss of
heterozygosity or trisomy can be detected.
[0306] For example, a normal heterozygous control has a ratio of
about 1:1 with respect to the two alleles that make up the
heterozygote. That is, each allele of the heterozygote can be
detected when a nucleic acid probe is used to detect the presence
of that allele. If the quantity of analytical output obtained by
the release of identifier nucleotide when the first and second
alleles are detected is expressed as a ratio, the relative amounts
of the first and second allele would be about the same for a sample
which is heterozygous at that locus. When a sample has lost
heterozygosity, one of the two alleles is not detectable. If the
first allele is lost, then the first allele will not be detected in
the sample if the sample is assayed using a nucleic acid probe for
the first allele. The second allele will, however, be present at a
similar amount as would be present in a known heterozygous control
sample, so assaying the sample with a nucleic acid probe for the
second allele will provide an analytical output. If the quantity of
the analytical output for the first and second alleles for a sample
having a loss of heterozygosity of the first allele is expressed as
a ratio, the ratio will be about 0:1, indicating the absence of the
first allele. Conversely, where the second allele is lost, the
ratio of the quantity of analytical output for the first and second
allele would be about 1:0, indicating the absence of the second
allele.
[0307] The presence of trisomy of an allele is detected in a
similar fashion. In a trisomy event, four outcomes are possible
with respect to a first and second allele. The trisomy can be
homozygous for the first allele, in which case three copies of the
first allele will be present and no copies of the second allele
will be present. Thus, the ratio of the quantity of analytical
output for the first and second allele will be 3:0. If the trisomy
is homozygous for the second allele, three copies of the second
allele will be present. The ratio of the quantity of analytical
output for the first and second allele will be 0:3. Two cases of
heterozygous trisomy are possible: two copies of allele one and one
copy of allele two, or one copy of allele one and two copies of
allele two. These two heterozygous trisomy outcomes can be detected
by determining the ratios of the quantity of analytical output for
the first and second alleles, preferably in comparison to a known
heterozygous control sample. If the ratio is 2:1, then the
heterozygous trisomy has two copies of the first allele and one
copy of the second allele. If the ratio is 1:2, then the
heterozygous trisomy has one copy of the first allele and two
copies of the second allele.
[0308] The use of an appropriate control, for example a
heterozygous control, allows the appropriate interpretation of the
ratios obtained from the analysis of a sample suspected of having a
loss of heterozygosity or of trisomy.
[0309] In the next step of some embodiments, separate reactions are
performed utilizing each probe. The probes are hybridized to the
target nucleic acid to form a probe nucleic acid-target nucleic
acid complex. In the probe nucleic acid-target nucleic acid
complex, the nucleotide at an interrogation position of the probe
is aligned with the nucleotide at the predetermined position in the
nucleic acid, so that base pairing occurs. The probe-target nucleic
acid complex is then reacted under conditions permitting
depolymerization of the probe from its 3' end.
[0310] Preferred conditions for depolymerization (depolymerization
conditions) are described herein. The nucleotides are then
detected. In some preferred embodiments, the nucleotides are
converted to ATP equivalents as described in reaction 4 and in the
Examples. In preferred embodiments, the ATP is detected by
luciferase or NADH detection systems.
[0311] These embodiments of the present invention permit
discrimination between nucleic acids from different species or
alleles, as NTPs are produced by depolymerization only when the
nucleotide at an interrogation position of the probe is
complementary to the nucleotide at the predetermined position of
the nucleic acid from the species. As described above, significant
depolymerization proceeds only if the base at an interrogation
position is complementary to the base at the predetermined position
in the target nucleic acid. The NTP concentration, including the
ATP concentration, differs when a mismatch is present as compared
to when a mismatch is not present. These differences can be
detected (e.g., by either an ATP or NADH detection system).
[0312] A method contemplated by the present invention has wide
applicability in assaying nucleic acids. In some aspects, an
endogenous nucleic acid is assayed to determine whether a
particular native or mutant sequence is present or absent. This
type of analysis is sometimes referred to as genotyping because the
genetic makeup of the subject from which the nucleic acid sample is
obtained is determined. Speciation, the identity of an organism,
such as the identification of a human, dog, chicken, bovine or the
like can be determined by use of species-specific nucleic acid
probes such as probes to selected regions of the gene encoding
cytochrome B.
[0313] Using a contemplated method, one can illustratively
determine whether a human patient, for example, has the Leiden V
mutation, a mutant .beta.-globin gene, the cystic fibrosis-related
gene in the region of the delta 508 allele, a mutation in a
prothrombin gene, congenital adrenal hyperplasia, a translocation
that takes place in the region of the bcr gene along with
involvement of a segment of the abl gene, the number of repeated
sequences in a gene such as are present in THO 1 alleles or the
TPOX alleles, as well as the loss of heterozygosity of the locus of
certain alleles as is found in certain cancers and also allelic
trisomy. Genomic typing can also be used to assay plant genomes
such as that of rice, soy or maize, and the genomes of microbes
such as Campylobacter jejuni, cytomegalovirus (CMV) or human
immunodeficiency virus (HIV) to determine whether a drug-resistant
strain is present in a sample.
[0314] In one illustrative application of a method of the
invention, detection of economically significant plant SNPs is
performed. In rice, amylose content is generally considered to be
the most important factor that determines rice processing and
cooking quality. A G T transition in the 5'-leader sequence of the
rice starch synthase pre-mRNA alters normal splicing of the
message. Reliable and cost-effective detection of economically
important SNPs such as the rice starch synthase SNP using a method
of the invention provides an important tool to the agricultural
industry.
[0315] The determination of an appropriate nucleic acid target
sequence useful for designing nucleic acid probes for use in a
method of the invention is within the skill of the art. Databases
of genetic sequences, such as Genbank, can be used to ascertain the
uniqueness of the selected nucleic acid target. Commercially
available software for designing PCR primers can be used to assist
in the design of probes for use in the invention.
[0316] Determination of Repeated Sequences
[0317] A process of the invention is useful for determining the
presence of repeated sequences in a nucleic acid sample. The
repeated known sequence present in a nucleic acid target sequence
typically has a length of 2 to about 24 bases per repeat. Di- and
tri-nucleotide repeats are well known in the art. An application of
this process is Single Tandem Repeat (STR) detection. Typically,
different alleles of the target nucleic acid have different numbers
of the repeated sequences, so the determination of the number of
repeats is useful in genotyping. Such methods have important
applications in the forensic sciences in identity testing. A method
for determining the number of repeated known sequences is as
follows.
[0318] Special nucleic acid probes are designed and obtained that
contain multiples of a known repeating sequence. Each probe
contains a different number of a repeated sequence that is
complementary to that of the nucleic acid target sequence. Each
probe has a 5'-terminal locker sequence that is complementary to
the non-repeated region of the target that is downstream of the
repeated region in the target. The probes typically have an
identifier nucleotide in the 3'-terminal region, but as described
herein, the release of nucleotides from the 3' terminus during a
depolymerization step of the invention can alternatively be
ascertained by the size of the remaining probe.
[0319] The use of a 5'-terminal locker sequence fixes the
5'-terminus of the probe relative to the repeated region. Thus, for
example when the probe has fewer repeats than the target, it is not
free to hybridize anywhere throughout the repeated region, but only
along the first matching group of repeats. in this embodiment, the
probe will be completely complementary to the target sequence, even
though it is shorter than the target. However, when the probe has
more repeats than the target, the probe extends into the adjacent
non-repeated region and is mismatched at its 3'-terminal
region.
[0320] In some cases, it is desirable to determine the number of
repeats by comparison with standard nucleic acid samples having
known numbers of repeats (and how they respond to the various
probes). In other cases, the number of repeats can be deduced by
the shape of the curve of a graph having its ordinate (x-) axis be
the number of repeats in the probe and its abscissa (y-) axis be
the analytical output indicating the number of nucleotides
released, such as light output in luminescence spectroscopic
analysis of all of the released nucleotide converted to ATP.
[0321] In such a graph, for example when the depolymerizing enzyme
is a template-dependent polymerase or exoIII, the output is greater
when the probe has the same or fewer repeats than the target,
relative to the output when the probe has more repeats than the
target. The S-shaped curve changes most rapidly after the number of
repeats in the probe surpasses the number of repeats in the target.
Thus, the derivative of the curve is greatest at that point.
Similar results when the depolymerizing enzyme preferentially
releases nucleotides from mismatched substrates, except that the
output is less when the probe has the same or fewer repeats than
the target.
[0322] Thus, in a preferred embodiment of a process to determine
the number of repeats of a known sequence, a plurality of
separately treated samples is provided. Each sample contains a
nucleic acid target sequence, containing a plurality of known
repeated sequences and a downstream non-repeated region on the
target relative to the repeated sequences. The sample is hybridized
with an above-described nucleic acid probe.
[0323] A treated reaction mixture is formed by admixing each
treated sample with a depolymerizing amount of an enzyme whose
activity is to release one or more nucleotides from the 3'-terminus
of a hybridized nucleic acid probe. The treated reaction mixture is
maintained for a time period sufficient to permit the enzyme to
depolymerize the hybridized nucleic acid probe and release an
identifier nucleotide.
[0324] The samples are analyzed for the presence or absence of
released identifier nucleotide to obtain an analytical output. The
analytical output from the sample whose probe contained the same
number of sequence repeats as present in the target nucleic acid is
indicative of and determines the number of sequence repeats present
in the nucleic acid target. The analytical output is obtained by
luminescence spectroscopy, mass spectroscopy, fluorescence
spectroscopy or absorption spectroscopy, including visualization of
the remaining probe, as described herein with regard to the general
method of the invention.
[0325] In one aspect of the method, the target nucleic acid is
homozygous with respect to the number of the repeated sequences at
the two alleles. In an alternative method of the invention, the
target nucleic acid is heterozygous for the repeated sequences.
[0326] In one method of the invention, an identifier nucleotide is
a nucleotide that is part of the region containing a repeated
sequence. In an alternative method of the invention, the nucleic
acid probe further comprises a second non-repeating sequence that
is located downstream of the repeated sequences in the probe (3' of
them). This second non-repeating sequence is complementary to a
non-repeating sequence located in the target nucleic acid 5' to its
repeated sequences. In this embodiment, it is contemplated that an
identifier nucleotide of the probe sequence is part of the region
containing the second non-repeating sequence. Thus, the identifier
nucleotide is present in a sequence containing 1 to about 20
nucleic acids that is complementary to a non-repeating sequence of
the target nucleic acid located in the probe 3' to the repeated
sequences.
[0327] Assays Using Hairpin Structures
[0328] Although it is preferred that the probes be constructed to
be free of hairpin structures, assays in which hairpin structures
are constructed are also useful. An embodiment of the invention,
such as demonstrated in Example 23, contemplates use of a hairpin
structure for determining the presence or absence of a nucleic acid
target sequence in a nucleic acid sample with a probe that is
hybridized to the target and then modified to be able to form a
hairpin structure. This embodiment comprises the following
steps.
[0329] A treated sample is provided that contains a nucleic acid
sample that may include a nucleic acid target sequence having an
interrogation position. The target sequence, if present in the
nucleic acid sample is hybridized with a nucleic acid probe. The
probe is comprised of at least two sections. The first section
contains the probe 3'-terminal about 10 to about 30 nucleotides.
These nucleotides are complementary to the target strand sequence
at positions beginning about 1 to about 30 nucleotides downstream
of the interrogation position. The second section of the probe is
located at the 5'-terminal region of the probe and contains about
10 to about 20 nucleotides of the target sequence. This same
sequence, therefore, exists in both the target and the probe in the
same 5' to 3' orientation. This sequence spans the region in the
target from the nucleotide at or just upstream (5') of the
interrogation position, to the nucleotide just upstream to where
the 3'-terminal nucleotide of the probe anneals to the target. An
optional third section of the probe, from zero to about 50,
preferably from zero to about 20, nucleotides in length and
comprising a sequence that does not hybridize with either the first
or second section, is located between the first and second sections
of the probe.
[0330] The probe of the treated sample is extended in a
template-dependent manner, by admixture with dNTPs and a
template-dependent polymerase, at least through the interrogation
position, thereby forming an extended probe/target hybrid. In a
preferred embodiment, the length of the probe extension is limited
by omission from the extension reaction of a dNTP complementary to
a nucleotide of the target sequence that is present upstream of the
interrogation position and absent between the nucleotide
complementary to the 3'-end of the interrogation position.
[0331] The extended probe/target hybrid is separated from any
unreacted dNTPs; i.e., purified at least to the degree needed to
use the extended probe strand to determine the presence or absence
of the interrogation region in the sample or the identity of the
base at the interrogation position. The extended probe/target
hybrid is denatured to separate the strands. The extended probe
strand is permitted to form a hairpin structure.
[0332] It is preferred that the polymerase enzyme utilized for an
extension reaction be a template-dependent polymerase that is free
of activity that adds a 3'-terminal deoxyadenosine in a
template-nonspecific manner. Thus, it is preferred to use other
than a polymerase such as Taq for a contemplated extension.
[0333] A treated reaction mixture is formed by admixing the hairpin
structure-containing composition with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of an extended probe hairpin structure. The
reaction mixture is maintained under depolymerizing conditions for
a time period sufficient for the depolymerizing enzyme to release
3'-terminus nucleotides, and then analyzed for the presence of
released identifier nucleotides. The analytical output indicates
the presence or absence of the nucleic acid target sequence. That
analytical output can be determined as discussed elsewhere
herein.
[0334] A still further embodiment of the invention, such as that
termed REAPER.TM. and demonstrated in Example 24 and FIG. 1, also
contemplates use of hairpin structures in determining the presence
or absence of a nucleic acid target sequence, or a specific base
within the target sequence, in a nucleic acid sample, and comprises
the following steps. A treated sample is provided that contains a
nucleic acid sample that may include a nucleic acid target sequence
hybridized with a first nucleic acid probe strand (FIG. 1A).
[0335] The hybrid is termed the first hybrid. The first probe is
comprised of at least two sections. The first section contains the
probe 3'-terminal about 10 to about 30 nucleotides that are
complementary to the target nucleic acid sequence at a position
beginning about 5 to about 30 nucleotides downstream of the target
interrogation position. The second section of the first probe
contains about 5 to about 30 nucleotides that are a repeat of the
target sequence from the interrogation position to about 10 to
about 30 nucleotides downstream of the interrogation position, and
does not hybridize to the first section of the probe. That is, the
second sequence is a repeat of the region in the target sequence
from the interrogation position downstream to the position where
the 3'-terminal nucleotide of the first probe aligns with the
target. An optional third section of the probe, located between the
first and second sections of the probe, is zero to about 50,
preferably to about 20, nucleotides in length and comprises a
sequence that does not hybridize to either the first or second
section.
[0336] The first hybrid in the treated sample is extended at the
3'-end of the first probe, thereby extending the first probe past
the interrogation position and forming an extended first hybrid
(FIG. 1B) whose sequence includes an interrogation position. The
extended first hybrid is comprised of the original target nucleic
acid and extended first probe. The extended first hybrid is then
denatured in an aqueous composition to separate the two nucleic
acid strands of the hybridized duplex and form an aqueous solution
containing a separated target nucleic acid and a separated extended
first probe.
[0337] A second probe, that is about 10 to about 2000, more
preferably about 10 to about 200, most preferably about 10 to about
30 nucleotides in length and is complementary to the extended first
probe at a position beginning about 5 to about 2000, preferably
about 5 to about 200, nucleotides downstream of the interrogation
position in extended first probe, is annealed to the extended first
probe, thereby forming the second hybrid (FIG. 1C). The second
hybrid is extended at the 3'-end of the second probe until that
extension reaches the 5'-end of the extended first probe, thereby
forming a second extended hybrid (FIG. 1D) whose 3'-region includes
an identifier nucleotide.
[0338] It is preferred that the polymerase enzyme utilized for an
extension reaction be a template-dependent polymerase that is free
of activity that adds a 3'-terminal deoxyadenosine in a
template-nonspecific manner. Thus, it is preferred to use other
than a polymerase such as Taq for a contemplated extension.
[0339] An aqueous composition of the extended second hybrid is
denatured to separate the two nucleic acid strands; i.e., the
extended second probe and the extended first probe. The aqueous
composition so formed is cooled to form a "hairpin structure" from
the separated extended second probe (FIG. 1E) when the target
sequence is present in the original nucleic acid sample. Thus, when
the target sequence is present in the original nucleic acid sample,
the 3'-terminal sequence of the second extended probe in the second
extended hybrid hybridizes with the sequence of the second extended
probe from a region comprising the interrogation position and
nucleotides downstream from the interrogation position of second
extended probe to the nucleotide position where the 3'-terminal
nucleotide of the original (first-named) probe annealed to the
original target.
[0340] A treated reaction mixture is formed by admixing the hairpin
structure-containing composition with a depolymerizing amount of an
enzyme whose activity is to release one or more nucleotides from
the 3'-terminus of a nucleic acid hybrid. The reaction mixture is
maintained under depolymerizing conditions for a time period
sufficient to release 3'-terminal region identifier nucleotides,
and then analyzed for the presence of released identifier
nucleotides. The analytical output indicates the presence or
absence of the nucleic acid target sequence. Again, the analytical
output can be determined by one of the several methods discussed
elsewhere herein.
[0341] As was the case in the previous embodiment, dNTPs are
utilized in the extension reactions. It is preferred that the
hairpin structures be separated from the dNTPs prior to
depolymerization to enhance the analysis for the identifier
nucleotide.
[0342] Kits
[0343] Other embodiments of the invention contemplate a kit for
determining the presence or absence of a predetermined endogenous
nucleic acid target sequence in a nucleic acid sample. Such a kit
comprises an enzyme whose activity is to release one or more
nucleotides from the 3' terminus of a hybridized nucleic acid probe
and at least one nucleic acid probe, said nucleic acid probe being
complementary to nucleic acid target sequence. The kit optionally
further comprises a nucleoside diphosphate kinase. Preferably, the
nucleoside diphosphate kinase is that encoded by Pyrococcus
furiosis. The kit optionally further comprises instructions for
detecting said nucleic acid by depolymerization. Preferably the
enzyme whose activity is to release nucleotides in the kit is a
template dependent polymerase that, in the presence of
pyrophosphate ions, depolymerizes hybridized nucleic acids whose
bases in the 3'-terminal region are matched with total
complementarity. Alternatively, the enzyme whose activity is to
release nucleotides in the kit exhibits a 3' to 5' exonuclease
activity, depolymerizing hybridized nucleic acids having one or
more mismatched bases at the 3' terminus of the hybridized
probe.
[0344] It is to be understood that such a kit is useful for any of
the methods of the present invention. The choice of particular
components is dependent upon the particular method the kit is
designed to carry out. Additional components can be provided for
detection of the analytical output, as measured by the release of
identifier nucleotide, or by detection of the remaining probe after
depolymerization. For example, ethidium bromide can be provided in
the kits of the invention for detection of a probe that has had
identifier nucleotide released from the 3'-terminal region.
[0345] The instructions present in such a kit instruct the user on
how to use the components of the kit to perform the various methods
of the present invention. These instructions can include a
description of the detection methods of the invention, including
detection by luminescence spectroscopy, mass spectrometry,
fluorescence spectroscopy, and absorbance spectroscopy.
[0346] In another embodiment, the invention contemplates a kit for
determining the presence or absence of at least one predetermined
nucleic acid target sequence in a nucleic acid sample comprising
the following components: an enzyme whose activity in the presence
of pyrophosphate is to release identifier nucleotide as a
nucleoside triphosphate from hybridized nucleic acid probe;
adenosine 5' diphosphate; pyrophosphate; a nucleoside diphosphate
kinase; and at least one nucleic acid probe, wherein the nucleic
acid probe is complementary to the predetermined nucleic acid
target sequence.
[0347] Preferably, the enzyme whose activity in the presence of
pyrophosphate is to release identifier nucleotides is selected from
the group consisting of the Tne triple mutant DNA polymerase,
Klenow exo-, Klenow, T4 DNA polymerase, Ath DNA polymerase, Taq DNA
polymerase and Tvu DNA polymerase. Preferably, the nucleoside
diphosphate kinase is that encoded by Pyrococcus furiosis.
[0348] The kit optionally comprises instructions for use.
[0349] In another embodiment, the invention contemplates a kit for
determining the presence or absence of a predetermined nucleic acid
target sequence in a nucleic acid sample comprising an enzyme whose
activity is to release one or more nucleotides from the 3' terminus
of a hybridized nucleic acid probe and instructions for use. Such a
kit optionally comprises a nucleoside diphosphate kinase.
Preferably, the nucleoside diphosphate kinase is that encoded by
Pyrococcus furiosis. The kit further optionally comprises a nucleic
acid probe complementary to the predetermined nucleic acid target
sequence.
[0350] In other embodiments of the present invention, nucleic acid
detection test kits are provided for performing a depolymerization
method contemplated by this invention, and particularly a
depolymerization detection method.
[0351] In one embodiment, the kit includes a vessel containing an
enzyme capable of catalyzing pyrophosphorolysis, including, but not
limited to Taq polymerase, Tne polymerase, Tne triple mutant
polymerase, Tth polymerase, Tvu polymerase, Ath polymerase, T4 DNA
polymerase, Klenow fragment, Klenow exo minus, E. coli DNA
polymerase I, AMV reverse transcriptase, MMLV reverse
transcriptase, or poly(A) polymerase. In another embodiment, the
kit contains a vessel that contains an exonuclease such as S1
nuclease, nuclease BAL 31, mung bean nuclease, exonuclease III and
ribonuclease H.
[0352] Either of the above enzyme types is utilized in a
contemplated method in a depolymerizing effective amount. That is,
the enzyme is used in an amount that depolymerizes the hybridized
probe to release an identifier nucleotide. This amount can vary
with the enzyme used and also with the temperature at which
depolymerization is carried out. An enzyme of a kit is typically
present in an amount of about 0.1 to 100 U/reaction; in
particularly preferred embodiments, the concentration is about 0.5
U/reaction. An amount of enzyme sufficient to carry out at least
one assay, with its controls is provided.
[0353] As noted elsewhere, the preferred analytical output for
determining the presence or absence of identifier nucleotide is
luminescence caused by the reaction of ATP with luciferin in the
presence of luciferase. A kit containing a pyrophosphorylation
enzyme for use in DNA detection using luminescence also preferably
includes a vessel containing NDPK and a vessel containing ADP.
Similarly, a kit containing an exonuclease enzyme for use in DNA
detection using luminescence also preferably includes a vessel
containing PRPP synthetase and a vessel containing ADP. The NDPK or
PRPP synthetase is provided in concentration of about 0.01 to 100
U/reaction, preferably about 0.1 to about 1.0 U/reaction.
[0354] Preferably, these reagents, and all of the reagents utilized
in the kits discussed herein, are free of contaminating ATP and
adenylate kinase. Some of the contaminants can be removed from the
enzymes by dialysis treatment.
[0355] Optionally, the kit contains vessels with reagents for
amplification of dNTPs or NTP to ATP. Amplification reagents
include, but are not limited to pyruvate kinase, adenylate kinase,
NMPK, NDPK, AMP (e.g., as the amplification enzymes and substrate),
and dCTP or AMP-CPP (e.g., as high-energy phosphate donors). In
particularly preferred embodiments, the kit can be packaged in a
single enclosure including instructions for performing the assay
methods. In some embodiments, the reagents are provided in
containers and are of a strength suitable for direct use or use
after dilution. In alternative preferred embodiments, a standard
set can also be provided in order to permit quantification of
results. In yet other preferred embodiments, test buffers for
optimal enzyme activity are included.
[0356] In yet other embodiments, a contemplated kit comprises a
nuclease, PRPP synthetase, PRPP, NDPK, and ADP together with
luciferase and luciferin. In preferred embodiments, the nuclease is
provided in a concentration of about 1 to 500 U/reaction; in
particularly preferred embodiments at a concentration of about 20
U/reaction. In a particularly preferred embodiment, the PRPP
synthetase is provided in concentration of about 0.01 U/reaction to
10 U/reaction, preferably about 0.1 U/reaction. In some preferred
embodiments, the kit includes all these reagents with luciferase
and luciferin being provided as a single reagent solution.
[0357] In other preferred embodiments, these reagents include, but
are not limited to, a high energy phosphate donor which cannot be
utilized by luciferase, preferably dCTP, and AMP together with
luciferase and luciferin. In alternative preferred embodiments, the
kit includes all these reagents with luciferase and luciferin being
provided in the same solution.
[0358] In still further embodiments of the present invention, the
kits described above can contain a probe or probes for
probe-mediated specific nucleic acid detection. In some
embodiments, the kit contains at least one nucleic acid probe for a
nucleic acid target of interest. In other embodiments, the kits
contain multiple probes, each of which contain a different base at
an interrogation position or which are designed to interrogate
different target DNA sequences.
[0359] In each of the embodiments, the kits contain instructions
for use in interrogating the identity of a specific base within a
nucleic acid target, for discriminating between two homologous
nucleic acid targets that differ by one or more base pairs, or for
determining whether a nucleic acid target contains a deletion or
insertion mutation. The types of nucleic acid probes that can be
included in the kits and their uses are described in greater detail
below.
EXAMPLE 1
Specific Detection of RNA: Comparison of Signals from RNA Species
that Match Probe Sequences in Reactions With and Without Added
Extraneous Target RNA
[0360] For the pyrophosphorylation reaction to be used to detect
specific target sequences, another requirement of the system is
that the probes should give a very similar signal in the presence
and absence of extraneous RNA. In this Example, the strength of the
signal of probes designed to detect target globin mRNA in the
presence of a large amount of yeast RNA is compared to the signal
seen in the absence of added yeast RNA.
[0361] Hybridization solutions containing various levels of yeast
RNA, Probe 6 (SEQ ID NO:1) or Probe 8 (SEQ ID NO:2) and target
globin mRNA (Gibco BRL, 18103-028) were assembled by adding 5 .mu.L
500 ng/.mu.L either probe 6 or probe 8 to 5 .mu.L 40 ng/.mu.L of
target globin mRNA and 10 .mu.L yeast RNA (Sigma Chemical Co.
R3629) in 1.times. TE buffer (10 mM Tris, 1 mM EDTA) to produce
solutions containing total amounts of yeast RNA of 0, 2, 20, 200,
400, and 800 ng. The solutions were heated at 50.degree. C. for 15
minutes and then permitted to cool to room temperature for 15.
[0362] The following master reaction mixture was assembled:
4 Nanopure water 346.5 .mu.L MMLV-RT 5X Reaction Buffer (Promega
132 .mu.L M195A) Sodium pyrophosphate (Promega M531) 16.5 .mu.L
NDPK (1 U/.mu.L) 33 .mu.L ADP (2 .mu.M) 33 .mu.L MMLV-RT (adjusted
to 100 U/.mu.L) 33 .mu.L (Promega, M1701)
[0363] Aliquots of the above master mix (18 .mu.L) was placed in
each of 18 tubes. After cooling 15 minutes, 2 .mu.L of the various
hybridization solutions containing probe 6 were added to the tubes
and the tubes were placed in a 37.degree. C. heating block.
[0364] After 15 minutes of incubation of the hybridization mixture
with the reaction master mix, 20 .mu.L of the solution were added
to 100 .mu.L of L/L reagent (Promega, F202A) and the light output
of the resulting reaction was measured using a Turner.RTM. TD-20/20
luminometer.
[0365] After the probe 6 data were collected, an identical set of
reactions was performed using the hybridization solutions
containing probe 8.
[0366] The following data were obtained:
5 Probe 6 Reactions Yeast RNA relative light units Average None 96
109 111 105.3 2 ng 98.4 85.0 118.5 100.7 20 ng 117.9 110.9 82.7
103.65 200 ng 56.4 110.1 93.2 86.6 400 ng 115.7 110.7 124.6 117 800
ng 127.6 128.7 143.1 133.1
[0367]
6 Probe 8 Reactions Yeast RNA relative light units Average None
105.8 97.0 82.3 95.0 2 ng 84.5 84.6 93.7 87.6 20 ng 99.6 111.7
104.9 105.4 200 ng 83.6 75.9 95.6 85.1 400 ng 94.7 97.2 81.9 91.2
800 ng 50.7 89.0 82.1 73.9
[0368] These data indicate that addition of very large amounts of
yeast RNA to the hybridization reaction does not greatly lower the
signal from hybridized probes for specific target RNA species.
7 Probe 6 SEQ ID 5'AGACTTCTCCTCACTGGACAGATGCACCAT3' NO:1 Probe 8
SEQ ID 5'GGGTCCATGGGTAGACAACCAGCAGC3' NO:2
EXAMPLE 2
Determination of the Presence of the Leiden Mutation of Factor
V
[0369] A synthetic first nucleic acid target of the Factor V gene
was designed to have the wild type sequence that contains a G at
position 32 of FV1 (SEQ ID NO:3). The complementary strand, FV2,
(SEQ ID NO:4) had 4 additional bases at its 3'-terminus. A second
synthetic nucleic acid target of Factor V was designed to have the
Leiden mutation, an A residue at position 32 of FV3 (SEQ ID NO:5).
The mutant complementary strand, FV4 (SEQ ID NO:6) also had 4
additional bases at its 3'-terminus. The nucleic acid target
oligonucleotides, FV1 to FV4, were separately dissolved at a
concentration of one mg/mL in water.
[0370] Nucleic acid probe FV5 (SEQ ID NO:7) was synthesized to be
totally complementary to one strand of the first target, FV1. The
probe was synthesized to place the complementary C residue at an
interrogation position penultimate to the 3'-terminal nucleotide of
the probe FV5, corresponding to the G at position 32 of FV1.
Similarly, a synthetic nucleic acid probe was prepared having
sequence FV6 (SEQ ID NO:8) that is totally complementary to one
strand of the second target, Factor V with the Leiden mutation,
FV3. The probe was synthesized to place the complementary T residue
at an interrogation position penultimate to the 3'-terminal
nucleotide of the probe FV6, corresponding to the A at position 32
of FV3. Nucleic acid probe stock solutions had a concentration of
one mg/mL in water.
[0371] The FV1 oligonucleotide was mixed with an equal amount of
its complementary strand, FV2, heated to 95.degree. C. for about 15
minutes and then cooled to room temperature to produce a first
sample containing a double stranded DNA segment including the first
nucleic acid target, corresponding to the wild type sequence of the
Factor V gene.
[0372] The FV3 oligonucleotide was mixed with an equal amount of
its complementary strand having FV4, heated to 95.degree. C. for
about 15 minutes, and then cooled to room temperature to produce a
second sample that included a double stranded DNA (dsDNA) segment
containing the second target, the sequence of the Factor V gene in
the region of the Leiden mutation.
[0373] One microliter of the dsDNA sample to be assayed for the
presence of the first or second target was admixed with 1L of a
nucleic acid probe and 18 .mu.L of water to form separate
hybridization compositions. Controls had 1 .mu.L of the dsDNA
sample and 19 .mu.L of water.
[0374] They were denatured by heating to 95.degree. C. for three
minutes, then maintained for 10 minutes under hybridizing
conditions (in a 37.degree. C. incubator) to form separate treated
samples.
[0375] A master mix was assembled containing 10.times. DNA
Polymerase Buffer (20 .mu.L; Promega, M195), sodium pyrophosphate
(5 .mu.L of 40 mM Na.sub.4P.sub.2O.sub.7 solution; Promega, C113),
Klenow Exo Minus (5 .mu.L; 5U; Promega, M218), NDPK (1 .mu.L of a
10U/.mu.L solution of NDPK [Sigma, N0379], dissolved in water), ADP
(2 .mu.L of a 10 .mu.M solution of ADP [Sigma, A5285] dissolved in
water), and water (67 .mu.L).
[0376] The hybridized, treated samples (20 .mu.L) were each mixed
with the master mix and maintained for 15 minutes at 37.degree. C.
to form a depolymerized sample.
[0377] The depolymerized sample was added to 100 .mu.L L/L reagent
(Promega, F202A), and the amount of light produced was read on a
Turner.RTM. TD20/20 luminometer. A total of 8 samples and two
controls were analyzed. The averaged results are shown below.
8 Average Assay Nucleic Acid Nucleic Acid Relative No. Target in
Sample Probe Light Units 1, 2 Factor V Factor V 1063 3, 4 Factor V
Factor V Leiden 88.8 5 Factor V none 8.652 6, 7 Factor V Leiden
Factor V 139.8 8, 9 Factor V Leiden Factor V Leiden 1016 10 Factor
V Leiden none 7.587
[0378] The data show that the light signal is about 10 fold greater
when the nucleic acid probe is exactly complementary to the nucleic
acid target (Assay Nos. 1, 2, 8 and 9) than when the nucleic acid
probe is partially complementary to the nucleic acid target with
the mismatch at the position penultimate to the 3'-terminal
nucleotide (Assay Nos. 3, 4, 6, and 7). The latter signal is in
turn about 10-fold greater than the light generated when there is
no probe to hybridize to the nucleic acid target (Assay Nos. 5 and
10).
9 FV1 5'CTAATCTGTAAGAGCAGATCCCTGGACAGGCGAGGAATACAGAGGGCAGC- A SEQ
ID NO:3 GACATCGAAGAGCT 3' FV2
5'AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTCGCCTGTCCAGGGATCTG SEQ ID NO:4
CTCTTACAGATTAGAGCT 3' FV3 5'CTAATCTGTAAGAGCAGATCCC-
TGGACAGGCAAGGAATACAGAGGGCAGCA SEQ ID NO:5 GACATCGAAGAGCT 3' FV4
5'AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTTGCCTGTCCAGGGATCTG SEQ ID NO:6
CTCTTACAGATTAGAGCT 3' FV5 5'CTGCTGCCCTCTGTATTCCTCG 3' SEQ ID NO:7
FV6 5'CTGCTGCCCTCTGTATTCCTTG 3' SEQ ID NO:8
EXAMPLE 3
Determination of the Presence or Absence of a Nucleotide Sequence
in a Sample Known to be Associated with the Factor V Leiden
Phenotype in Humans with Additional Interrogation Probes
[0379] In this Example, another pair of probes, complementary to
the opposite template strand as those used in Example 2, were used
to detect the gene sequences of the wild type Factor V gene and the
Leiden allele of this gene. Oligonucleotides FV7 (SEQ ID NO:9) and
FV8 (SEQ ID NO:10) were dissolved at 1 mg/mL in water.
Oligonucleotides FVl (SEQ ID NO:3), FV2 (SEQ ID NO:4), FV3 (SEQ ID
NO:5) and FV4 (SEQ ID NO:6) were used as targets, and the following
solutions were assembled.
10 Target Probe Water Solution (.mu.L) (.mu.L) (.mu.L) 1 and 2 1
FV1 + FV2 1 FV7 18 3 and 4 1 FV1 + FV2 1 FV8 18 5 1 FV1 + FV2 none
19 6 and 7 1 FV3 + FV4 1 FV7 18 8 and 9 1 FV3 + FV4 1 FV8 18 10 1
FV3 + FV4 none 19
[0380] The above solutions were heated to 95.degree. C. for three
minutes, then placed in a 37.degree. C. incubator for 10 minutes.
The following master mix was assembled:
11 10X DNA Polymerase Buffer (Promega M195) 20 .mu.L 40 mM Sodium
Pyrophosphate (Promega C113) 5 .mu.L 10 U/.mu.l Klenow Exo Minus
(Promega M218) 5 .mu.L NDPK (Sigma, NO379 at 10 U/.mu.L in water) 1
.mu.L ADP (Sigma A5285, 10 .mu.M in water) 2 .mu.L Water 67 .mu.L
100 .mu.L
[0381] Twenty microliters of master mix were added to each of the
heated nucleotide mixes after incubation at 37.degree. C. for 10
minutes. The resulting reactions were incubated for 15 minutes at
37.degree. C. and then added to 100 .mu.L L/L reagent (Promega,
F202A) and the light produced was immediately read using a
Turner.RTM. TD20/20 luminometer.
[0382] The following results were obtained.
12 Relative Reaction Light Units 1 217.9 2 237.9 3 47.76 4 48.33 5
5.903 6 18.79 7 19.19 8 186.8 9 181.5 10 5.837
[0383] These data show that probe FV7 gave a much stronger signal
than FV8 on DNA containing a sequence corresponding the native
Factor V gene, and thus can be used to detect this DNA sequence in
a sample. Probe FV8 gave a much stronger signal than FV7 on DNA
containing a sequence encoding the Factor V gene in the region of
the Leiden mutation.
13 FV1 5'CTAATCTGTAAGAGCAGATCCCTGGACAGGCGAGGAATACAGAGGGCAG- CA SEQ
ID NO:3 GACATCGAAGAGCT 3' FV2
5'AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTCGCCTGTCCAGGGATCTG SEQ ID NO:4
CTCTTACAGATTAGAGCT 3' FV3 5'CTAATCTGTAAGAGCAGATCCC-
TGGACAGGCAAGGAATACAGAGGGCAGCA SEQ ID NO:5 GACATCGAAGAGCT 3' FV4
5'AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTTGCCTGTCCAGGGATCTG SEQ ID NO:6
CTCTTACAGATTAGAGCT 3' FV7 5'GACAAAATACCTGTATTCCTCG 3' SEQ ID NO:9
FV8 5'GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:10
EXAMPLE 4
Detection of a Sequence in the Cystic Fibrosis Gene in the Region
of the Delta 508 Mutation
[0384] In this Example, an assay was performed to detect a sequence
that encodes a segment of the cystic fibrosis gene spanning the
mutation known as the delta F508 allele.
[0385] Oligonucleotides CF1 (SEQ ID NO:ll) and CF2 (SEQ ID NO:12)
were synthesized and redissolved in water at a concentration of 50
pmol/.mu.L. These primers were used to produce an amplified segment
of the human chromosomal DNA by PCR amplification. PCR reactions
contained 20 ng human genomic DNA, 50 pmol each primer, 1.times.
Promega Taq Reaction Buffer with 1.5 mM MgCl2 (Promega, M188A), 200
.mu.M dNTPs, and 1.25 U Taq DNA Polymerase (Promega M186A). Cycling
conditions were 1.times.2 minutes at 94.degree. C., 35.times.[0.5
minutes at 94.degree. C., 1 minute at 60.degree. C., 1 minute at
72.degree. C.], 1.times.7 minutes at 72.degree. C., 4.degree. C.
soak. The amplified DNA was purified using Wizard PCR Preps
(Promega A7170) by mixing 25 .mu.L PCR product with 1 mL resin and
washing with 3.times.1 mL 80% isopropanol. This DNA was used to
represent wild type human DNA encoding the cystic fibrosis gene
spanning the delta F508 mutation.
[0386] Oligonucleotides CF6 (SEQ ID NO:16) and CF7 (SEQ ID NO:17)
were dissolved in water at a concentration of 1 mg/mL, mixed and
annealed to form a double strand DNA segment as described for
oligonucleotide FV1 and FV2 above. This DNA was used to represent
human DNA encoding the delta F508 mutation at this locus.
[0387] Oligonucleotide probes CF3 (SEQ ID NO:13), CF4 (SEQ ID
NO:14) and CF5 (SEQ ID NO:15) were prepared. The sequence of probe
CF3 is completely complementary to wild type cystic fibrosis gene.
The sequence of probe CF4 was identical to that of probe CF3 except
for the 3'-terminal nucleotide that is complementary to the
nucleotide present in the delta F508 mutation, and thus was
completely complementary to one strand of that mutant sequence. The
sequence of probe CF5 was completely complementary to the second
strand of that F508 mutant sequence, and also therefore differed
from a total complement of probe CF3 at the 3'-terminal nucleotide.
The probes were separately dissolved in water to a concentration of
1 mg/mL. The following solutions were assembled.
14 Probe Target Water Solution (.mu.L) (.mu.L) (.mu.L) 1 and 2 1,
CF3 4, Purified, 15 Amplified DNA 3 and 4 1, CF4 4, Purified, 15
Amplified DNA 5 and 6 1, CF5 4, Purified, 15 Amplified DNA 7 none
4, Purified, 16 Amplified DNA 8 and 9 1, CF3 1, Annealed 18 (CF6 +
CF7) 10 and 11 1, CF4 1, Annealed 18 (CF6 + CF7) 12 and 13 1, CF5,
1, Annealed 18 (CF6 + CF7) 14 none 1, Annealed 19 (CF6 + CF7)
[0388] These solutions were heated at 95.degree. C. for 3 minutes
and then placed in a 37.degree. C. incubator for 10 minutes.
[0389] A master mix was assembled as described in Example 3 and 20
.mu.L of this solution were added to each of solutions 1-14 above.
They were incubated for another 15 minutes at 37.degree. C.
[0390] The solutions were added to 100 .mu.L of L/L reagent
(Promega F202A)) and the light produced by the reactions was
immediately measured using a Turner.RTM. TD 20/20 luminometer. The
following results were obtained.
15 Relative Net Solution Light Units Average RLU* 1 1527 .sup. 762
2 1683 3 766.7 (-48.5) 4 823.5 5 893.2 .sup. 44.1 6 881.0 7 843.4 8
72.73 .sup. 11.0 9 80.05 10 310.9 .sup. 306.5 11 302.1 12 439.0
.sup. 434.4 13 429.8 14 65.90 *The net value is calculated by
averaging the duplicate samples and subtracting the target alone
value measured for the different sets.
[0391] These data show that probe CF3 provided much higher signals
with wild type DNA than were provided by either of probes CF4 or
CF5 that had a mismatched nucleotide at the site of the mutation.
In addition, both probes CF4 and CF5 that were completely
complementary to the mutant sequence provided much higher signals
with DNA encoding the delta F508 mutant than with wild type
DNA.
16 CF1 5'CATTCACAGTAGCTTACCCA 3' SEQ ID NO:11 CF2
5'GCAGAGTACCTGAAACAGGA 3' SEQ ID NO:12 CF3 5'CATCATAGGAAACACCAAG 3'
SEQ ID NO:13 CF4 5'CATCATAGGAAACACCAAT 3' SEQ ID NO:14 CF5
5'GGCACCATTAAAGAAAATATCATT 3' SEQ ID NO:15 CF6
5'CTGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATGATGAATATAG SEQ ID NO:16
CF7 5'CTATATTCATCATAGGAAACACCAATGATATTTTCTTTAATGGTGCCAG 3' SEQ ID
NO:17
EXAMPLE 5
Detection of a Sequence in the Cystic Fibrosis Gene in the Region
of the Delta 508 Mutation including a Sample Containing Both the
Normal and Delta F508 Alleles
[0392] This Example demonstrates an assay that detects a sequence
encoding a segment of the cystic fibrosis gene spanning the
mutation known as the delta F508 allele. The assay is illustrated
using the wild type human sequence of this gene in this region and
using a sample that has both alleles. The results here demonstrate
that the assay can discriminate between homozygotes for these
alleles, and can be used to detect heterozygote samples in which
both alleles are present together, as would be the case with a
carrier for a wide variety of genetic diseases.
[0393] Oligonucleotides CF8 (SEQ ID NO:18) and CF9 (SEQ ID NO:19),
a synthetic wild type target, were dissolved in water and annealed
as described for Example 2. CF6 (SEQ ID NO:16) and CF7 (SEQ ID
NO:17) were also used as targets. CF3 (SEQ ID NO:13) and CF4 (SEQ
ID NO:14) were used as probes. The following solutions were
assembled.
17 Probe Target(s) Water Solution (.mu.L) (.mu.L) (.mu.L) 1 and 2
1, CF3 1, Annealed 18 (CF8 + CF9) 3 and 4 1, CF4 1, Annealed 18
(CF8 + CF9) 5 none 1, Annealed 19 (CF8 + CF9) 6 and 7 1, CF3 1,
Annealed 17 (CF6 + CF7) and 1, Annealed (CF8 + CF9) 8 and 9 1, CF4
1, Annealed 17 (CF6 + CF7) and 1, Annealed (CF8 + CF9) 10 none 1,
Annealed 18 (CF6 + CF7) and 1, Annealed (CF8 + CF9) 11 and 12 1,
CF3 1, Annealed 18 (CF6 + CF7) 13 and 14 1, CF4 1, Annealed 18 (CF6
+ CF7) 15 none 1, Annealed 19 (CF6 + CF7)
[0394] The above solutions were heated at 95.degree. C. for 3
minutes, then placed in a 37.degree. C. incubator for 10 minutes. A
master mix was made as in Example 3 and 20 .mu.L of this solution
were then added to each of tubes 1-15. The tubes were incubated at
37.degree. C. for an additional 15 minutes, then the solutions were
added to 100 .mu.L of L/L reagent (Promega F202A) and the light
produced by the reactions was read immediately using a Turner.RTM.
TD20/20 luminometer. The following data were obtained.
18 Relative Adjusted Net Sample Light Units Light Value* 1 310.1
307.9 2 342.3 3 22.45 4.41 4 23.02 5 18.29 6 400.2 346.98 7 393.7 8
332.3 269.38 9 306.4 10 49.97 11 96.67 37.68 12 109.1 13 371.3
305.4 14 369.8 15 65.22 *This value was calculated by averaging the
duplicate reactions and subtracting the value measured for the
appropriate target alone control reaction.
[0395] These data again show that probe CF3 provided a much
stronger signal with normal (wild type; homozygous) DNA than did
probe CF4, and probe CF4 provided a much stronger signal with the
homozygous delta F508 mutation target than did probe CF3. In
addition, when both targets were present in the sample, as in a
heterozygote, signals were provided from both probes to indicate
the presence of a heterozygote. Thus, the analytical output from
this method illustrated whether the nucleic acid target sequence in
a nucleic acid sample was homozygous or heterozygous, and when
homozygous, which of the alleles was present.
19 CF3 5'CATCATAGGAAACACCAAG 3' SEQ ID NO:13 CF4
5'CATCATAGGAAACACCAAT 3' SEQ ID NO:14 CF6
5'CTGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATGATGAATATAG 3' SEQ ID NO:16
CF7 5'CTATATTCATCATAGGAAACACCAATGATATTTTCTTTAATGGTGCCAG 3' SEQ ID
NO:17 CF8 5'CTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG- ATGAATATAG
3' SEQ ID NO:18 CF9 5'CTATATTCATCATAGGAAACACCAA-
AGATGATATTTTCTTTAATGGTGCCAG 3' SEQ ID NO:19
EXAMPLE 6
Detection of DNA Sequences Corresponding to the Prothrombin Gene in
the Region of a Single Nucleotide Polymorphism
[0396] An assay for the presence or absence of a mutation in the
human prothrombin gene is illustrated in this Example. This SNP is
characterized by a G to A substitution in the prothrombin gene.
[0397] Oligonucleotides PT1 (SEQ ID NO:20), PT2 (SEQ ID NO:21), PT3
(SEQ ID NO:22), and PT4 (SEQ ID NO:23) were synthesized and
dissolved in water to a concentration of 1 mg/mL. A sample of PT1
and PT2 were then diluted to 0.3 ng in water for use in the
solutions below, and the following solutions were made.
20 Probe Target Water Solution (.mu.L) (.mu.L) (.mu.L) 1 and 2 1,
PT3 1, PT1 18 3 and 4 1, PT4 1, PT1 18 5 none 1, PT1 19 6 and 7 1,
PT3 1, PT2 18 8 and 9 1, PT4 1, PT2 18 10 none 1, PT2 19
[0398] These solutions were heated at 95.degree. C. for three
minutes then placed in a 37.degree. C. incubator for 10
minutes.
[0399] A master mix was assembled as in Example 3 and 20 .mu.L of
this solution were added to each of solutions 1-10, above, and all
solutions were incubated for 15 minutes at 37.degree. C. After this
incubation, these solutions were added to 100 .mu.L of L/L reagent
(Promega F202A) and the light produced by the solutions immediately
read using a Turner.RTM. TD 20/20 luminometer. The following
results were obtained.
21 Sample Relative Light Units 1 240.9 2 253.3 3 56.10 4 55.88 5
5.88 6 29.61 7 31.49 8 738.0 9 646.8 10 6.21
[0400] These data demonstrate the probe PT3 provided much higher
signals with a wild type target (PT1) than does probe PT4, but that
probe PT4 provided a much higher signal with mutant template (PT2)
than did PT3.
22 PT1 5'TCCCAATAAAAGTGACTCTCAGCGAGCCTCAATGCTCCCAGTGCTATTCA 3' SEQ
ID NO:20 PT2 5'TCCCAATAAAAGTGACTCTCAGCAAGCCTCAATGCTCCCAG- TGCTATTCA
3' SEQ ID NO:21 PT3 5'GGAGCATTGAGGCTCG 3' SEQ ID NO:22 PT4
5'GGAGCATTGAGGCTTG 3' SEQ ID NO:23
EXAMPLE 7
Determination of SNPs in DNA Isolated from Plant Materials
[0401] A process of the invention is used here to determine the
genotype of rice DNAs at a known SNP site.
[0402] A particular target produced by amplification of a segment
of the rice genome is interrogated in this Example. It was found
that this target produces high background signal values if nothing
is done to eliminate one strand of the amplified DNA target and did
not exhibit discrimination between two primers that were designed
to detect a SNP present in some rice strains. This Example
illustrates how one can purposefully destroy one of the amplified
DNA strands and interrogate the other strand. For this case in
particular, such manipulations result in greatly reduced background
light signals from the target, permitting clear determination of
the interrogation signals.
[0403] Probes RS1 (SEQ ID NO:24) and RS2 (SEQ ID NO:25) were
dissolved at a concentration of 50 pmole/.mu.L in water. Probe RS1
contained phosphothioate linkages at the first four 5'-terminal
linkages that are not cleaved by the enzyme used in the reaction.
DNA was isolated from rice and was at a concentration of 10
.mu.g/mL.
[0404] Five coded DNA samples and two DNA samples of known genotype
(the "GI" allele and the "T" allele) were obtained and subjected to
amplification with probes RS1 (SEQ ID NO:24) and RS2 (SEQ ID
NO:25). The DNA was then treated with T7 Exonuclease 6 for 15
minutes at 37.degree. C. and purified. The resulting purified DNA
was subjected to pyrophosphorylation reactions using probes RS3
(SEQ ID NO:26), RS4 (SEQ ID NO:27), or no probe and the reaction
products added to L/L reagent (Promega, F202A) and light production
measured.
[0405] The following results were obtained:
23 Relative Light Units Measured WT Probe Variant Probe DNA
Analyzed (RS3 G Allele) (RS4 T Allele) No Probe #1 784.5 307.5
229.9 #2 286.3 882.7 227.9 #3 291.5 862.4 202.9 #4 560.4 195.5
158.2 #5 706.8 235.5 187.7 G Allele 810.7 250.0 189.2 T Allele
416.6 1121 243.4
[0406] Net Light Units, Ratio and Called Genotype
24 Net Light Units* DNA WT Variant Called Analyzed Probe Probe
Ratio** Genotype #1 554.6 77.6 7.1 G Allele #2 58.4 654.8 0.09 T
Allele #3 88.6 659.5 0.13 T Allele #4 402.2 37.3 10.8 G Allele #5
519.1 47.8 10.9 G Allele G Allele 621.5 60.8 10.2 G Allele Std.
Deviation T Allele 173.2 877.6 0.20 T Allele Std. Deviation *Net
light units = total light units--no primer values. **Ratio = Net
light units WT primer/net light units variant primer
[0407] After these results were obtained, the identity of the DNA
samples was uncoded and all the called genotypes agreed with the
previously determined genotype of these samples. These results
demonstrate the assay described in this Example can be used to
determine SNPs in plant DNA and that removal of one DNA strand of a
sample can help eliminate high background signals from a template,
permitting SNPs to be determined.
[0408] RS1 5'C*C*C*A*ACACCTTACAGAAATTAGC 3' SEQ ID NO:24 (*
signifies the presence of a phosphorothioate linkage between the
indicated bases.)
[0409] RS2 5'TCTCAAGACACAAATAACTGCAG 3' SEQ ID NO:25
[0410] RS3 5'AGAACATCTGCAAGG 3' SEQ ID NO:26
[0411] RS4 5'AGAACATCTGCAAGT 3' SEQ ID NO:27
EXAMPLE 8
Discrimination of Repeated DNA Sequences Using
Pyrophosphorylation-Based Assay Methods
[0412] This Example illustrates an assay for determining the number
of repeats of a four base pair sequence in a DNA. Discrimination of
such repeat sequences has been found to be very useful for
identification of forensic samples. The probes in this set, TR1
(SEQ ID NO:31), TR2 (SEQ ID NO:32) and TR3 (SEQ ID NO:33) were
designed to exactly match known alleles of the THO 1 locus with 6,
7, and 8 repeats respectively, of a CATT sequence.
[0413] Probes TR1-TR3 were suspended in water to a concentration of
1 mg/mL. Targets that were homozygous for THO 1 alleles with 6, 7
and 8 repeats were amplified using the protocol in the Gene
Print.TM. System instructions (Promega). These targets were named
allele 6 (SEQ ID NO:28), allele 7 (SEQ ID NO:29) and allele 8 (SEQ
ID NO:30), respectively. Gel-purified targets were PCR amplified
and then further purified using the Wizard.TM. PCR Clean-up system
(Promega, A7170) and the concentration of the DNA measured by
DNAQuant (Promega). These targets were adjusted to a concentration
of 1 .mu.g/mL and to 3.3 .mu.g/mL by the addition of deionized
water. The following solutions containing probes were assembled in
a final volume of 20 .mu.L by the addition of water.
25 Solution Probe Target (ng) #1 -- allele 6, 1 #2 TR1 allele 6, 1
#3 TR2 allele 6, 1 #4 TR3 allele 6, 1 #5 -- allele 7, 1 #6 TR1
allele 7, 1 #7 TR2 allele 7, 1 #8 TR3 allele 7, 1 #9 -- allele 8, 1
#10 TR1 allele 8, 1 #11 TR2 allele 8, 1 #12 TR3 allele 8, 1 #13 --
allele 6, 3.3 #14 TR1 allele 6, 3.3 #15 TR2 allele 6, 3.3 #16 TR3
allele 6, 3.3 #17 -- allele 7, 3.3 #18 TR1 allele 7, 3.3 #19 TR2
allele 7, 3.3 #20 TR3 allele 7, 3.3 #21 -- allele 8, 3.3 #22 TR1
allele 8, 3.3 #23 TR2 allele 8, 3.3 #24 TR3 allele 8, 3.3
[0414] These solutions were heated at 95.degree. C. for 3 minutes,
then permitted to cool by incubation at room temperature for 10
minutes.
[0415] The following master mix was made.
26 Amount Component (.mu.L)/reaction 10X DNA Polymerase Buffer 4 40
mM Sodium Pyrophosphate 0.5 10 .mu.M ADP 0.4 Klenow exo-, 10
U/.mu.L 0.5 NDPK, 1 U/.mu.L 0.2 Nanopure water 14.4
[0416] This solution was mixed and 20 .mu.L of this solution were
added to solutions 1-24 above, and the resulting solutions
incubated for 15 minutes at 37.degree. C. After this incubation, 4
.mu.L of the resulting solution were added to 100 .mu.L of L/L
reagent(Promega, F202A) and the light produced was immediately
measured using a Turner.TM. TD20/20 luminometer. The following data
were obtained.
27 Relative Solution Light Units #1 3.97 #2 50.79 #3 5.94 #4 6.03
#4 3.79 #5 67.23 #7 28.94 #8 6.73 #9 3.19 #10 49.52 #11 30.99 #12
30.63 #13 8.62 #14 256.90 #15 16.74 #16 13.83 #17 6.73 #18 206.5
#19 110.2 #20 15.35 #21 6.49 #22 271.9 #23 150.5 #24 154.8
[0417] The values from the no probe reactions above were subtracted
from the values for the various probe/target matches and the
resulting values are shown in the table below.
28 Allele TR1 TR2 TR3 Assayed Probe Probe Probe Relative Light
Units With 1 ng of Target Allele 6 44.46 -0.01 -0.44 Allele 7 61.07
23.17 0.44 Allele 8 43.97 25.83 24.95 Relative Light Units With 3.3
ng of Target Allele 6 245.08 4.88 1.73 Allele 7 196.58 100.24 5.14
Allele 8 262.22 140.78 144.83
[0418] If, in the repeat region, the probe contains the same number
of repeats as the target or fewer, no mismatching bases should be
present at the 3' end of the probe and a relatively strong signal
is obtained. The TR1 probe shows such a signal with targets
containing 6, 7 or 8 repeats. However, if the probe contains more
repeats in this region than are present in the target, mismatched
bases are expected at the 3' end of the probe that should greatly
reduce the signal developed. As expected, the TR3 probe gave a
strong signal against the allele 8 target, but gave a much weaker
signal against the allele 7 and 6 targets. Because the signal
generated using the various probes can be used to determine the
number of repeated units in the repeat region, this method can be
used to determine the alleles present in samples.
[0419] Using the above method, probes containing the same number or
fewer repeated sequences as the target produced similar light
output. When more repeats were present in the probe than the
target, low analytical outputs were observed. The number of repeats
in the target could thus accurately be determined by an indicative
change in the analytical output, here, luminescence, between the
separately assayed samples.
29 Allele 6 5'GGTGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAGG-
AAGAGGCCAATGGG 3' SEQ ID NO:28 Allele 7
5'GGTGAATGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAGGAAGAGGCCAATGGG 3'
SEQ ID NO:29 Allele 8 5'GGTAGGTGAATGAATGAATGAATGAATGAA-
TGAATGAATGAGGGAAATAAGGGAGGAAGAGGCCAATGGG 3' SEQ ID NO:30 TR1
5'CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTCATTCATTCATTCACC 3' SEQ
ID NO:31 TR2 5'CCCATTGGCCTGTTCCTCCCTTATTTCC-
CTCATTCATTCATTCATTCATTCATTCATTCACC 3' SEQ ID NO:32 TR3
5'CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTCATTCATTCATTCATTCATTCACC
3' SEQ ID NO:33
EXAMPLE 9
Discrimination of Repeated DNA Sequences Using Pyrophosphorylation
Based Assay Methods Using Another Class of Probes
[0420] A surprising result is presented in this Example that
demonstrates that a class of probes that should only produce
signals with targets of a certain class, essentially give
equivalent signals with additional targets. Although these results
do not match the predicted results, they can still be used to
determine the allelic composition of samples.
[0421] The probes in the Example above were designed to hybridize
to alleles of THO 1 that are used for genotyping humans. They were
designed to hybridize as illustratively shown below for probe TR2
with targets of three alleles.
[0422] Hybridization of probe TR2 (top strand) with an Allele 6
target (bottom strand):
30 CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT(CATT).sub.4CATTCATTC ACC
GGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA(GTAA).sub.4GTAAGTG- G
[0423] Hybridization of probe TR2 (top strand) with an Allele 7
target (bottom strand):
31 CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT(CATT).sub.4CATTCATTC ACC
GGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA(GTAA).sub.4GTAAGTA- AG TGG
[0424] Hybridization of probe TR2 (top strand) with an Allele 8
target (bottom strand):
32 CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT(CATT).sub.4CATTCATTC AAC
GGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA(GTAA).sub.4GTAAGTA- AG
TAAGTGGATGG 5'
[0425] As described in Example 8, when the target contains fewer
repeats than the probe, mismatched bases can occur at the 3' end of
the probe, creating a double strand DNA region that is a very poor
substrate for the pyrophosphorylation reaction. These predictions
were verified with the results obtained.
[0426] In this Example, a new form of probe is used in the
reactions. These probes are designed to extend beyond the repeat
region and hybridize to the target following this DNA segment when
they are hybridized to the allele with the correct number of repeat
segments. The predicted hybridization segments for the allele 7
probe (TR6) with the allele 6, 7, and 8 targets are shown
below.
[0427] Hybridization of probe TR6 (top strand) with an Allele 6
target (bottom strand):
33 CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT(CATT).sub.4CATTCATTC ACC
GGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA(GTAA).sub.4GTAAGTG- GA TGG
[0428] Hybridization of probe TR6 (top strand) with an Allele 7
target (bottom strand):
34 CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT(CATT).sub.4CATTCATTC ACC
GGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA(GTAA).sub.4GTAAGTA- AG TGGATGG
5'
[0429] Hybridization of probe TR6 (top strand) with an Allele 8
target (bottom strand):
35 CCCATTGGCCTCTTCCTCCCTTATTTCCCTCATT(CATT).sub.4CATTCATTC ACC
GGGTAACCGGAGAAGGAGGGAATAAAGGGAGTAA(GTAA).sub.4GTAAGTA- AG
TAATGGAGTGG 5'
[0430] As shown above, probe TR6 was designed to form a product
without 3' end mismatches with only allele 7. Thus, this probe was
expected to only give a strong signal with the allele 7 target.
[0431] In order to test the actual signals that such probes would
give with various targets, probes TR4 (SEQ ID NO:34), TR5 (SEQ ID
NO:35), TR6 (SEQ ID NO:36), TR7 (SEQ ID NO:37) and TR8 (SEQ ID
NO:38) were dissolved in water to a concentration of 1 mg/mL. These
probes were used with the targets Allele 6 (SEQ ID NO:28), Allele 7
(SEQ ID NO:29) and Allele 8 (SEQ ID NO:30) to generate the
following solutions. The final volume of these solutions was
adjusted to 20 mL by the addition of water. The probes were used at
a concentration of 1 .mu.g/reaction.
36 Soln. Probe Target (ng) #1 -- Allele 6, 1 #2 TR4 Allele 6, 1 #3
TR5 Allele 6, 1 #4 TR6 Allele 6, 1 #5 TR7 Allele 6, 1 #6 TR8 Allele
6, 1 #7 -- Allele 7, 1 #8 TR4 Allele 7, 1 #9 TR5 Allele 7, 1 #10
TR6 Allele 7, 1 #11 TR7 Allele 7, 1 #12 TR8 Allele 7, 1 #13 --
Allele 8, 1 #14 TR4 Allele 8, 1 #15 TR5 Allele 8, 1 #16 TR6 Allele
8, 1 #17 TR7 Allele 8, 1 #18 TR8 Allele 8, 1 #19 -- Allele 6, 3.3
#20 TR4 Allele 6, 3.3 #21 TR5 Allele 6, 3.3 #22 TR6 Allele 6, 3.3
#23 TR7 Allele 6, 3.3 #24 TR8 Allele 6, 3.3 #25 -- Allele 7, 3.3
#26 TR4 Allele 7, 3.3 #27 TR5 Allele 7, 3.3 #28 TR6 Allele 7, 3.3
#29 TR7 Allele 7, 3.3 #30 TR8 Allele 7, 3.3 #31 -- Allele 8, 3.3
#32 TR4 Allele 8, 3.3 #33 TR5 Allele 8, 3.3 #34 TR6 Allele 8, 3.3
#35 TR7 Allele 8, 3.3 #36 TR8 Allele 8, 3.3
[0432] These solutions were heated at 95.degree. C. for 3 minutes,
then cooled at room temperature for 10 minutes. A master mix was
assembled and added to these solutions as in the previous Example.
The resulting solutions were then heated at 37.degree. C. for 15
minutes and were sampled as in the previous Example. The samples
were added to L/L reagent (Promega, F202A) and the light output was
immediately measured as in the previous Example. The following
results were obtained.
37 Target -- TR4 TR5 TR6 TR7 TR8 Relative Light Units From
Reactions Containing Probes With 1 ng of Target Allele 6 2.14 47.40
33.87 11.45 7.57 7.98 Allele 7 2.06 53.00 30.97 30.43 12.38 10.41
Allele 8 2.51 21.30 27.54 30.99 39.04 14.84 (none) (nd) 2.28 2.30
2.59 3.12 3.43 Expected -- A 5 A 6 A 7 A 8 A 9 Allele Detected
Relative Light Units From Reactions Containing Probes With 3.3 ng
of Target Allele 6 8.52 282.6 291.5 90.62 61.34 46.49 Allele 7
12.23 276.2 237.8 286.4 103.4 74.92 Allele 8 10.33 170.6 242.5
264.4 264.9 111.9 (none) (nd) 3.56 3.11 3.25 3.63 3.60 Expected --
A 5 A 6 A 7 A 8 A 9 Allele Detected
[0433] Surprisingly, these probes did not provide the expected
detection pattern. For example, probe TR6 was expected to only give
a strong signal with a target with allele 7 (A 7). Although the
probe did show a lower signal with allele 6 (A 6) than with allele
7 (90.6 vs. 286.4 units, respectively), very little difference was
seen between the signals with alleles 7 and 8 (A 8) (286.4 vs.
264.4 units respectively). In general, all the probes exhibited
substantially equal reactivity with any target that had the same
number of repeated units or greater than the number of repeated
units in the probe. These same probes showed lower signals with
targets having fewer repeat units than those present in the probe,
with the signal strength seen decreasing as the difference in the
number of repeat units increased. Thus, although these probes
clearly did not provide the expected signal patterns, they can be
used to determine THO 1 alleles.
38 Allele 6 5' GGTGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAG-
GAAGAGGC SEQ ID NO:28 CAATGGG 3' Allele 7 5'
GGTGAATGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAGGAAG SEQ ID NO:29
AGGCCAATGGG 3' Allele 8 5' GGTAGGTGAATGAATGAATGAAT-
GAATGAATGAAGAATGAGGGAAATAAG SEQ ID NO:30 GGAGGAAGAGGCCAATGGG 3' TR4
5' CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO:34
CATTCATTCACC 3' TR5 5'
CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATTCATT SEQ ID NO:35
CATTCATTCACC 3' TR6 5' CCCATTGGCCTGTTCCTCCCTTATTTC-
CCTCATTCATTCATTCATT SEQ ID NO:36 CATTCATTCATTCACC 3' TR7 5'
CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO:37
CATTCATTCATTCATTCATTCACC 3' TR8 5'
CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO:38
CATTCATTCATTCATTCATTCATTCACC 3'
EXAMPLE 10
Additional Probes for Detection of THO 1 Alleles
[0434] Additional probes are used in this Example to demonstrate
that the creation of additional mismatches between THO 1 allele
targets and probes can result in the formation of probe/target
combinations that provide strong signals with essentially one THO 1
allele.
[0435] Probes TR 9 (SEQ ID NO:39), TR10 (SEQ ID NO:40) and TR11
(SEQ ID NO:41) were dissolved at 1 mg/mL and assembled into
reactions with target at 3 ng/reaction with allele 6 (SEQ ID
NO:28), allele 7 (SEQ ID NO:29), and allele 8 (SEQ ID NO:30) of THO
1 and without any target as described in the Example above. These
solutions were heated and cooled as in the previous Example. The
resulting solutions were treated with master mix, incubated, added
to L/L reagent (Promega, F202A) and the light produced measured as
in the previous Example. The following results were obtained.
39 Relative Light Units Probe Probe Probe Target TR9 TR10 TR11 none
Allele 6 59.74 35.86 75.78 8.96 Allele 7 51.73 2.32 15.85 10.54
Allele 8 58.58 25.37 33.67 9.85 (none) 47.27 34.24 3.676 (nd)
[0436] The values for the probe alone and target alone reactions
were subtracted from the values for the combined reactions and are
shown in the table below.
40 Relative Light Units Target Probe TR9 Probe TR10 Probe TR11
Allele 6 3.51 -7.34 63.14 Allele 7 -6.08 -22.46 1.63 Allele 8 1.46
-18.72 20.14
[0437] Increasing the number of mismatched bases between the probe
and target lowers the signal value measured, and in many cases
decreases the values seen below those attributable from background
reactions. In particular, probes TR9, which has a mismatch of 2
base pairs, and TR10, which has an A to C mutation 3 bases from the
end of the probe, do not exhibit the ability to detect THO 1
alleles. However, probe TR11, which has a A to G change 3 bases
from the end of the probe, produced a measurable signal with the
allele 6 target that is greater than the signals seen with the
other targets.
[0438] Probes TR12 (SEQ ID NO:42) and TR13 (SEQ ID NO:43) were then
used as above. The following data were obtained.
41 Relative Light Units Target Probe TR12 Probe TR13 Allele 6 9.7
9.8 Allele 7 5.0 7.1 Allele 8 10.4 12.6 (none) 3.0 2.9
[0439] These probes, having additional mismatches four base pairs
from the 3' end of the probe, only provided very low light signals
and apparently did not discriminate between the alleles of THO 1.
Thus, these data suggest that probes that can provide
allele-specific signals can be identified by designing probes with
base pair mismatches placed in the probe sequence near the 3' end
of the probe.
42 Allele 6 5' GGTGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAG-
GAAGAGGC SEQ ID NO:28 CAATGGG 3' Allele 7 5'
GGTGAATGAATGAATGAATGAATGAATGAATGAGGGAAATAAGGGAGGAAG SEQ ID NO:29
AGGCCAATGGG 3' Allele 8 5' GGTAGGTGAATGAATGAATGAAT-
GAATGAATGAATGAATGAGGGAAATAAG SEQ ID NO:30 GGAGGAAGAGGCCAATGGG 3'
TR9 5' CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO:39
CATTCATTCATTCAGC 3' TR10 5'
CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO:40
CATTCATTCATTCCCC 3' TR11 5'
CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO:41
CATTCATTCATTCGCC 3' TR12 5'
CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO:42
CATTCATTCATTGACC 3' TR13 5'
CCCATTGGCCTGTTCCTCCCTTATTTCCCTCATTCATTCATT SEQ ID NO:43
CATTCATTCATTAACC 3'
EXAMPLE 11
Discrimination of Repeated DNA Sequences Using
Pyrophosphorylation-based Assay Methods-III
[0440] In this Example, PCR targets spanning between 6 to 13 copies
of the TPOX four nucleotide short tandem repeat were discriminated
with probes specific for the number of repeats using a
pyrophosphorylation based assay. The targets were prepared by
standard PCR amplification of each of the (Promega, DC5111) TPOX
bands that were previously gel-purified. The PCR cycling parameters
were 94.degree. C., 1 minute (94.degree. C., 15 seconds; 60.degree.
C., 30 seconds; 72.degree. C., 60 seconds).times.35, 68.degree. C.,
10 minutes. The correct size of the PCR products was confirmed on a
4% polyacrylamide gel electrophoresis. The PCR products were
purified using Wizard.RTM. PCR Purification System (Promega, A7170)
and resuspended in water to a concentration of 10 ng/.mu.L. The
interrogation sequence probes were P6 (SEQ ID NO:44), P7 (SEQ ID
NO:45), P8 (SEQ ID NO:46), P9 (SEQ ID NO:47), P10 (SEQ ID NO:48),
P11 (SEQ ID NO:49), P12 (SEQ ID NO:50) and P13 (SEQ ID NO:51).
[0441] Targets containing between 6 and 13 TGAA repeats were each
interrogated with each of the interrogation probes listed above.
The target alleles used were A6 (SEQ ID NO:52), A7 (SEQ ID NO:53),
A8 (SEQ ID NO:54), A9 (SEQ ID NO:55), A10 (SEQ ID NO:56), A11 (SEQ
ID NO:57), A12 (SEQ ID NO:58) and A13 (SEQ ID NO:59), respectively.
The probes were at a final concentration of 2.5 .mu.M in the
solution, 10 ng of target were used per reaction and the volume was
increased to 20 .mu.L with water. The solutions were heated at
95.degree. C. for 2 minutes, then cooled at room temperature over
10 minutes.
[0442] Twenty microliters of master mix were added to each solution
(14.7 .mu.L water, 4 .mu.L 10.times. DNA polymerization buffer, 5
.mu.L 40 mM NaPPi, 0.4 .mu.L 10 .mu.M ADP, 0.2 .mu.L NDPK (1
U/.mu.L), 0.2 .mu.L Klenow exo- (10 U/.mu.L)) and they were further
incubated at 37.degree. C. for 15 minutes. Then, 4 .mu.L of the
solution were added to 100 .mu.L of L/L reagent and the light
output read with a Turner.RTM. TD20/20 luminometer. The relative
light units (rlu) obtained are reported below:
43 Raw rlu numbers Probe Target P6 P7 P8 P9 P10 P11 P12 P13 None A6
57.62 16.80 15.65 28.51 23.37 25.31 26.70 47.48 14.08 A7 23.29
73.44 22.39 37.28 20.31 25.01 26.29 44.24 25.06 A8 25.04 20.82
54.63 35.78 20.69 21.02 22.99 37.51 18.37 A9 28.83 21.60 25.03
85.98 30.71 28.55 29.54 50.86 21.48 A10 27.69 25.53 30.30 42.38
61.04 30.21 27.71 46.30 32.80 A11 30.29 35.07 30.67 51.20 40.37
69.92 39.12 58.52 30.00 A12 35.36 25.43 29.71 45.14 28.44 38.76
63.31 57.05 40.24 A13 39.35 27.67 29.92 42.56 33.59 32.80 36.04
84.37 32.70 None 8.67 6.29 9.15 27.98 14.18 16.51 16.22 32.98
4.66
[0443] The above values were adjusted for background and the
negative numbers converted to zero to provide the data in the table
below.
44 Probe Target P6 P7 P8 P9 P10 P11 P12 P13 A6 40 1 0 0 0 0 1 5 A7
0 47 0 0 0 0 0 0 A8 3 1 32 0 0 0 0 0 A9 3 0 0 41 0 0 0 1 A10 0 0 0
0 19 0 0 0 A11 0 3 0 0 1 28 0 0 A12 0 0 0 0 0 0 12 0 A13 3 0 0 0 0
0 0 23
[0444] The data indicate that the interrogation probes can
recognize the presence of the related homozygote alleles of the
TPOX locus. Similarly, heterozygote targets were assayed with the
same set of interrogation probes. Ten nanograms of each purified
PCR target were included in each interrogation reaction. The
reaction conditions were identical to those for the homozygote
targets described above. The rlu values obtained are reported
below.
45 Raw rlu numbers Probe Target P6 P7 P8 P9 P10 P11 P12 P13 None
A6, 91.09 36.85 40.90 50.88 41.93 41.33 83.69 83.69 31.78 A13 A11,
55.94 48.13 54.35 62.64 50.92 84.19 80.33 66.41 54.62 A12 A9, 61.75
43.49 47.71 93.76 77.91 37.18 39.11 59.74 33.37 A10 A7, A8 35.66
76.04 62.03 36.03 27.92 30.07 31.92 51.40 50.52 None 6.29 4.87 6.22
20.83 10.89 11.86 12.41 28.64 4.32
[0445] The values were adjusted for background and the negative
numbers converted to zero to provide the data in the table
below.
46 Probe Target P6 P7 P8 P9 P10 P11 P12 P13 A6, A13 57.35 4.53 7.23
2.59 3.58 2.01 6.45 27.59 A11, A12 0 0 0 0 0 22.03 17.62 0 A9, A10
26.42 9.58 12.45 43.88 37.97 0 0 2.05 A7, A8 0 24.98 9.62 0 0 0 0
0
[0446] As can be seen from the results in the table above, this
method accurately identified each of the heterozygote targets,
although probe P6 also identified one false positive for an unknown
reason.
[0447] Interrogation Probe Sequences:
47 P6 5' GGCACTTAGGGAACCCTCAC TGAA TGAA TGAA TGAA SEQ ID NO:44 TGAA
TGAA TATT 3' P7 5' GGCACTTAGGGAACCCTCAC TGAA TGAA TGAA TGAA SEQ ID
NO:45 TGAA TGAA TGAA TATT 3' p8 5' GGCACTTAGGGAACCCTCAC TGAA TGAA
TGAA TGAA SEQ ID NO:46 TGAA TGAA TGAA TGAA TATT 3' P9 5'
GGCACTTAGGGAACCCTCAC TGAA TGAA TGAA TGAA SEQ ID NO:47 TGAA TGAA
TGAA TGAA TGAA TATT 3' P10 5' GCACTTAGGGAACCCTCAC TGAA TGAA TGAA
TGAA SEQ ID NO:48 GAA TGAA TGAA TGAA TGAA TGAA TATT 3' P11 5'
GGCACTTAGGGAACCCTCAC TGAA TGAA TGAA TGAA SEQ ID NO:49 TGAA TGAA
TGAA TGAA TGAA TGAA TGAA TATT 3' P12 5' GGCACTTAGGGAACCCTCAC TGAA
TGAA TGAA TGAA SEQ ID NO:50 TGAA TGAA TGAA TGAA TGAA TGAA TGAA TGAA
TATT 3' P13 5' GGCACTTAGGGAACCCTCAC TGAA TGAA TGAA TGAA SEQ ID
NO:51 TGAA TGAA TGAA TGAA TGAA TGAA TGAA TGAA TGAA TATT 3' Target
Alleles: A6: 5' GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID
NO:52 ATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGG
CCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTG
TTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTC
TTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3'
A7: 5' GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO:53
ATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGG
CCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTG
TTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTC
TTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3'
A8: 5' GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO:54
ATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGG
CCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTG
TTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTC
TTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3'
A9: 5' GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO:55
ATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGAAGGG
CCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTG
TTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTC
TTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3'
A10: 5' GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO:56
ATGAATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGACAGA
AGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACTTGTG
TTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTC
TTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3'
A11: 5' GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO:57
ATGATGAATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACAAGGA
CAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCCGACT
TGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAATCTC
TTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG TAGAGTCAACCTCA 3'
A12: 5' GGCACTTAGGGAACCCTCACTGAATGAATGAATGAATGA SEQ ID NO:58
ATGAATGAATGAATGAATGAATGAATGAATGTTTGGGCAAATAAACGCTGACA
AGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCACAGCCC
GACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGATGAA
TCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGGACAG
TAGAGTCAACCTCA 3' A13: 5' GGCACTTAGGGAACCCTCACTGAA- TGAATGAATGAATGA
SEQ ID NO:59 ATGAATGAATGAATGAATGAATGAATGAATGAATGTTT-
GGGCAAATAAACGCT
GACAAGGACAGAAGGGCCTAGCGGGAAGGGAACAGGAGTAAGACCAGCGCA- CA
GCCCGACTTGTGTTCAGAAGACCTGGGATTGGACCTGAGGATTCAATTTTGGA
TGAATCTCTTAATTAACCTGTGTGGTTCCCAGTTCCTCCCCTGAGCGCCCAGG
ACAGTAGAGTCAACCTCA 3'
EXAMPLE 12
Interrogation for Loss of Heterozygosity
[0448] In certain types of disease states such as some cancers,
there is a change in the heterozygosity of the locus of certain
alleles. For example, a non-cancerous cell may be heterozygous at a
particular locus. In a cancer cell, however, one of the two alleles
may be lost or deleted at the particular locus. This is referred to
as loss of heterozygosity.
[0449] This type of loss of heterozygosity (LOH) reaction was
created by PCR-amplifying 25 ng (1 .mu.L) and 50 ng (2 .mu.L) of
two E. coli targets (W3110, DH5.alpha.) with probes 10730 (SEQ ID
NO:60) and 10731 (SEQ ID NO:61) as described below. These probes
span the .DELTA.M15 93 bp deletion present in DH5.alpha. DNA, but
not present in W3110 DNA. The number of PCR cycles was optimized so
amplification of the "heterozygote" target (1 .mu.L W3110 and 1
.mu.L DH5.alpha.) produced one-half the amount of DNA in each band
as did amplification of the "homozygote" target (2 .mu.L W3110 or 2
.mu.L DH5.alpha.) under the same amplification conditions.
[0450] PCR targets spanning the locus of interest were created in
duplicate as follows:
[0451] 2 .mu.L E. coli genomic DNA, W3110 or DH5.alpha. for
homozygote sample (50 ng); 1 .mu.L each W3110 and DH5.alpha. for
the heterozygote sample ; 1 .mu.L of W3110 or DH5.alpha. for the
LOH sample.
48 5 .mu.l 10X Taq buffer with 15 mM MgCl.sub.2 (Promega, M188A)
0.5 .mu.L Probe 10730 (50 pmol) 0.5 .mu.L Probe 10731 (50 pmol) 1
.mu.L 10 mM dNTPs 1 .mu.L Taq DNA Polymerase (Promega, M186A) 40
.mu.L water
[0452] PCR cycling parameters were 96.degree. C., 1 minute;
(94.degree. C., 15 seconds; 60.degree. C., 30 seconds; 72.degree.
C., 45 seconds).times.20; 72.degree. C., 45 seconds. The PCR
reaction was purified with 500 .mu.L Wizard.TM. PCR Purification
Resin (Promega, A7181) according to manufacturer instructions and
eluted with 25 .mu.L water.
[0453] The duplicate DNA targets (1 .mu.L) were then interrogated
in duplicate, with 1 .mu.g (200 pmol) probe 10732 (SEQ ID NO:62), a
sequence common to both W3110 and DH5.alpha.; 1 .mu.g (200 pmol)
probe 10733 (SEQ ID NO:63), a sequence completely matching only
W3110 DNA; and 1 .mu.g (200 pmol) probe 10734 (SEQ ID NO:64), a
sequence completely matching only DH5 DNA. Four microliters of the
interrogation reaction were combined with 100 .mu.l L/L reagent
(Promega, F202A) and the light output measured.
49 hetero- homozygotes zygotes LOH oligo W1 W2 D1 D2 W D W1 W2 D1
D2 alone No oligo 93 131 59 115 129 83 101 59 63 71 -- 10732 91 372
542 480 447 403 307 53 362 352 4 10732 95 465 536 494 479 419 295
257 364 349 4 10733 95 373 112 95 251 191 218 173 67 76 6 10733 86
353 108 88 245 187 204 158 77 68 6 10734 185 212 427 384 337 264
181 161 182 263 111 10734 179 199 409 378 318 258 159 127 182 282
111
[0454] The deletion-specific interrogation oligonucleotide (10734)
gave high background. In general these data show the utility of the
technology for determination of LOH. However, two samples, the
first W/W homozygote and first D LOH, give aberrant data for an
unknown reason.
50 10730 5' CACTTTATGCTTCCGGCTCGTATG 3' (lacZ) SEQ ID NO:60 10731
5' GGGATAGGTTACGTTGGTGTAGATGG (lacZ) SEQ ID NO:61 10732 5'
GTTGGGAAGGGCGATCGGTG 3' (common lac probe) SEQ ID NO:62 10733 5'
GGGATGTGCTGCAAGGCGATT 3' (wt lac probe) SEQ ID NO:63 10734 5'
GGATTCACTGGCCGTCGTGG 3' (deletion lac probe) SEQ ID NO:64
EXAMPLE 13
Interrogation For Loss of Heterozygosity--CMV
[0455] The use of an interrogation assay to determine loss of
heterozygosity with a synthetic cytomegalovirus (CMV) target is
demonstrated in this Example.
[0456] The CMV target was chosen because the interrogating probe
oligonucleotides (9211 (SEQ ID NO:71) and 9212 (SEQ ID NO:72) had
been previously used and well characterized. Oligonucleotides 10800
(SEQ ID NO:65) and 10801 (SEQ ID NO:66) were annealed to produce a
synthetic target, "A", representing a fragment of the CMV genome.
Likewise, oligonucleotides 10803 (SEQ ID NO:67) and 10805 (SEQ ID
NO:68) were annealed to produce a synthetic target, "G"
representing a fragment of the CMV genome. Targets A and G are
identical except at one nucleotide position where they have the
nucleotide resulting in their name. Both targets have SacI
overhangs.
[0457] The targets were cloned into the SacI restriction site of
pZERO-2 plasmid (Invitrogen) and transformed into TOP10 E. coli
cells (Invitrogen). The presence of the correct nucleotide sequence
in the A and G clones was confirmed by sequencing. However, the G
clone was found to contain an unintended mutation at the nucleotide
position three bases in from the 5' end of the region that anneals
to the interrogation probes. Because this mismatch is near the 5'
end of the interrogation probe annealing sequence, it should not
affect the interrogation results.
[0458] The following five target solutions were created with the A
and G clones:
[0459] 1. Hetero: 125 pg A and 125 pg G/microliter
[0460] 2. LOH A: 125 pg A and no G/microliter
[0461] 3. LOH G: no A and 125 pg G/microliter
[0462] 4. Mix Ag: 125 pg A and 62 pg G/microliter
[0463] 5. Mix Ga: 62 pg A and 125 pg G/microliter
[0464] These target solutions were PCR amplified with the JH67 (SEQ
ID NO:69) and 11077 (SEQ ID NO: 70) probes in the following
reaction:
[0465] 2 .mu.Target solution
[0466] 1 .mu.L Probes JH67 and 11077 (50 pmol each)
[0467] 1 .mu.L 10 mM dNTPs
[0468] 5 .mu.L 10.times. Taq buffer
[0469] 1 .mu.L Taq DNA polymerase
[0470] 40 .mu.L water
[0471] The PCR cycling parameters were: 96.degree. C., 1 minute;
(94.degree. C., 15 seconds; 60.degree. C., 30 seconds; 72.degree.
C., 45 seconds).times.15; 72.degree. C., 45 seconds. The entire PCR
reaction was then purified with 500 .mu.L Wizard.TM. PCR
purification resin (Promega, A7170) according to manufacturer's
instructions. The DNA was eluted with 30 .mu.L TE buffer. A
standard interrogation reaction with 6 .mu.L target and 1 .mu.g
interrogation probe, was performed with the exception that 2 units
of Klenow exo- were used per reaction. Four microliters of the
final reaction were combined with 100 .mu.L of L/L reagent and the
relative light units measured.
51 Oligo Heterozygote LOH A LOH G Mix Ag Mix Ga Alone No oligo 30
40 65 29 34 51 19 59 26 41 -- A oligo 279 340 74 329 27 27 258 309
50 164 5.2 308 372 76 339 20 26 351 330 83 167 5.2 G oligo 302 324
37 91 285 272 127 106 245 302 6.3 278 325 30 87 256 187 113 124 215
357 6.3 A:G ratio 1.01 1.10 2.26 3.76 0.09 0.11 2.54 2.78 0.29 0.50
G:A ratio 0.99 0.91 0.44 0.27 11.59 8.71 0.39 0.36 3.46 1.99
[0472] These data illustrate that LOH can be determined using this
method with appropriate interrogation probes.
52 10800 5'
CGTGTATGCCACTTTGATATTACACCCATGAACGTGCTCATCGACGTGAACCCGC- ACAACGAGCT
3' SEQ ID NO:65 10801 5'
CGTTGTGCGGGTTCACGTCGATGAGCACGTTCATGGGTGTAATATCAAAGTGGCATACACGAGCT
3' SEQ ID NO:66 10803 5' CGTGTATGCCACTTTGATATTACACCCGTGAACGTGCTC-
ATCGACGTGAACCCGCCAAACGAGCT 3' SEQ ID NO:67 10805 5'
CGTTGTGCGGGTTCACGTCGATGAGCACGTTCACGGGTGTAATATCAAAGTGGCATACACGAGCT
3' SEQ ID NO:68 JH67 5' TCACACAGGAAACAGCTATGACCATG 3' SEQ ID NO:69
11077 5' GCAAGGCGATTAAGTTGGGTAACG 3' (M13 forward probe) SEQ ID
NO:70 9211 5' CACTTTGATATTACACCCATG 3' SEQ ID NO:71 9212 5'
CACTTTGATATTACACCCGTG 3' SEQ ID NO:72
EXAMPLE 14
Multiplex Analysis of Congenital Adrenal Hyperplasia (CAH) Gene
[0473] Congenital adrenal hyperplasia (CAH) is a group of autosomal
recessive diseases resulting from a wide range of mutations in the
steroid 21-hydroxylase (CYP21) gene that contains 10 exons. There
is a high level of nucleic acid homology (98% in exons, 96% in
introns) between CYP21, the functional gene, and CYP21P, the
nonfunctional pseudogene. The many types of mutations in this gene
that can lead to disease include complete gene deletions, large
gene conversions, single point mutations, and a small 8 bp deletion
[See, White, et al., Hum. Mutat., 3:373-378, (1994)].
[0474] The majority of the CAH disease-causing mutations are
sequences present in the nonexpressed CYP21P pseudogene, and arise
in the CYP21 gene through recombination between CYP21P and CYP21.
Thus, one mutation detection strategy specifically detects the
CYP21 gene, and not the CYP21P pseudogene. The frequency of
disease-carrying alleles in the population is about 1 in 50.
[0475] The CAH target was interrogated for a variety of mutations
using Klenow exo- and yeast NDPK, and the results were compared to
a similar analysis using Tne triple mutant thermostable DNA
polymerase and a thermostable Pfu NDPK. Both wild type CAH PCR
products, mutant synthetic targets, and a pseudogene PCR product
amplified from the cloned CYP21P pseudogene were utilized as
targets in this assay. They are listed below.
[0476] Primer pairs used in PCR amplification and the resulting
products are as follows.
53 Size PCR Primers Segment Segment Amplified 10912 + 10909 1400 bp
5' end CYP21 11461 + 11480 918 bp 5' end CYP21 10910 + 11286 1492
bp 3' end CYP21 11535 + 11286 1496 bp 3' end CYP21 10912 + 10911
2680 bp pseudogene (CYP21P)
[0477] Synthetic targets and interrogation oligos utilized are
listed below.
[0478] PCR reactions were assembled to amplify regions of the CAH
gene with 4 different probe sets, using undigested human genomic
DNA (Promega, G3041) as target (25 ng per reaction). For
amplification of the pseudogene, human genomic DNA was predigested
with the restriction enzyme Bcl I, which specifically cleaves the
CYP21 gene upstream of the forward PCR probe, thus permitting only
amplification of CYP21P [Krone, Clinical Chem. 44(10):2075-2082
(1998)].
[0479] The 2680 bp PCR product was amplified from 50 ng of digested
DNA and subsequently cloned into the plasmid vector PGEM-T Easy
(Promega, A1380) following the manufacturer's protocol. A clone was
selected and sequenced (USB Sequenase kit, US70770) to confirm it
was indeed the pseudogene. The cloned CYP21P gene in the PGEM-T
Easy vector was used in subsequent amplifications to obtain pure
pseudogene PCR product for mutation interrogation analysis (100 pg
of plasmid per PCR reaction).
[0480] The use of thermostable enzymes to interrogate the CAH gene
has also permitted the interrogation of up to 6 multiple sites
within one reaction. The method used in this Example is
illustrative of routine studies carried out in screening
laboratories where usual results show the presence of an expected
gene (or the absence of a mutant gene) in almost all of the
samples, and only rarely shows the presence of a mutant gene. In
the case illustrated here, a qualitative result is provided from
which the exact mutation present can be determined in a subsequent
assay.
[0481] Thus, equal volumes of the CAH wild type (WT) 918 bp and
1496 bp PCR product) were combined (to thus span the entire CAH
gene) and interrogated either separately at each mutation site, or
as a multiplexed group. The discrimination ratio was good both in
the separate reactions for the combined PCR products, as well as
the multiplexed reaction. In addition, the multiplexed reaction
using the CAH wild type PCR products and either 6 wild type
interrogation oligo probes or 6 mutant interrogation oligo probes
was combined with an equimolar amount of synthetic target (mutant
synthetic target for each mutation site; 0.2 pmoles either PCR
product or synthetic target), to simulate a heterozygote
sample.
54 Tne/Pfu Tne/Pfu Tne/Pfu Probe Mutant NDPK, NDPK, NDPK for
Synthetic Target No WT Mutant Mutation Target DNA Oligo Oligo Oligo
Site Added CAH WT 172.7 553.0 180.2 1 918 bp + 1496 bp Same 172.7
535.7 184.0 2 Same 172.7 494.8 182.0 3 Same 172.7 486.7 148.7 4
Same 172.7 471.7 187.9 5 Same 172.7 317.5 179.7 6 Same 172.7 297.5
246.4 7 Same 523.7 1929.0 499.5 1, 2, 3, 4, 5 and 6 Same 506.0
1882.0 2234.0 1 1 Same 525.4 1848.0 1505.0 2 2 Same 535.9 1735.0
2877.0 3 3 Same 547.5 1880.0 4879.0 4 4 Same 552.4 2000.0 3864.0 5
5 Same 482.9 1938.0 2189.0 6 6 Same 514.5 1791.0 4192.0 2 + 4 2 + 4
Same 537.6 1752.0 3427.0 5 + 6 5 + 6
[0482] Because of the large size of the CAH gene and the large
number of different mutations that may be present, the use of the
thermostable enzymes, and thus the increased stringency of the
detection procedure, was found to be highly advantageous with this
complex target. Mutation sites that interrogated poorly using
Klenow exo- and yeast NDPK at 37.degree. C., were more successfully
interrogated when using the Tne triple mutant polymerase and Pfu
NDPK at elevated temperatures. In addition, use of the thermostable
enzymes permitted the multiplexing of numerous wild type or mutant
interrogation oligos in the same interrogation assay, to obtain the
rapid screening for mutations that may be present.
55 11143 5' CGGAGCCTCCACCTCCCG SEQ ID NO:73 CAH interrogator oligo
6 (wild type) for mutation site 1 11085 5' CACCCTCCAGCCCCCAGC 3'
SEQ ID NO:74 CAH interrogator oligo 2 (pseudogene/mutant) for
mutation site 2 11084 5' CGGAGCCTCCACCTCCTG 3' SEQ ID NO:75 CAH
interrogator oligo 1 (pseudogene/mutant) for mutation site 1 11086
5' CCTCACCTGCAGCATCAAC 3' SEQ ID NO:76 CAH interrogator oligo 3
(pseudogene/mutant) for mutation site 3 11144 5' CACCCTCCAGCCCCCAAC
3' SEQ ID NO:77 CAH interrogator oligo 7 (wild type) for mutation
site 2 11145 5' CCTCACCTGCAGCATCATC 3' SEQ ID NO:78 CAH
interrogator oligo 8 (wild type) for mutation site 3 11087 5'
CCTGGAAGGGCACTT 3' SEQ ID NO:79 CAH interrogator oligo 4
(pseudogene/mutant) for mutation site 4 11146 5' CCTGGAAGGGCACGT 3'
SEQ ID NO:80 CAH interrogator oligo 9 (wild type) for mutation site
4 11088 5' GATTCAGCAGCGACTGTA 3' SEQ ID NO:81 CAH interrogator
oligo 5 (pseudogene/mutant) for mutation site 5 11147 5'
GATTCAGCAGCGACTGCA 3' SEQ ID NO:82 CAH interrogator oligo 10 (wild
type) for mutation site 5 11287 5' CGAGGTGCTGCGCCTGCG 3' SEQ ID
NO:83 CAH interrogation oligo 11 (wild type) for mutation site 6
11288 5' CGAGGTGCTGCGCCTGTG 3' SEQ ID NO:84 CAH interrogation oligo
12 (pseudogene/mutant) for mutation site 6 11641 5'
GGGATCACATCGTGGAGATG 3' SEQ ID NO:85 CAH interrogation oligo 23
(wild type) for mutation site 7 11642 5' GGGATCACAACGAGGAGAAG 3'
SEQ ID NO:86 CAH interrogation oligo 24 (pseudogene/mutant) for
mutation site 7
EXAMPLE 15
Detection of Chromosomal DNA Without Amplification: I
[0483] In theory, direct detection of a single copy gene in
chromosomal DNA should be possible if enough DNA can be assayed.
The amount of human genomic DNA needed can be calculated as
follows: 1 ( 1 .times. 10 - 9 g D N A ) ( 5 .times. 10 9 bases /
genome ) ( 1 .times. 10 3 bases specific target ) = approximately 5
mg of D N A
[0484] However, as the amount of DNA interrogated increases,
nonspecific DNA signal from this DNA also increases. Therefore,
chromosomal DNA in amounts approaching even 1 .mu.g of DNA would
produce very high background.
[0485] Increasing the copies of target DNA per chromosome is one
way to overcome this limitation. Many such sequences are known. The
absolute sequence of the repeated DNA in different species can vary
as does the number of copies of the sequence in the genome. For
example, there are estimated to be 500-1000 copies of a sequence
known as the rep sequence in the E. coli chromosome. The Alu
sequence is present in the haploid human chromosome in
approximately 300,000 copies. The estimated amount of human
chromosomal DNA needed to detect the Alu sequence is: 2 5 .times.
10 - 3 grams DNA ( single copy gene requirement ) 3 .times. 10 5
copies per genome = 1.7 .times. 10 - 8 grams ( or about 17 ng of
DNA )
[0486] In this example, probes to two regions of the Alu sequence
(Alu 1 oligonucleotide 11597 (SEQ ID NO:87) and Alu 2
oligonucleotide 11598 (SEQ ID NO:88)) were used to demonstrate that
direct detection of chromosomal DNA is achievable.
[0487] The genomic DNA (4.2 .mu.g) was digested to completion (5
hours, 37.degree. C.) with 40 units of Sph I restriction enzyme,
which leaves a 3' overhang on the digested fragments. Either 40 ng
or 80 ng of the digested genomic DNA was annealed to 1.0 .mu.g of
the interrogation probes 11597 and 11598 in separate reactions, and
11597 and 11598 in the same reaction with water added to a final
volume of 20 .mu.L. A negative control, without an interrogation
probe, was also assembled. The solutions were heated at 92.degree.
C. for 3 minutes and cooled at room temperature for 15 minutes.
[0488] Twenty microliters of master mix, described below, were
added to each annealing reaction and the tubes were further
incubated at 37.degree. C. for 20 minutes, then stored on ice. Four
microliters of the reaction were added to 100 .mu.L of L/L reagent
(Promega F120B) in quadruplicate samples, and relative light units
(rlu) measured on a Turner.RTM. TD20/20 luminometer. The rlu
results are reported below.
56 Master Mix: 200 .mu.L 10X DNA Polymerase Buffer 25 .mu.L 40 mM
NaPPi 25 .mu.L Klenow exo- 10 .mu.L NDPK 1 U/.mu.L 20 .mu.L ADP 10
.mu.M 720 .mu.L water
[0489]
57 Rxn* 1 Rxn 2 Rxn 3 Rxn 4 average Net Std Dev* No DNA 3.372 3.342
3.306 3.249 3.317 0 0.052898 alu1 only 3.92 3.625 3.756 3.799 3.775
0.458 0.12174 alu2 only 23.18 25.47 24.58 25.19 24.61 21.29
1.020082 40 ng DNA 20.63 21.98 23.91 22.3 22.21 18.39 1.347504 alu1
+ 40 ng DNA 53.12 57.05 52.52 36.5 49.80 46.03 9.089798 alu2 + 40
ng DNA 99.23 91.26 55.9 85.59 83.00 58.39 18.90995 80 ng DNA 38.57
44.34 42.96 46.33 43.05 39.73 3.291454 alu1 + 80 ng DNA 89.25 68.01
91.43 96.14 86.21 82.44 12.46776 alu2 + 80 ng DNA 156.2 156.6 149.9
143.7 151.6 127.0 6.095353 alu1 + alu2 30.65 23.82 32.57 27.60
28.66 25.34 3.820881 alu1 + alu2 + 66.49 101.1 104.9 104.3 94.1975
65.81 18.54682 40 ng DNA *Std Dev = 1 standard deviation, Rxn =
reaction
[0490]
58 11597 5' AGACCCCATCTCTAA 3' (Alu 1) SEQ ID NO:87 11598 5'
GCCTGGGTGACAGAGCA 3' (A1u 2) SEQ ID NO:88
EXAMPLE 16
Detection of Chromosomal DNA Without Amplification: II
[0491] Another method to detect the presence of the Alu sequence is
to perform single probe extension reactions is described
herein.
[0492] This Example illustrates use of a different type of
oligonucleotide probe that is used to form a hairpin structure in
the interrogation technology of this invention. This study
demonstrates a method for eliminating the need for adding a probe
specific to the interrogation site to the interrogation
reaction.
[0493] Here, the oligonucleotide probe anneals to the target strand
downstream of (3' to) the interrogation position in the target
strand. The oligonucleotide has at its 5' end an unannealed region
of nucleotides followed by about 5 to about 20 nucleotides that are
identical to the interrogation region on the target strand. The
annealed 3' end of the oligonucleotide is then extended through the
interrogation position of the target strand creating what is
referred to as extended probe. The hybrid is denatured and a
hairpin structure formed between the extended probe strand and the
5' end of the oligonucleotide probe. This region is then assayed in
a standard interrogation reaction to determine if a mismatch is
present or not.
[0494] Four probes were designed to represent different types of
hairpin formations that an extended probe strands may assume. These
probes are 10207 (SEQ ID NO:177), 10208 (SEQ ID NO:178), 10209 (SEQ
ID NO:179), and 10212 (SEQ ID NO:180).
[0495] These probes are predicted to form the following
self-hybridized secondary structures when allowed to self-anneal:
2
[0496] Probes are designed to bind to a target Alu sequence and be
extended. Following extension, the probe can form a "hairpin"
structure--forming a stretch of double strand DNA. This DNA is then
detected in a "probeless" pyrophosphorylation assay. If the
extended probe sequence extends beyond the segment of the probe
designed to form one segment of the hairpin, the product is not
expected to be detected because the product has a 3' overhang. In
order to prevent such a situation, the probes that have been
designed can be used in reactions missing one of the four DNA
bases. By performing the reactions in this way, the probe is not
extended beyond the region of hybridization. Scheme 1 illustrates
how two such probes hybridize to an Alu sequence. 3
[0497] The predicted extended products of these probes and the
secondary structure of the hairpins that the extended products can
form are shown in Scheme 2, below. 4
59 Oligo A 5' CTCCAGCCTCGGTGACAGAGCAAGACCCTGTCTCAAAAAAAAAG-
CCTCGGTGCAGGGTCTTGCTCTGT 3' SEQ ID NO:93 Oligo B 5'
CTCCAGCCTGAGCAACACAGCAAGACCCTGTCTCAAAACAAAACGCCTGAGCCAGGGTCTTGCTGTGTT
3' SEQ ID NO:94
EXAMPLE 17
Speciation--Detection of Mitochondrial DNA Specific to Various
Animals
[0498] In this example, a segment of mitochondrial DNA comprising a
segment of the cytochrome B gene was amplified from a variety of
animal species using PCR primers 11590 (SEQ ID NO:95) and 11589
(SEQ ID NO:96) (PNAS 86:6196-6200). These PCR primers were diluted
in 10 mM Tris, pH 7.5, to a final concentration of 0.22
.mu.g/.mu.L. The genomic DNAs used were bovine (Clontech, 6850-1),
chicken (Clontech, 6852-1), dog (Clontech, 6950-1) and human
(Promega, G1521).
[0499] The PCR reactions were assembled to include 5 .mu.L
10.times. buffer with 15 mM MgCl.sub.2 (Promega, M188J), 1 .mu.L
dNTPs 10 mM (Promega, C144G), 2 .mu.L primer 11590, 2 .mu.L primer
11589, 0.5 .mu.L Taq polymerase 5U/.mu.L (Promega, M186E), and 38.5
.mu.L water. To each tube was then added 1 .mu.L (100 ng) of
genomic DNA. The PCR cycling parameters were (15 seconds,
94.degree. C.; 15 seconds, 55.degree. C.; 30 seconds, 72.degree.
C.).times.30. The size of PCR products was confirmed by running an
aliquot on an agarose gel and visualizing with ethidium bromide
(EtBr) staining. The PCR products were then separated from free
nucleotides (Promega, A7170) and an aliquot run on an agarose gel.
All samples produced a PCR product of the same size.
[0500] Each PCR DNA was then used in an assay to determine if it
could be specifically identified with a species-specific probe. One
microliter of interrogation probe (1 .mu.g/.mu.L) and 17 .mu.L
water were combined with 2 .mu.L of the appropriate PCR product and
heated at 91.degree. C. for 3 minutes, then cooled at room
temperature for 15 minutes. Twenty microliters of master mix
(described below) were added to each tube and each was further
incubated at 37.degree. C. for 15 minutes. Four microliters of the
solutions were then added to 100 .mu.L L/L reagent (Promega F120B),
and the relative light output (rlu) measured on a Turner.RTM.
TD20/20 luminometer. The rlu average values from two reactions,
minus the DNA background values, along with the standard deviation
values are listed below.
60 Master mix: 312 .mu.L 10X DNA pol buffer (Promega M195A) 39
.mu.L NaPPi 40 mM (Promega E350B) 39 .mu.L Klenow exo minus
(Promega M128B) 15.6 .mu.L NDPK 1 U/.mu.L 31.2 .mu.L ADP 10 .mu.M
(Sigma) 1123 .mu.L water (Promega AA399)
[0501]
61 Averages from 2 reactions. Net light units are calculated by
subtracting the DNA background Standard Deviations No Human Chicken
Cow Dog No Human Chicken Cow Dog Probe DNA DNA DNA DNA DNA DNA DNA
DNA DNA DNA comzoo -0.096 44.5 14.25 119.7 124.6 0.654 7.000 23.33
3.465 8.63 huzoo1 1.771 38 -40 -51.85 -63.55 0.137 38.96 31.74
6.505 12.52 huzoo2 -0.889 101.6 -23.35 -0.05 -48.75 0.761 3.959
0.141 8.768 2.19 chzoo1 43.07 -30.4 34.05 -2.75 -31.15 7.078 1.909
6.364 2.687 8.70 chzoo2 -0.361 57.6 50.7 33.05 -3.25 0.075 43.77
29.34 12.59 21.43 cozoo2 1.925 90.95 125.1 202.6 132.5 0.208 20.08
13.22 8.627 19.30 dozoo2 0.966 71.8 158.7 0.180 9.546 1.98
[0502] The data demonstrate that the primers detect the
mitochondrial PCR product. Both of the human-specific probes (11576
(SEQ ID NO:97) and 11583 (SEQ ID NO:98)) were shown to be specific
for human mitochondrial DNA. The common probe, 11582 (SEQ ID
NO:99), detected all of the species, but was less efficient with
chicken DNA. The chicken-specific probe, 11577 (SEQ ID NO:100), was
specific for chicken mitochondrial DNA, but the other
chicken-specific probe, 11584 (SEQ ID NO:101), detected all the
species except dog. The cow-specific probe, 11588 (SEQ ID NO:102),
gave the best detection signal for cow DNA, but also detected the
other species. The dog-specific probe, 11586 (SEQ ID NO:103), was
assayed only with dog and cow DNA, but detected the dog DNA better
than cow DNA. A cleaner PCR product provides DNA with less
background.
62 11590 zooamp2 5' AAACTGCAGCCCCTCAGAA SEQ ID NO:95
TGATATTTGTCCTCA 3' 11589 zooamp1 5' AAAAAGCTTCCATCCAACA SEQ ID
NO:96 TCTCAGCATGATGAAA 3' 11576 huzoo1 5' CCAGACGCCTCA 3' SEQ ID
NO:97 11583 huzoo2 5' ACCTTCACGCCA 3' SEQ ID NO:98 11582 comzoo 5'
TGCCGAGACGT 3' SEQ ID NO:99 11577 chzoo1 5' GCAGACACATCC 3' SEQ ID
NO:100 11584 chzoo2 5' GGAATCTCCACG 3' SEQ ID NO:101 11588 cozoo2
5' ACATACACGCAA 3' SEQ ID NO:102 11586 dozoo2 5' ATATGCACGCAA 3'
SEQ ID NO:103
EXAMPLE 18
Trisomy Detection
[0503] Detection of a simulated trisomy sample is demonstrated in
this Example. The nucleic acid probes and targets were previously
described in Example 13. These include the CMV probes 9211 (SEQ ID
NO:71) and 9212 (SEQ ID NO:72), the p-ZERO-2 clone of the
double-stranded synthetic CMV wild type target CMV-A (10800 (SEQ ID
NO:65) and 10801 (SEQ ID NO:66)), and the p-ZERO-2 clone of the
double-stranded synthetic CMV mutant target CMV-G (10803 (SEQ ID
NO:67) and 10805 (SEQ ID NO:68)).
[0504] Each p-ZERO-2 plasmid (1 .mu.g) was digested to completion
with Pst I restriction enzyme at 37.degree. C. for 1 hour. Ten
microliters of the digest were then further diluted with 20 .mu.L
of water.
[0505] The following Master Mix was assembled.
63 60 .mu.l 10X DNA polymerase buffer (Promega M195A) 7.5 .mu.l 40
mM NaPPi (Promega C113) 1.5 .mu.l 10 U/.mu.L Klenow exo- (Promega
M128A) 3 .mu.L NDPK 6 .mu.L 10 .mu.M ADP (Promega A5285) 225 .mu.L
water (Promega AA399)
[0506] The following solutions were assembled using the digested
and diluted templates.
64 Simulated Solution Template Probe (.PI.L) Water phenotype rlu 1
1.PI.L CMV-A 1.PI.L 9211 18 homoZ* A 53.27 2 1.PI.L CMV-A 1.PI.L
9212 18 homoZ A 7.19 3 1.PI.L CMV-G 1.PI.L 9211 18 homoZ G 5.78 4
1.PI.L CMV-G 1.PI.L 9212 18 homoZ G 63.84 5 1.PI.L CMV-A 1.PI.L
9211 17 heteroZ* 1:1 54.08 1.PI.L CMV-G 6 1.PI.L CMV-A 1.PI.L 9212
17 heteroZ 1:1 61.54 1.PI.L CMV-G 7 2.PI.L CMV-A 1.PI.L 9211 16
trisomy 90.87 1.PI.L CMV-G 2:1 (A:G) 8 2.PI.L CMV-A 1.PI.9212 16
trisomy 64.74 1.PI.L CMV-G 2:1 (A:G) 9 1.PI.L CMV-A 1.PI.L 9211 16
trisomy 50.86 2.PI.L CMV-G 1:2 (A:G) 10 1.PI.L CMV-A 1.PI.L 9212 16
trisomy 111.0 2.PI.L CMV-G 1:2 (A:G) * homoZ = homozygous; heteroZ
= heterozygous.
[0507] The solutions were heated at 95.degree. C. for 3 minutes
then cooled for 10 minutes at room temperature. Then, 20 .mu.l
master mix were added, and the solutions further heated at
37.degree. C. for 15 minutes. Four microliters of the solutions
were then added to 100 .mu.L of L/L reagent (Promega F202A) and the
relative light output measured immediately on a Turner.RTM. TD20/20
luminometer. The rlu values are listed above.
[0508] The rlu values demonstrate that the 1:2 (A:G) template mix
exhibits a 1:2 rlu ratio, whereas the heterozygous 1:1 A:G rlu
ratio is close to 1:1. A contemplated method is thus shown to be
useful in detecting trisomy.
[0509] CMV Interrogation Oligos
65 Wild Type CMV-A Probe: (9211) 5' CACTTTGATATTACACCCATG 3' SEQ ID
NO:71 Mutant CMV-G probe: (9212) 5' CACTTTGATATTACACCCGTG 3' SEQ ID
NO:72 10800 5' CGTGTATGCCACTTTG
ATATTACACCCATGAACGTGCTCATCGACGTGAACCCGCA CAACGAGCT 3' SEQ ID NO:65
10801 5' CGTTGTGCGGGTTCAC GTCGATGAGCACGTTCATGGGTGTAATATCAAAGTGGCAT
ACACGAGCT 3' SEQ ID NO:66 10803 5' CGTGTATGCCACTTTG
ATATTACACCCGTGAACGTGCTCATCGACGTGAACCCG- CA CAACGAGCT 3' SEQ ID
NO:67 10805 5' CGTTGTGCGGGTTCAC
GTCGATGAGCACGTTCACGGGTGTAATATCAAAGTGGCAT ACACGAGCT 3' SEQ ID
NO:68
EXAMPLE 19
Analysis of SNP Heterozygosity Level in DNA Isolated from Plant
Materials
[0510] Eight different rice DNA samples, with varying amounts of
two alleles differing at an SNP site, were analyzed to determine
the ability of pyrophosphorylation to detect the degree of
heterozygosity in a plant sample. The DNA genotypes (G and T) are
described in Example 7.
[0511] Eight coded heterozygous rice DNA samples and two homozygous
rice DNA samples were obtained (Texas A&M, Crop Biotechnology
Center) and PCR amplified with primers RS1 (SEQ ID NO:24) and RS2
(SEQ ID NO:25) as described in Example 7. The resulting PCR
products were then treated with T7 Exonuclease 6 and purified as
described in Example 7. The resulting DNA was interrogated by
combining 4 .mu.L of the PCR product with 150 pmoles of
interrogation oligonucleotide and water to a final volume of 20
.mu.L. This solution was incubated at 95.degree. C. for 2 minutes,
then at 37.degree. C. for 10 minutes. Interrogation
oligonucleotides used were RS3 (SEQ ID NO:26), RS4 (SEQ ID NO:27),
and none. Twenty microliters of master mix were then added and the
solution further incubated at 37.degree. C. for 15 minutes. These
solutions were then combined with 100 .mu.L of L/L reagent
(Promega, F202A) and light output measured in a Berthold Eg&G
microlumat plus luminometer. The relative light units (rlu) were
corrected for no oligonucleotide background and are listed
below:
66 G (RS3) T (RS4) Sample Allele Allele % G % T 1 100,366 119,046
45.7 54.3 2 83,428 163,241 33.8 66.2 3 90,309 90,628 49.9 50.1 4
168,074 35,835 82.4 17.6 5 173,422 31,403 84.7 15.3 6 166,516
13,692 92.4 7.6 7 171,933 17,384 90.8 9.2 8 103,047 1,724 98.4
1.6
[0512] These G:T ratios were further confirmed by the following
study. The eight heterozygote samples and the two homozygote
samples were re-amplified as previously described, but the PCR
reaction also included 1 .mu.L of .sup.32PdATP and .sup.32PdCTP.
Ten microliters of the resulting PCR product were then digested
with 1 .mu.L restriction endonuclease AccI in 2.5 mM MgCl.sub.2 in
the PCR buffer for one hour at 37.degree. C. AccI cuts the G allele
PCR product into a 120 bp doublet, but will not cut the 240 bp
fragment from the T allele PCR product. The digest was run on a 10%
acrylamide TBE gel, dried for one hour at 65.degree. C. and
resulting bands quantified for one hour on a
[0513] Molecular Dynamics Fluoroimager screen. The following values
were obtained.
67 Sample % G % T 1 47.6 52.4 2 35.8 64.2 3 57.2 42.8 4 77.6 22.3 5
81.1 18.9 6 89.2 10.8 7 85.4 14.6 8 nd nd
[0514] No values were generated for sample 8 because the PCR
reaction was not successful. The correlation coefficient between
the two data sets was 0.992036.
68 RS1 5' CCCAACACCTTACAGAAATTAGC 3' SEQ ID NO:24 RS2 5'
TCTCAAGACACAAATAACTGCAG 3' SEQ ID NO:25 RS3 5' AGAACATCTGCAAGG 3'
SEQ ID NO:26 RS4 5' AGAACATCTGCAAGT 3' SEQ ID NO:27
EXAMPLE 20
Interrogation using Fluorescent Labels
[0515] This example demonstrates that nucleotides released from the
3'-terminus of a probe hybridized to a target nucleic acid of
interest by a process of the invention can be detected by mass
spectrometry or by fluorimetric HPLC and thereby provide evidence
of the presence or absence of the target nucleic acid in a nucleic
acid sample or of a specific base at an interrogation position of
the target.
[0516] The interrogation probe is designed to have a fluorescent
label attached to the 5'-terminal nucleotide. Fluorescent tags,
such as fluorescein or rhodamine, can be incorporated into the
probe during synthesis with a fluorescent molecule attached to the
phosphoramadite nucleotide present at the 5'-end of the
oligonucleotide that will be used as a probe (Glen Research).
[0517] Probes PT5 (SEQ ID NO:104) and PT6 (SEQ ID NO:105) are used
to PCR-amplify a region of human genomic DNA spanning about 500
base pairs encoding the prothrombin gene. Probe PT5 has
phosphorothioate linkages between the first five bases at the 5'
end The PCR cycling parameters were as follows: 94.degree. C., 2
minutes; (94.degree. C., 30 seconds; 600C, 1 minutes; 700C, 1
minutes).times.40; 70.degree. C., 5 minutes. Fifty units of T7 gene
6 Exonuclease (USB Amersham) were added to 25 .mu.L of the PCR
reaction and the solution was incubated for 30 minutes at
37.degree. C. Magnetic silica (Promega, A1330) was used to remove
free nucleotides from the solution and the remaining DNA was eluted
with 100 .mu.L of water. The PCR product is treated with T7 gene 6
Exonuclease (USB Amersham) and purified from free nucleotides as
described above.
[0518] The prothrombin interrogation probes are 11265 (SEQ ID
NO:106), that is totally complementary to a segment of the mutant
prothrombin sequence, and 11266 (SEQ ID NO:107), that is totally
complementary to a segment of the wild-type prothrombin sequence.
Each of these probes has a destabilizing mutation eight bases from
the 3' end. And each of these probes has a label at its 5'-end,
incorporated during synthesis of the probe as described above.
[0519] The purified PCR product is interrogated in separate
reactions with each of the two interrogation probes (wild-type and
mutant). Interrogation reactions, with the target molecule in molar
excess over the probe molecule, for each of the interrogation
probes are assembled as follows: 40 .mu.L PCR product 15 pmol
Interrogation oligo water is added to a final volume of 50
.mu.L.
[0520] The reactions are incubated at 95.degree. C. for 3 minutes
and then at 37.degree. C. for 15 minutes.
[0521] Fifty microliters of master mix are then added. The
composition of the master mix containing Klenow exo- is described
in Example 1 with the exception that there is no ADP and no NDPK.
The reaction then proceeds at 37.degree. C. for 15 minutes. The
hybrid is then denatured by incubating the reaction at 95.degree.
C. for 3 minutes, adding 100 .mu.L water to dilute the separated
strands and placing the resulting denatured solution tube on
ice.
[0522] The solutions are then split in half and analyzed using two
different methods. In one method, the size of the labeled probe in
the solutions is analyzed by silicon desorption ionization mass
spectroscopy (Wei, J. et al. Nature. 399:243-246, 1999). This
method is sensitive to femtomole and attomole levels of analyte.
The samples are prepared for spectrometry as described in that
paper. Essentially, analytes are dissolved in a deionized
water/methanol mixture (1:1) at concentrations typically ranging
from 0.001 to 10.0 .mu.M. Aliquots (at least 0.5 to 1.0 .mu.L,
corresponding to at least 0.5 femtomol to 100 picomol analyte) of
solution are deposited onto the porous surfaces and allowed to dry
before mass spectrometry analysis.
[0523] These studies are performed on a Voyager DE-STR,
time-of-flight mass spectrometer (PerSeptive Biosystems) using a
pulsed nitrogen laser (Laser Science) operated at 337 nm. Once
formed, ions are accelerated into the time-of-flight mass analyzer
with a voltage of 20 kV. Other liquid chromatography-mass
spectrometry (LC-MS) instrumentation may also be used for analysis
(Niessen W. J. Chromatog. A 794:407-435 (1998) In a second method,
the size of the denatured labeled probe strand in the solution is
analyzed by HPLC using a fluorescence detector as described in
Jain, et al. Biochem. Biophys. Res. Commun. 200:1239-1244 (1994) or
Levitt, B. et al. Anal. Biochem. 137:93-100 (1984). The size of the
denatured labeled probe strand is confirmed on an ABI 377.
[0524] The size of the labeled probe strand present in the
denatured solution indicates whether or not a nucleotide was
released from the 3'-terminus of the probe, and therefore whether a
match or mismatch base pair existed at the 3' terminus of the
probe/template hybrid. For the denatured solution containing
wild-type probe, the observance of a labeled probe that is shorter
than the length of the original probe indicates that there is a
matched base at the 3'-terminus of at least one allele in the
original sample and therefore, that at least one allele in the
original sample is wild-type. For the denatured solution containing
mutant probe, the observance of a labeled probe that is shorter
than the length of the original probe indicates that there is a
matched base at the 3'-terminus of at least one allele in the
original sample and therefore, that at least one allele in the
original sample is wild-type. In both cases, the analytical output
can be quantified to determine whether the genotype is homozygous
or heterozygous at that locus.
69 PT5 5' ATAGCACTGGGAGCATTGAGGC 3' SEQ ID NO:104 PT6 5'
GCACAGACGGCTGTTCTCTT 3' SEQ ID NO:105 11265 5' GTGATTCTCAGCA 3' SEQ
ID NO:1O6 11266 5' GTGATTCTCAGCG 3' SEQ ID NO:107
EXAMPLE 21
Determination of the Minimum Cell Population That Can be Detected
in a Complex Cell Mixture
[0525] This example was designed to illustrate that a small cell
population can be detected in a background of other cells using a
contemplated process. Thus, RNA was isolated from a cell mixture
that contained increasing numbers of K562 cells (ATCC CCL 243) in a
constant background of 1.times.10.sup.6 NIH-3T3 cells (ATCC CRL
1658). The RNA was then used for RT-PCR of the bcr/abl
translocation transcript, which is specific to the K562 cells due
to chromosomal translocation. A translocation takes place in the
region of the bcr gene along with involvement of a segment of the
abl gene. The NIH-3T3 cells possess no translocation and are thus
wild type for the bcr and abl transcripts.
[0526] NIH-3T3 cells were washed with phosphate-buffered saline and
centrifuged at 2000.times. g for 5 minutes to pellet the cells. The
supernatant was removed and the cell pellet was resuspended in SV
RNA lysis buffer (Promega Corp., #Z3100) to a final concentration
of 1.times.10.sup.6 cells/175 .mu.L of lysis buffer. Similarly,
K562 cells were washed in phosphate-buffered saline and centrifuged
at 2000.times. g for 5 minutes to pellet the cells. The supernatant
was removed and the cell pellet was resuspended in SV RNA lysis
buffer to a final concentration of 1.times.10.sup.6 cells in 175
.mu.L of lysis buffer. The cell lysates were passed through an
18-gauge needle to shear the genomic DNA.
[0527] The cell lysates were combined as listed below, and SV lysis
buffer was added to a final volume of 350 .mu.L. Sample 2 used a
1:1000 dilution of the K562 lysate, samples 3, 4, and 5 used a
1:100 dilution of the K562 lysate, and samples 6 through 10 used
undiluted lysate.
70 Number Volume (.PI.L) Number Volume Sample NIH-3T3 NIH-3T3 K562
(.PI.L) K562 1 1 .times. 10.sup.6 175 0 0 2 1 .times. 10.sup.6 175
10 1.75 3 1 .times. 10.sup.6 175 100 1.75 4 1 .times. 10.sup.6 175
500 8.75 5 1 .times. 10.sup.6 175 1000 17.5 6 1 .times. 10.sup.6
175 1 .times. 10.sup.4 1.75 7 1 .times. 10.sup.6 175 5 .times.
10.sup.4 8.75 8 1 .times. 10.sup.6 175 1 .times. 10.sup.5 17.5 9 1
.times. 10.sup.6 175 5 .times. 10.sup.5 87.5 10 1 .times. 10.sup.6
175 1 .times. 10.sup.6 175
[0528] Total RNA was then isolated from the combined lysates listed
above using the SV Total RNA Isolation System according to
manufacturer's instructions (Promega, Z3100). The RNAs were each
eluted into 100 .mu.L water and stored at -20.degree. C. until
used.
[0529] The resulting RNA concentration of each sample was
determined by UV spectroscopy of 10 .mu.L of the appropriate sample
mixed with 300 .mu.L water. In set A, the samples were a
combination of NIH-3T3 cells and K562 cells as listed above. In set
B, the samples were only K562 cells in the number and volume as
listed above. The spectroscopy results are listed below. Absorbance
at OD260 measures the amount of DNA, the absorbance at OD280
measures the amount of protein, and 260/280 ratio determines the
purity of the RNA with a high purity sample having a 260/280 ratio
of about 2.0. concentration
71 concentration Sample OD260 OD280 260/280 (ng/mL) 1A 0.104 0.051
2.04 124 2A 0.103 0.047 2.19 124 3A 0.113 0.055 2.05 135 4A 0.088
0.042 2.09 106 5A 0.110 0.056 1.96 132 6A 0.112 0.054 2.07 134 7A
0.091 0.043 2.12 109 8A 0.103 0.050 2.06 124 9A 0.124 0.057 2.17
249 10A 0.162 0.078 2.07 194 1B 0.008 0.006 1.33 na* 2B 0.005 0.004
1.25 na 3B 0.002 0.004 0.50 na 4B 0.001 0.003 0.33 na 5B 0.001
0.001 1.00 na 6B 0.002 0.002 1.00 na 7B 0.002 0.003 0.67 na 8B
0.001 0.000 na na 9B 0.037 0.018 2.05 44 10B 0.067 0.303 2.23 80
*na = too low to be accurately measured.
[0530] The very low values for 1B through 8B indicate the lack of
sensitivity for spectroscopy at very low nucleic acid
concentrations as well known in the art.
[0531] RT-PCR reactions for bcr-abl product, which is K562 specific
due to the chromosomal translocation, were set up using the RT-PCR
system (Promega, A1250) according to manufacturer's instructions
with 5 .mu.L total RNA and the amplification primers 9261 and 10860
(below) to provide a 200 base pair RT-PCR product. The 10860
oligonucleotide has three phosphorothioate linkages at the 5'
terminus. The RT-PCR cycling parameters were 48.degree. C. 45
minutes, 95.degree. C. 2 minutes, 40.times.(94.degree. C. 30
seconds, 65.degree. C. 1 minute, 68.degree. C. 1 minute),
68.degree. C. 7 minutes, 4.degree. C. soak.
72 9261 5' GGAGCTGCAGATGCTGACCAAC 3'. SEQ ID NO:108 amplification
primer 10860 5' GCTACTGGCCGCTGAAGGGC 3' SEQ ID NO:109 amplification
primer 11065 5' GCTGACCATCAATAAGGAAG 3' SEQ ID NO:110 interrogation
primer
[0532] Five microliters of the resulting amplification products
were run on a 1.5% agarose gel. The 200 bp bcr/abl band was
detectable in samples 3B through 10B and 3A through 10A. Therefore,
the bcr/abl translocation using the method described above, was
easily detected in 100 K562 cells in a background of
1.times.10.sup.6 NIH-3T3 cells.
[0533] In a separate set of reactions, the products of the RT-PCR
reaction (25 .mu.L) were treated with 0.5 units of T7 gene 6
exonuclease for 30 minutes at 37.degree. C. Samples were then
purified using MagneSil.TM. magnetic silica particles.
[0534] Thus, twenty-five microliters of PCR product was combined
with 1.5 mg of particles in 150 .mu.L of binding buffer (0.4 M
guanidine thiocyanate+0.08 M potassium acetate). The compositions
were incubated for 2 minutes at room temperature. The particles
were captured by placing the tube on a magnetic stand and the
supernatant was removed. The particles were washed four times with
150 .mu.L of 70% ethanol. The particles were resuspended in 50
.mu.L of water and incubated for 2 minutes at room temperature.
Then, 150 .mu.L of binding buffer (no particles) was added and the
tube incubated for 2 minutes at room temperature. The particles
were captured and washed three times with 150 .mu.L of 70% ethanol.
The final wash was removed and the particles were permitted to air
dry for 10 minutes at room temperature. The particles were then
resuspended in 100 .mu.L of water and incubated for 2 minutes at
room temperature. The particles were captured and the supernatant
containing the purified PCR product was transferred to a clean
tube. When 5 .mu.L of each purified RT-PCR product were used for an
interrogation assay, a graph of the statistical data indicated that
10 K562 cells could be detected because the -4 sigma value of the
10-cell interrogation assay does not intersect with the +4 sigma
value for the 0 cell control interrogation reaction. Thus, using
this technique, 0.001% of a mixed cell population could be
detected.
[0535] The following master mix was assembled:
73 10X buffer (Promega, M190) 20 .mu.L 25 mM MgCl.sub.2 20 .mu.L
water 51 .mu.L 40 mM NaPPi 5 .mu.L 10 uM ADP 2 .mu.L 5 U/.mu.L Tne
triple mutant 1 .mu.L polymerase (1 U/reaction) 0.5 U/.mu.L Pfu
NDPK 1 .mu.L (0.1 U/reaction)
[0536] The sample (5 .mu.L) and the 11065 interrogation oligo (1
.mu.g) were combined in water to a final volume of 20 .mu.L. They
were heated to 95.degree. C. for 3 minutes, then incubated for 10
minutes at 60.degree. C. Then 20 .mu.L master mix was added and the
reaction incubated for 15 minutes at 60.degree. C. Then 100 .mu.L
of L/L were added, and the light output (average rlu) was measured
on a Turner.RTM. TD 20/20 luminometer. The bachground RLU was
subtracted and averaged results from 3 readings are listed
below.
74 K562 cell NIH-3T3 bcr/abl Detection number cell number Average
rlu Sigma 0 1 .times. 10.sup.6 82.26 0.47 10 1 .times. 10.sup.6
162.43 9.39 100 1 .times. 10.sup.6 793.23 34.43 500 1 .times.
10.sup.6 1512.72 86.33 1000 1 .times. 10.sup.6 2042.83 116.02 10000
1 .times. 10.sup.6 2792.50 68.42
EXAMPLE 22
Automation of PCR Clean-up and ATP Measurement Steps of an
Interrogation Reaction
[0537] The ability to automate the PCR clean-up and the ATP
measurement steps of an interrogation reaction are demonstrated in
this example. The robot is also capable of automating the addition
of the interrogation master mix and permitting for the incubation
time prior to the luciferase reaction; however, that is not
demonstrated in this particular example.
[0538] The PCR procedure detailed in Example 7, using the same PCR
amplification primers, the same SNP interrogation primers and the
same amplification profile, is used here to determine the genotype
of 189 rice genomic DNAs at a known SNP site. After the PCR
reactions were completed, 25 .mu.L of each of the 50 .mu.L PCR
reactions was transferred to a well of a Dynex.TM. V-bottom 96-well
plate and placed on a Biomek.TM. 2000 robot (Beckman). T7
Exonuclease 6 (1 .mu.L) was added to each well and the plate
incubated at 37.degree. C. for 30 minutes. The robot was programmed
and completed the following steps as listed below:
[0539] (1) The robot mixed magnetic silica particles (Promega) in a
guanidine thiocyanate solution by pipetting up and down in a
trough.
[0540] (2) 180 .mu.L of the particles were drawn into a set of 8
micropipette tips and transferred to column one of the plate. This
same set of 8 tips transferred 180 .mu.L of the particles to each
column of the plate.
[0541] (3) Separate tips were used to mix the contents of the wells
by pipetting up and down four times. In this step, a tip unique for
every well was used.
[0542] (4) The robot moved the plate to a station containing a
magnetic pin array where the particles were captured on the side of
the well and the supernatant removed as waste and placed in a
trough.
[0543] (5) The robot performed three 70% ethanol washes, using a
new set of tips - a unique tip for every well.
[0544] (6) After removal of the last wash solution, the robot
gripper arm then moved the plate off the magnet to a separate
station where it added 50 .mu.L water to each well.
[0545] (7) The water was mixed with the particles by pipetting up
and down and then 150 .mu.L 0.4 M guanidine thiocyanate, 0.08 M
potassium acetate were added to each well. One row of 8 tips was
used to dispense the solution, whereas a unique tip for each well
was used to mix the contents of the well.
[0546] (8) The robot moved the plate back to the magnetic pin
array.
[0547] (9) The contents of the wells were mixed by pipetting up and
down, the particles were captured on the wall of the well and the
supernatant was removed to the waste trough.
[0548] (10) Three rounds of 100 .mu.L 70% ethanol washes were
performed.
[0549] (11) After the final wash was removed, the particles were
permitted to air dry for 10 minutes.
[0550] (12) The gripper arm of the robot then removed the plate
from the magnet to a separate stand.
[0551] (13) 100 .mu.L water were added to each well and the
contents of the well mixed with a tip unique to each well, by
pipetting up and down.
[0552] (14) The plate was put on the magnet and the water
containing the DNA, separated from the particles, was transferred
to a clean plate.
[0553] At this point a multichannel pipetter was used to manually
transfer 10 .mu.L of the contents of each well to a luminometer 96
well plate and the interrogation oligo (150 pmoles in 10 .mu.L) was
added to each well. Then, 10 .mu.L of 0.6 N NaOH were added to each
well and the plate incubated for 5 minutes at room temperature to
denature the double stranded DNA. Then 10 .mu.L of 0.1 M Tris pH
7.3 were added to neutralize the solution and permit the
interrogation oligonucleotide to anneal to the template DNA, if it
were capable of annealing The plate was transferred to a
Berthold.TM. luminometer and warmed to 37.degree. C. for 5 minutes.
The luminometer then automatically added 100 .mu.L of L/L reagent.
The data were transferred to the Excel.RTM. spreadsheet for
analysis. The data are documented below. Of a total of 192 samples,
189 samples were able to be distinguished as homozygote wild type
or mutant, or heterozygotes by the use of this method.
75 Total Samples Called: 189 WT Homozygotes 108 Mutant Homozygotes
74 Heterozygotes 7 No Calls 3 % Samples Called 97.4 Average WT RLUs
289791.96 Average mutant RLUs 294958.30 Control Values: Control WT
Interrogation 1100 rlu Control Mutant Interrogation 1300 rlu
[0554]
76 RS1 5' C*C*C*AACACCTTACAGAAATTAGC 3' SEQ ID NO:24 (* signifies
the presence of a phosphorothioate linkage between the indicated
bases.) RS2 5' TCTCAAGACACAAATAACTGCAG 3' SEQ ID NO:25 RS3 5'
AGAACATCTGCAAGG 3' SEQ ID NO:26 RS4 5' AGAACATCTGCAAGT 3' SEQ ID
NO:27
[0555]
77 WT Mutant No Adj. WT Adj. Mutant I.D. Monoplex Monoplex Interr.
Average Average Ratio Call A1 199749 507274 185645 14104.00
321629.00 0.0420 Mutant Homozygote A2 458265 95890 92591 365674.00
3299.00 0.9911 WT Homozygote A3 759556 161615 160051 599505.00
1564.00 0.9974 WT Homozygote A4 498118 351711 138786 359332.00
212925.00 0.6279 Heterozygote A5 195485 53873 86935 108550.00
-33062.00 1.4380 WT Homozygote A6 49647 244903 46486 3161.00
198417.00 0.0157 Mutant Homozygote A7 71513 307323 59663 11850.00
247660.00 0.0457 Mutant Homozygote A8 322849 80613 63968 258881.00
16645.00 0.9396 WT Homozygote A9 277232 52362 52128 225104.00
234.00 0.9990 WT Homozygote A10 54809 293085 44761 10048.00
248324.00 0.0389 Mutant Homozygote A11 302564 43711 43365 259199.00
346.00 0.9987 WT Homozygote A12 271769 57301 45478 226291.00
11823.00 0.9503 WT Homozygote B1 294153 949381 289250 4903.00
660131.00 0.0074 Mutant Homozygote B2 141633 575781 137968 3665.00
437813.00 0.0083 Mutant Homozygote B3 713771 571635 128947
584824.00 442688.00 0.5692 Heterozygote B4 184624 98392 98706
85918.00 -314.00 1.0037 WT Homozygote B5 293297 53877 49345
243952.00 4532.00 0.9818 WT Homozygote B6 232345 34581 29098
203247.00 5483.00 0.9737 WT Homozygote B7 325601 142317 138329
187272.00 3988.00 0.9791 WT Homozygote B8 62215 281539 48648
13567.00 232891.00 0.0550 Mutant Homozygote B9 328210 67103 65440
262770.00 1663.00 0.9937 WT Homozygote B10 270590 74764 72931
197659.00 1833.00 0.9908 WT Homozygote B11 317326 54672 54072
263254.00 600.00 0.9977 WT Homozygote B12 343235 71537 64609
278626.00 6928.00 0.9757 WT Homozygote C1 756526 164558 161686
594840.00 2872.00 0.9952 WT Homozygote C2 567636 133446 121643
445993.00 11803.00 0.9742 WT Homozygote C3 659442 283021 149965
509477.00 133056.00 0.7929 WT Homozygote C4 165142 652352 139950
25192.00 512402.00 0.0469 Mutant Homozygote C5 260593 48904 46766
213827.00 2138.00 0.9901 WT Homozygote C6 70456 332594 66055
4401.00 266539.00 0.0162 Mutant Homozygote C7 268615 55585 53627
214988.00 1958.00 0.9910 WT Homozygote C8 265788 52417 50841
214947.00 1576.00 0.9927 WT Homozygote C9 218246 65177 60855
157391.00 4322.00 0.9733 WT Homozygote C10 31062 30995 28923
2139.00 2072.00 0.5080 Signal/Bkg Low C11 407704 65466 62189
345515.00 3277.00 0.9906 WT Homozygote C12 82010 351069 56481
25529.00 294588.00 0.0797 Mutant Homozygote D1 653788 366405 360959
292829.00 5446.00 0.9817 WT Homozygote D2 184581 681894 176450
8131.00 505444.00 0.0158 Mutant Homozygote D3 764581 327686 217446
547135.00 110240.00 0.8323 WT Homozygote D4 706754 216238 179087
527667.00 37151.00 0.9342 WT Homozygote D5 69660 327874 67779
1881.00 260095.00 0.0072 Mutant Homozygote D6 56895 303900 50699
6196.00 253201.00 0.0239 Mutant Homozygote D7 373792 82464 79207
294585.00 3257.00 0.9891 WT Homozygote D8 344957 64456 57610
287347.00 6846.00 0.9767 WT Homozygote D9 302895 75707 74534
228361.00 1173.00 0.9949 WT Homozygote D10 382257 76015 69250
313007.00 6765.00 0.9788 WT Homozygote D11 62715 326270 54740
7975.00 271530.00 0.0285 Mutant Homozygote D12 72334 429302 63801
8533.00 365501.00 0.0228 Mutant Homozygote E1 291395 974002 284355
7040.00 689647.00 0.0101 Mutant Homozygote E2 846719 380934 208940
637779.00 171994.00 0.7876 WT Homozygote E3 268317 436356 238130
30187.00 198226.00 0.1322 Mutant Homozygote E4 1025425 215357
225516 799909.00 -10159.00 1.0129 WT Homozygote E5 385114 84055
76639 308475.00 7416.00 0.9765 WT Homozygote E6 349652 72824 65685
283967.00 7139.00 0.9755 WT Homozygote E7 57669 257988 53889
3780.00 204099.00 0.0182 Mutant Homozygote E8 350706 81980 73186
277520.00 8794.00 0.9693 WT Homozygote E9 187804 59978 57690
130114.00 2288.00 0.9827 WT Homozygote E10 419892 74603 64973
354919.00 9630.00 0.9736 WT Homozygote E11 114204 44254 45919
68285.00 -1665.00 1.0250 WT Homozygote E12 72764 431146 59266
13498.00 371880.00 0.0350 Mutant Homozygote F1 182251 133298 140425
41826.00 -7127.00 1.2054 Signal/Bkg Low F2 1085040 263816 262330
822710.00 1486.00 0.9982 WT Homozygote F3 1149638 288799 277169
872469.00 11630.00 0.9868 WT Homozygote F4 1298679 369636 343923
954756.00 25713.00 0.9738 WT Homozygote F5 421495 90313 85890
335605.00 4423.00 0.9870 WT Homozygote F6 248386 43837 50870
197516.00 -7033.00 1.0369 WT Homozygote F7 395810 79728 73931
321879.00 5797.00 0.9823 WT Homozygote F8 437260 84282 76015
361245.00 8267.00 0.9776 WT Homozygote F9 92817 419474 70407
22410.00 349067.00 0.0603 Mutant Homozygote F10 69232 176622 65238
3994.00 111384.00 0.0346 Mutant Homozygote F11 405233 79452 73190
332043.00 6262.00 0.9815 WT Homozygote F12 347235 60861 49639
297596.00 11222.00 0.9637 WT Homozygote G1 661728 199046 197228
464500.00 1818.00 0.9961 WT Homozygote G2 1316486 379375 354161
962325.00 25214.00 0.9745 WT Homozygote G3 372004 1299117 359896
12108.00 939221.00 0.0127 Mutant Homozygote G4 295045 1059830
288259 6786.00 771571.00 0.0087 Mutant Homozygote G5 102109 404399
94908 7201.00 309491.00 0.0227 Mutant Homozygote G6 374825 93518
83355 291470.00 10163.00 0.9663 WT Homozygote G7 91658 439103 85492
6166.00 353611.00 0.0171 Mutant Homozygote G8 389514 101115 90483
299031.00 10632.00 0.9657 WT Homozygote G9 352032 65176 59725
292307.00 5451.00 0.9817 WT Homozygote G10 61225 229948 40071
21154.00 189877.00 0.1002 Mutant Homozygote G11 97339 513948 82923
14416.00 431025.00 0.0324 Mutant Homozygote G12 94130 526318 83889
10241.00 442429.00 0.0226 Mutant Homozygote H1 529590 1465949
532127 -2537.00 933822.00 -0.0027 Mutant Homozygote H2 443487
1315342 414585 28902.00 900757.00 0.0311 Mutant Homozygote H3
1372124 437675 473109 899015.00 -35434.00 1.0410 WT Homozygote H4
385710 1401153 334935 50775.00 1066218.00 0.0455 Mutant Homozygote
H5 417722 93776 87874 329848.00 5902.00 0.9824 WT Homozygote H6
98909 433581 87379 11530.00 346202.00 0.0322 Mutant Homozygote H7
96824 333184 80419 16405.00 252765.00 0.0609 Mutant Homozygote H8
518543 115426 101354 417189.00 14072.00 0.9674 WT Homozygote H9
423527 96796 84702 338825.00 12094.00 0.9655 WT Homozygote H10
434588 102160 97889 336699.00 4271.00 0.9875 WT Homozygote H11
428308 83111 76995 351313.00 6116.00 0.9829 WT Homozygote H12
387833 82556 72483 315350.00 10073.00 0.9690 WT Homozygote 0.00 Not
Determined 0.0000 A1 58027 222183 57684 343.00 164499.00 0.0021
Mutant Homozygote B1 185071 51454 55494 129577.00 -4040.00 1.0322
WT Homozygote C1 295696 122251 125871 169825.00 -3620.00 1.0218 WT
Homozygote D1 57147 151617 58288 -1141.00 93329.00 -0.0124 Mutant
Homozygote E1 160272 365059 152760 7512.00 212299.00 0.0342 Mutant
Homozygote F1 140035 323425 127593 12442.00 195832.00 0.0597 Mutant
Homozygote G1 340699 125807 126213 214486.00 -406.00 1.0019 WT
Homozygote H1 42236 164029 38652 3584.00 125377.00 0.0278 Mutant
Homozygote A2 44991 208056 38228 6763.00 169828.00 0.0383 Mutant
Homozygote B2 70844 252510 58554 12290.00 193956.00 0.0596 Mutant
Homozygote C2 121370 327497 105696 15674.00 221801.00 0.0660 Mutant
Homozygote D2 276654 97372 94479 182175.00 2893.00 0.9844 WT
Homozygote E2 320433 129999 123842 196591.00 6157.00 0.9696 WT
Homozygote F2 78482 204964 73273 5209.00 131691.00 0.0380 Mutant
Homozygote G2 125103 335824 104245 20858.00 231579.00 0.0826 Mutant
Homozygote H2 240462 45557 42355 198107.00 3202.00 0.9841 WT
Homozygote A3 243958 55075 52528 191430.00 2547.00 0.9869 WT
Homozygote B3 91970 264544 85181 6789.00 179363.00 0.0365 Mutant
Homozygote C3 336897 119084 117108 219789.00 1976.00 0.9911 WT
Homozygote D3 271665 58558 59833 211832.00 -1275.00 1.0061 WT
Homozygote E3 337079 125908 122511 214568.00 3397.00 0.9844 WT
Homozygote F3 143218 353952 128180 15038.00 225772.00 0.0624 Mutant
Homozygote G3 347214 132332 133270 213944.00 -938.00 1.0044 WT
Homozygote H3 154490 40370 43119 111371.00 -2749.00 1.0253 WT
Homozygote A4 33118 153268 24474 8644.00 128794.00 0.0629 Mutant
Homozygote B4 332295 135622 128922 203373.00 6700.00 0.9681 WT
Homozygote C4 285837 91412 87076 198761.00 4336.00 0.9787 WT
Homozygote D4 318153 70585 65242 252911.00 5343.00 0.9793 WT
Homozygote E4 239939 297176 111888 128051.00 185288.00 0.4087
Heterozygote F4 276763 99403 89728 187035.00 9675.00 0.9508 WT
Homozygote G4 138492 74565 72105 66387.00 2460.00 0.9643 WT
Homozygote H4 346257 108363 96379 249878.00 11984.00 0.9542 WT
Homozygote A5 265294 76739 76917 188377.00 -178.00 1.0009 WT
Homozygote B5 275221 88736 92304 182917.00 -3568.00 1.0199 WT
Homozygote C5 98000 299991 97809 191.00 202182.00 0.0009 Mutant
Homozygote D5 71935 307011 67942 3993.00 239069.00 0.0164 Mutant
Homozygote E5 125117 344353 127235 -2118.00 217118.00 -0.0099
Mutant Homozygote F5 337438 139681 141316 196122.00 -1635.00 1.0084
WT Homozygote G5 320827 109590 110836 209991.00 -1246.00 1.0060 WT
Homozygote H5 284119 254549 83420 200699.00 171129.00 0.5398
Heterozygote A6 196486 40731 34709 161777.00 6022.00 0.9641 WT
Homozygote B6 91086 284419 73381 17705.00 211038.00 0.0774 Mutant
Homozygote C6 287819 109940 109470 178349.00 470.00 0.9974 WT
Homozygote D6 324772 96989 90618 234154.00 6371.00 0.9735 WT
Homozygote E6 104800 22690 16390 88410.00 6300.00 0.9335 WT
Homozygote F6 326617 108807 100688 225929.00 8119.00 0.9653 WT
Homozygote G6 325606 108051 105410 220196.00 2641.00 0.9881 WT
Homozygote H6 272423 300350 88598 183825.00 211752.00 0.4647
Heterozygote A7 205580 132345 52452 153128.00 79893.00 0.6571
Heterozygote B7 128452 310516 113483 14969.00 197033.00 0.0706
Mutant Homozygote C7 104698 305524 93208 11490.00 212316.00 0.0513
Mutant Homozygote D7 307564 86991 85063 222501.00 1928.00 0.9914 WT
Homozygote E7 275408 101068 97206 178202.00 3862.00 0.9788 WT
Homozygote F7 120699 303920 76423 44276.00 227497.00 0.1629 Mutant
Homozygote G7 323578 103479 104487 219091.00 -1008.00 1.0046 WT
Homozygote H7 251697 203978 60296 191401.00 143682.00 0.5712
Heterozygote A8 80230 276995 74846 5384.00 202149.00 0.0259 Mutant
Homozygote B8 137609 300532 104219 33390.00 196313.00 0.1454 Mutant
Homozygote C8 48060 108280 46704 1356.00 61576.00 0.0215 Mutant
Homozygote D8 100470 264735 91454 9016.00 173281.00 0.0495 Mutant
Homozygote E8 292829 122176 116160 176669.00 6016.00 0.9671 WT
Homozygote F8 105447 326479 96111 9336.00 230368.00 0.0389 Mutant
Homozygote G8 147067 362978 136719 10348.00 226259.00 0.0437 Mutant
Homozygote H8 332952 153555 91114 241838.00 62441.00 0.7948 WT
Homozygote A9 244720 70456 75204 169516.00 -4748.00 1.0288 WT
Homozygote B9 112223 322330 106362 5861.00 215968.00 0.0264 Mutant
Homozygote C9 118996 327770 95704 23292.00 232066.00 0.0912 Mutant
Homozygote D9 94361 270031 81552 12809.00 188479.00 0.0636 Mutant
Homozygote E9 142083 342389 134979 7104.00 207410.00 0.0331 Mutant
Homozygote F9 343667 131080 132731 210936.00 -1651.00 1.0079 WT
Homozygote G9 138858 369893 129933 8925.00 239960.00 0.0359 Mutant
Homozygote H9 143881 66587 63328 80553.00 3259.00 0.9611 WT
Homozygote A10 252930 67971 63808 189122.00 4163.00 0.9785 WT
Homozygote B10 111801 306491 101836 9965.00 204655.00 0.0464 Mutant
Homozygote C10 332112 116649 113212 218900.00 3437.00 0.9845 WT
Homozygote D10 113247 339818 98752 14495.00 241066.00 0.0567 Mutant
Homozygote E10 158838 328193 116442 42396.00 211751.00 0.1668
Mutant Homozygote F10 339981 125573 117205 222776.00 8368.00 0.9638
WT Homozygote G10 111672 311430 104189 7483.00 207241.00 0.0348
Mutant Homozygote H10 362138 145457 106673 255465.00 38784.00
0.8682 WT Homozygote A11 253647 157356 95627 158020.00 61729.00
0.7191 No Call B11 164237 359643 145067 19170.00 214576.00 0.0820
Mutant Homozygote C11 339294 124660 120341 218953.00 4319.00 0.9807
WT Homozygote D11 347411 112399 111482 235929.00 917.00 0.9961 WT
Homozygote E11 353768 137003 142014 211754.00 -5011.00 1.0242 WT
Homozygote F11 347503 131462 129637 217866.00 1825.00 0.9917 WT
Homozygote G11 136856 327747 115035 21821.00 212712.00 0.0930
Mutant Homozygote H11 358593 150488 113576 245017.00 36912.00
0.8691 WT Homozygote A12 73492 208038 67210 6282.00 140828.00
0.0427 Mutant Homozygote B12 299590 106653 97203 202387.00 9450.00
0.9554 WT Homozygote C12 23190 73050 19140 4050.00 53910.00 0.0699
Mutant Homozygote D12 106633 318290 92331 14302.00 225959.00 0.0595
Mutant Homozygote E12 367938 170961 159625 208313.00 11336.00
0.9484 WT Homozygote F12 136109 348590 126033 10076.00 222557.00
0.0433 Mutant Homozygote G12 315191 119810 58932 256259.00 60878.00
0.8080 WT Homozygote H12 264473 60564 55720 208753.00 4844.00
0.9773 WT Homozygote
EXAMPLE 23
Self-Annealing Interrogation Probe
[0556] This Example illustrates use of a different type of
oligonucleotide probe that is used to form a hairpin structure in
the interrogation technology of this invention. This study
demonstrates a method for eliminating the need for adding a probe
specific to the interrogation site to the interrogation
reaction.
[0557] Here, the oligonucleotide probe anneals to the target strand
downstream of (3' to) the interrogation position in the target
strand. The oligonucleotide has at its 5' end an unannealed region
of nucleotides followed by about 5 to about 20 nucleotides that are
identical to the interrogation region on the target strand. The
annealed 3' end of the oligonucleotide is then extended through the
interrogation position of the target strand creating what is
referred to as extended probe. The hybrid is denatured and a
hairpin structure formed between the extended probe strand and the
5' end of the oligonucleotide probe. This region is then assayed in
a standard interrogation reaction to determine if a mismatch is
present or not.
[0558] Four probes were designed to represent different types of
hairpin formations that an extended probe strands may assume. These
probes are 10207 (SEQ ID NO:89), 10208 (SEQ ID NO:90), 10209 (SEQ
ID NO:91), and 10212 (SEQ ID NO:92).
[0559] These probes are predicted to form the following
self-hybridized secondary structures when allowed to self-anneal:
5
[0560] A 5 .mu.L (5 .mu.g) aliquot of each of the four probes was
diluted to 100 .mu.L with nanopure water. They were then
sequentially diluted 1:10 to a final dilution factor of 1:100,000.
Twenty microliters of the diluted probes were heated, in separate
tubes, at 95.degree. C. for 3 minutes and cooled to room
temperature for 10 minutes to permit self-annealing. Twenty
microliters of Master Mix, as described in Example 1, were then
added to each tube and the tubes were incubated at 37.degree. C.
for 15 minutes. Ten microliters of the solutions were added to 100
.mu.L of L/L reagent (Promega, F202A) and relative light units
measured immediately with a Turner.RTM. TD20/20 luminometer. The
no-probe control resulted in 57.24 relative light units and the
remaining probe results are reported below in relative light units
(rlu).
78 Log Probe dilution 10207 10208 10209 10212 -5 44.89 56.22 57.57
57.80 -4 85.21 64.56 58.26 63.15 -3 297.7 70.53 79.12 82.65 -2
970.5 108.4 80.06 106.7
[0561] Probe 10207 worked as an efficient target for interrogation
as expected, with probe 10208 providing the anticipated negative
results. Probe 10212 has only a three base match so it may be
un-extended, thus resulting in the low values. Probe 10209 likely
has the 3' terminal nucleotide unannealed when the hairpin forms
due to the mismatch at the third nucleotide in from the 3' end.
Such an unannealed 3' terminal nucleotide would account for the low
rlu values.
79 10207 5' ATGAACGTACGTCGGATGAGCACGTTCAT 3' SEQ ID NO:89 10208 5'
GTGAACGTACGTCGGATGAGCACGTTCAT 3' SEQ ID NO:90 10209 5'
ATAAACGTACGTCGGATGAGCACGTTCAT 3' SEQ ID NO:91 10212 5'
ATAAACGTACGTCGGATGAGCACG 3' SEQ ID NO:92
EXAMPLE 24
Interrogation with a Self-Annealing Primer II
[0562] This example and FIG. 1 illustrate use of a different type
of oligonucleotide probe, a "REAPER.TM." probe in a process of this
invention. This example demonstrates a method for eliminating the
need for adding a probe specific to the interrogation site to the
interrogation reaction.
[0563] Here, the oligonucleotide first probe (SEQ ID NO:112), at
its 3'-end, anneals to the target strand (SEQ ID NO:111) at a
position downstream of (3' to) the interrogation position in the
target strand (FIG. 1A). The probe has at its 5'-end an unannealed
region of nucleotides including about 5 to about 20 nucleotides
that are identical to a region on the target strand including the
interrogation position. This region of identity is present in the
same orientation on both the target and the probe strands.
[0564] The annealed 3'-end of the probe is then extended through
the interrogation position of the target strand forming what is
referred to as a first extended probe and an extended first hybrid
as is illustrated in FIG. 1B (SEQ ID NO:113). The extended first
hybrid is denatured and a second probe (SEQ ID NO:114) is annealed
to the first extended probe to form a second hybrid. This second
probe is complementary to the first extended probe strand at a
region downstream of the interrogation position on the first
extended probe strand (FIG. 1C).
[0565] The second probe is then extended and a second extended
hybrid is formed as illustrated in FIG. 1D. The second extended
hybrid is comprised of the first extended probe and second extended
probe (SEQ ID NO:115).
[0566] The strands of the second extended hybrid are denatured and
permitted to renature to form a hairpin structure. Upon hairpin
formation, the first extended probe forms a hairpin structure that
has a 3'-overhang, whereas the second extended probe forms a
hairpin structure that contains a 5'-overhang that provides a
substrate for depolymerization. The second extended probe strand is
then depolymerized and the analytical output obtained as described
elsewhere herein. The analytical output determines the presence or
absence of the original target strand or of a particular base in
the original target strand as is also discussed elsewhere
herein.
[0567] SEQ ID NO:111 oligonucleotide is diluted to 1 mg/mL in
water. This solution is labeled 111. SEQ ID NO:112 oligonucleotide
is diluted to 1 mg/mL in water and this solution is labeled 112.
One microliter of each solution 111 and 112 is combined with 18
.mu.L water. The solution is heated to 95.degree. C. for 5 minutes
then is cooled at room temperature for 10 minutes to permit
oligonucleotides of SEQ ID NOs:111 and 112 to anneal.
[0568] To this solution are added dNTP mixture to a final
concentration of 0.25 mM for each DNTP, 10.times. Klenow buffer to
a final concentration of 1.times., and 5 U of Klenow enzyme. The
tube with these components is incubated at 37.degree. C. for 30
minutes. The extended first hybrid DNA so formed (containing SEQ ID
NO: 113) is purified (Qiagen, Mermaid system) and eluted into 50
.mu.l of water.
[0569] To this solution of the purified extended first hybrid is
added 1 .mu.L SEQ ID NO:114 oligonucleotide (1 mg/mL) as second
probe. The solution is then heated to 95.degree. C. for 5 minutes
and is cooled at room temperature to permit 114 and 113 to anneal
as illustrated in FIG. 1C to form the second hybrid. To this
solution are added a DNTP mixture to a final concentration of 0.25
mM for each dNTP, 10.times. Klenow buffer to a final concentration
of 1.times., and 5 U of Klenow enzyme. The tube with these
components is incubated at 37.degree. C. for 30 minutes to form a
second extended hybrid that contains a second extended probe
(oligonucleotide SEQ ID NO: 115).
[0570] The SEQ ID NO:115/113 second extended hybrid DNA (FIG. 1D)
formed is purified (Qiagen, Mermaid system) to separate the
extended hybrid from the unreacted dNTPs and eluted into 50 .mu.L
water. (Alternatively, the original 112 oligo is biotinylated at
it's 5'-end and this biotin is then also present in strand of SEQ
ID NO:113. This biotinylated strand 113 is then denatured from
strand 115 and removed from the solution with streptavidin coated
paramagnetic particles according to the manufacturer's instructions
(Promega, Z5481) and the 115 hairpin structure is allowed to form
as below).
[0571] This hybrid solution is then heated to 95.degree. C. for 5
minutes diluted to 100 .mu.L with water and is cooled on ice for 10
minutes to permit hairpin structure formation.
[0572] The following master mix is assembled and mixed.
80 Component Amount 10X DNA Pol Buffer 200 .mu.L (Promega, M195A)
Klenow exo- (1 U/.mu.L) 12.5 .mu.L (Promega M218B) 40 mM Sodium
Pyrophosphate 25 .mu.L (Promega C350B) NDPK (1 U/.mu.L) 10 .mu.L 10
.mu.M ADP (Sigma A5285) 20 .mu.L Water 732.5 .mu.L
[0573] Twenty microliters of this master mix are added to 20 .mu.L
of the above hairpin-containing solutions after cooling, and the
resulting mixtures are heated at 37.degree. C. for 15 minutes.
After this incubation, duplicate 4 .mu.L samples of the solution
are removed, added to 100 .mu.L of L/L Reagent (Promega, F202A) and
the light produced by the reaction is measured immediately using a
Turner TD20/20 luminometer. A positive analytical output at levels
over background (no enzyme) indicates that a matched base was
present at the 3'-terminus of the hairpin structure and this
further indicates the presence of the target strand, and for this
particular example, it also indicates the presence of a G base at
the interrogation position of the target.
81 5' CCCGGAGAGACCTCCTTAAGGGGCCATATTATTTCGTCGATTCCAGTGTT SEQ ID
NO:111 GGCCAAACGGAT 3' 5' GGGGCCATATTATTTCGCCGTTT-
GGCCAACACTGGAATCGA 3' SEQ ID NO:112 5'
GGGGCCATATTATTTCGCCGTTTGGCCAACACTGGAATCGACGAAATAAT SEQ ID NO:113
ATGGCCCCTTAAGGAGGTCTCTCCGGG 3' 5' CCCGGAGAGACCTCCT 3' SEQ ID NO:114
5' CCCGGAGAGACCTCCTTAAGGGGCCATATTATTTCGTCGATTCC- AGTGTT SEQ ID
NO:115 GGCCAAACGGCGAAATAATATGGCCCC 3'
EXAMPLE 25
Interrogation With a Self-Annealing Primer: III
[0574] In this example, the method described in Example 23 is used
to detect the presence of E. coli DNA in a sample. A Reaper.TM.
probe (12028) was designed to hybridize to 20 nucleotides of the
lac Z gene of E. coli DNA. This region of the lac Z gene is
designated SEQ ID NO:117. It is important that the unextended
primer does not self-anneal and extend upon itself to form primer
dimers or a high level of background noise would result. The primer
was designed to minimize this possibility.
82 12028 5' TTACCCAACTTAATCAGGGGGATGTGCTGCAAGGC 3' SEQ ID NO:116
lacZ segment 5' . . . CGTTACCCAACTTAATCGCCTTGCAGCA- CATCCCCCTT . .
. 3' SEQ ID NO:117
[0575] The following reaction was set up to hybridize
oligonucleotide 12028 to a complementary region of the E. coli lac
Z gene. The 12028 oligonucleotide is present in great molar excess
in the reaction, so multiple extension rounds are performed to
ensure that all of the 12028 oligonucleotide is extended to make
SEQ ID NO:116. The extension is performed in the absence of dCTP to
halt the extension by the polymerase after addition of 15 bases.
Oligonucleotide 12028 was diluted with nanopure water to a final
concentration of 100 .mu.g/mL.
83 1 and 2 3 and 4 5 and 6 10X Thermophilic 2 .mu.L 2 .mu.L 2 .mu.L
buffer 25 mM MgCl.sub.2 2 .mu.L 2 .mu.L 2 .mu.L 1 mg/mL E.coli B
DNA (Sigma D4889) 2 .mu.L -- 2 .mu.L 100 .mu.g/mL 12028 -- 1 .mu.L
1 .mu.L 100 .mu.M dGTP, 10 .mu.L 10 .mu.L 10 .mu.L dATP, TTP water
4 .mu.L 5 .mu.L 3 .mu.L
[0576] The solutions were incubated for 3 minutes at 95.degree. C.
to denature the DNA. Then 1 .mu.L of Taq DNA Polymerase (Promega,
M1861) at 5 U/.mu.L was added and the incubation at 95.degree. C.
proceeded for an additional 2 minutes followed by (95.degree. C. 30
seconds, 55.degree. C. 30 seconds, 72.degree. C. 1 min).times.35
cycles, and a 4.degree. C. soak. The tube was then removed and
twenty microliters of the solutions were transferred to a fresh
tube. One microliter of 1 U/.mu.L Shrimp Alkaline Phosphatase (SAP)
was added to each tube and they were then incubated at 37.degree.
C. for 30 minutes, followed by an incubation at 65.degree. C. for
15 minutes to remove the terminal phosphate from the residual
nucleotides and the 5' end of the oligonucleotides in the solution.
The tubes were spun briefly in a microcentrifuge to collect the
solution at the bottom of then tube and they were then heated at
95.degree. C. for 3 minutes to denature the DNA. The tubes were
then permitted to cool at room temperature for 10 minutes, and
permit the stem loop structure to form.
[0577] To remove the free nucleotide from the solution, the
contents of each tube were separately applied to a Sephadex.RTM.
G-25 column, prespun according to manufacturer's instructions
(Pharmacia), and then centrifuged at 3000 rpm for 2 minutes. Twenty
microliters of the eluted solution were added to 5 .mu.L master mix
(50 .mu.L 10.times. DNA Polymerase buffer, 6.25 .mu.L 40 mM NaPPi,
10 .mu.L 10 U/.mu.L Klenow exo minus, 2 .mu.L 1 U/.mu.L NDPK, 5 pL
10 .mu.M ADP and 27 .mu.L water), mixed and incubated for 20
minutes at 37.degree. C. Then 20 .mu.L were removed and added to
100 .mu.L of L/L Reagent (Promega Corp.) The light output was
measured immediately in a Turner.RTM. TD20/20 luminometer. The
results in relative light units (rlu) are listed below.
84 Solution rlu 1. 129 2. 105 3. 5 4. 10 5. 150 6. 164
[0578] The background rlu values in solutions 3 and 4 (primer
alone) are very low, indicating good removal of the free
nucleotides. The higher background in solutions 1 and 2 must
therefore result from the E.coli DNA. It appears that the
efficiency of extension of the 12028 oligonucleotide is in the
range of 20-25%. This might be increased through the use of higher
nucleotide and/or primer concentrations. 12028/lacZ (SEQ ID
NO:116/117) hybrid:
85 lacZ 3' . . . TTCCCCCTACACGACGTTCCGCTAATTCAACCCATTGC . . . 5'
12028 AGGGGGATGTGCTGCAAGGC 3' C T A ATTCAACCCATT 5'
[0579] Extended 12028/lacZ (SEQ ID NO:118/117) hybrid with the
nucleotides added by extension underlined:
86 lacZ 3' . . . TTCCCCCTACACGACGTTCCGCTAATTCAACCCATTGC . . . 5'
12028 AGGGGGATGTGCTGCAAGGCGATTAAGTTCGGTAA 3' C T A ATTCAACCCATT
5'
[0580] Extended 12028 (SEQ ID NO:118) secondary structure with the
nucleotides added by extension underlined:
87 G-G-G-G-A G T A G 5' TTACCCAACTTAATC T 3' AATGGGTTGAATTAG G C C
G T G-A-A-C-G
EXAMPLE 26
Detection of DNA Sequences in the Genome of Campylobacter
jejuni
[0581] Oligonucleotides 11453 (SEQ ID NO:119) and 11454 (SEQ ID
NO:120) are exactly complementary and can be annealed, thereby
forming a synthetic target representing a 70 bp segment of
Campylobacter jejuni. These two oligonucleotides were diluted in
nanopure water to a final concentration of 10 .mu.g/mL. Four
microliters of each were then mixed with 232 .mu.L 10 mM Tris pH
7.3 to yield a target solution of 0.3 .mu.g/mL of DNA.
Oligonucleotides 11451 (SEQ ID NO:121) and 11450 (SEQ ID NO:122)
are Campylobacter jejuni interrogation probes that bind to opposite
strands of the bacterial genome represented in the synthetic
target. Oligonucleotide 11451 anneals to oligonucleotide 11454.
oligonucleotide 11450 anneals to oligonucleotide 11453.
[0582] The following solutions were assembled in triplicate and
nanopure water added to a final volume of 20 .mu.L.
88 0.3 ng 1 .mu.g Solution Target Probe rlu 1. + 11451 391 2. +
11450 241 3. + none 28 4. - 11451 248 5. - 11450 30 6. - none
24
[0583] The assembled solutions were incubated at 92.degree. C. for
5 minutes, then cooled at room temperature for 10 minutes. Master
mix was prepared as in Example 1 using 10 units Klenow exo-
polymerase and 4 units NDPK. Twenty microliters of master mix were
added to each tube and incubated at 37.degree. C. for 15 minutes.
Five microliters of each solution were then combined with 100 .mu.L
of L/L reagent (Promega F202A) and light output measured
immediately on a Turner.RTM. TD20/20 luminometer. The average
relative light units (rlu) are recorded in the table above Using
each of the interrogation probes with the target appears to give
strong net signal. The top probe (11451) however, gives very strong
signal alone, possibly due to hairpin formation, and is less
suitable for interrogation. The bottom interrogation probe (11450)
is the better for interrogation.
89 11453 5' CTTGAAGCATAGTTCTTGTTTTTAAACTTTGTCCATCTTGAGCCGC- TTGA
SEQ ID NO:119 GTTGCCTTAGTTTTAATAGT 3' 11454 5'
ACTATTAAAACTAAGGCAACTCAAGCGGCTCAAGATGGACAAAGTTTA SEQ ID NO:120
AAAACAAGAACTATGCTTCAAG 3' 11451 5' AGTTCTTGTTTTTAAACTTTGTCCATCTTG
3' SEQ ID NO:121 11450 5' CAAGATGGACAAAGTTTAAAAACAAGAACT 3' SEQ ID
NO:122
EXAMPLE 27
Detection of E. coli Repetitive Sequence without Nucleic Acid
Amplification
[0584] In this Example repetitive sequence in E. coli is detected
without the need for amplification of the target sequence prior to
pyrophosphorylation. This target sequence is denoted as
`colirep`.
[0585] Oligonucleotide 11707 (SEQ ID NO:123) is totally
complementary to a segment of colirep DNA sequence. Twelve
microliters of oligonucleotide 11707 solution (1 mg/mL) were
combined with 204 .mu.L of water to make solution A. Another
solution was prepared by combining 4 .mu.L of 11707 (1 mg/mL) with
204 .mu.L water and 8 .mu.L 10 mM Tris, pH 8.0 to make solution B.
The E. coli is Sigma cat#D4889, E. coli Strain B ultra pure.
[0586] Four nanograms (2 .mu.L) E. coli DNA were combined with 18
.mu.L solution A and with 18 .mu.L solution B in separate tubes.
Similarly, 40 ng E. coli DNA was combined with 18 .mu.L solution A
and with 18 .mu.L solution B in separate tubes. These solutions
were then incubated at 92.degree. C. for 3 minutes and cooled at
room temperature for 15 minutes. The following master mix was
assembled:
90 10X DNA Polymerase buffer 240 .mu.L 40 mM NaPPi 30 .mu.L Klenow
exo- (10 U/.mu.L) 30 .mu.L NDPK (1 U/.mu.L) 12 .mu.L 10 .mu.M ADP
(Sigma) 24 .mu.L water 864 .mu.L
[0587] Twenty microliters of master mix were added to each reaction
and they were then incubated at 37.degree. C. for 15 minutes. One
hundred microliters of L/L Reagent were then added and the relative
light output (rlu) immediately measured with a Turner.RTM. TD 20/20
luminometer. The rlu were:
91 Solution rlu-1 rlu-2 rlu-3 Average Tris 2.85 3.562 3.059 3.157
11707 (A) 13.69 12.13 13.67 13.16 11707 (B) 7.473 7.234 6.981 7.259
40 ng DNA + Tris 75.62 75.52 73.24 74.79 40 ng DNA + 97.71 134.2
105.1 112.3 11707 (A) 40 ng DNA + 81.46 87.56 76.28 81.77 11707 (B)
4 ng DNA + Tris 6.719 8.084 5.882 6.895 4 ng DNA + 24.50 25.97
25.17 25.21 11707 (A) 4 ng DNA + 15.69 17.22 16.99 16.63 11707
(B)
[0588] The data reflect that oligonucleotide probe 11707 can detect
E. coli DNA without amplification by a process of the
invention.
[0589] Interrogation oligonucleotide: 11707 5' AGTGACTGGGG 3' SEQ
ID NO:123
[0590] From the foregoing, it will be observed that numerous
modifications and variations can be effected without departing from
the true spirit and scope of the present invention. It is to be
understood that no limitation with respect to the specific examples
presented is intended or should be inferred. The disclosure is
intended to cover by the appended claims modifications as fall
within the scope of the claims.
Sequence CWU 1
1
123 1 30 DNA rabbit probe to globin mRNA 1 agacttctcc tcactggaca
gatgcaccat 30 2 26 DNA rabbit probe to globin mRNA 2 gggtccatgg
gtagacaacc agcagc 26 3 65 DNA Homo sapiens target for human Factor
V gene 3 ctaatctgta agagcagatc cctggacagg cgaggaatac agagggcagc
agacatcgaa 60 gagct 65 4 69 DNA Homo sapiens target for human
Factor V gene 4 agctcttcga tgtctgctgc cctctgtatt cctcgcctgt
ccagggatct gctcttacag 60 attagagct 69 5 65 DNA Homo sapiens target
for human Factor V Leiden gene 5 ctaatctgta agagcagatc cctggacagg
caaggaatac agagggcagc agacatcgaa 60 gagct 65 6 69 DNA Homo sapiens
target for human Factor V Leiden gene 6 agctcttcga tgtctgctgc
cctctgtatt ccttgcctgt ccagggatct gctcttacag 60 attagagct 69 7 22
DNA Homo sapiens probe for human Factor V gene 7 ctgctgccct
ctgtattcct cg 22 8 22 DNA Homo sapiens probe for human Factor V
gene 8 ctgctgccct ctgtattcct tg 22 9 22 DNA Homo sapiens probe for
human Factor V gene 9 gacaaaatac ctgtattcct cg 22 10 22 DNA Homo
sapiens probe for human Factor V gene 10 gacaaaatac ctgtattcct tg
22 11 20 DNA Homo sapiens probe for human cystic fibrosis gene 11
cattcacagt agcttaccca 20 12 20 DNA Homo sapiens probe for human
cystic fibrosis gene 12 gcagagtacc tgaaacagga 20 13 19 DNA Homo
sapiens probe for human cystic fibrosis gene 13 catcatagga
aacaccaag 19 14 19 DNA Homo sapiens probe for human cystic fibrosis
gene 14 catcatagga aacaccaat 19 15 24 DNA Homo sapiens probe for
human cystic fibrosis gene 15 ggcaccatta aagaaaatat catt 24 16 49
DNA Homo sapiens probe for human cystic fibrosis gene 16 ctggcaccat
taaagaaaat atcattggtg tttcctatga tgaatatag 49 17 49 DNA Homo
sapiens probe for human cystic fibrosis gene 17 ctatattcat
cataggaaac accaatgata ttttctttaa tggtgccag 49 18 52 DNA Homo
sapiens target for wild-type cystic fibrosis gene 18 ctggcaccat
taaagaaaat atcatctttg gtgtttccta tgatgaatat ag 52 19 52 DNA Homo
sapiens target for mutant cystic fibrosis gene 19 ctatattcat
cataggaaac accaaagatg atattttctt taatggtgcc ag 52 20 50 DNA Homo
sapiens target for human prothrombin wild-type gene 20 tcccaataaa
agtgactctc agcgagcctc aatgctccca gtgctattca 50 21 50 DNA Homo
sapiens target for human prothrombin mutant gene 21 tcccaataaa
agtgactctc agcaagcctc aatgctccca gtgctattca 50 22 16 DNA Homo
sapiens probe for human prothrombin gene 22 ggagcattga ggctcg 16 23
16 DNA Homo sapiens probe for human prothrombin gene 23 ggagcattga
ggcttg 16 24 23 DNA Artificial Sequence Description of Artificial
Sequenceprobe for Rice genome (with phosphorothioate linkages) 24
cccaacacct tacagaaatt agc 23 25 23 DNA Artificial Sequence
Description of Artificial Sequenceprobe for Rice genome 25
tctcaagaca caaataactg cag 23 26 15 DNA Artificial Sequence
Description of Artificial Sequenceprobe for "G" allele of Rice 26
agaacatctg caagg 15 27 15 DNA Artificial Sequence Description of
Artificial Sequenceprobe for "T" allele of Rice 27 agaacatctg caagt
15 28 58 DNA Artificial Sequence Description of Artificial
SequenceTHO locus with 6x CATT repeat 28 ggtgaatgaa tgaatgaatg
aatgaatgag ggaaataagg gaggaagagg ccaatggg 58 29 62 DNA Artificial
Sequence Description of Artificial SequenceTHO locus with 7x CATT
repeat 29 ggtgaatgaa tgaatgaatg aatgaatgaa tgagggaaat aagggaggaa
gaggccaatg 60 gg 62 30 70 DNA Artificial Sequence Description of
Artificial SequenceTHO locus with 8x CATT repeat 30 ggtaggtgaa
tgaatgaatg aatgaatgaa tgaatgaatg agggaaataa gggaggaaga 60
ggccaatggg 70 31 58 DNA Artificial Sequence Description of
Artificial Sequenceprimer for THO1 locus with 6x CATT repeats 31
cccattggcc tgttcctccc ttatttccct cattcattca ttcattcatt cattcacc 58
32 62 DNA Artificial Sequence Description of Artificial
Sequenceprobe for THO1 locus with 7x CATT repeats 32 cccattggcc
tgttcctccc ttatttccct cattcattca ttcattcatt cattcattca 60 cc 62 33
66 DNA Artificial Sequence Description of Artificial Sequenceprobe
for THO1 locus with 8x CATT repeats 33 cccattggcc tgttcctccc
ttatttccct cattcattca ttcattcatt cattcattca 60 ttcacc 66 34 54 DNA
Artificial Sequence Description of Artificial Sequenceprobe for
THO1 locus with 5x CATT repeats 34 cccattggcc tgttcctccc ttatttccct
cattcattca ttcattcatt cacc 54 35 58 DNA Artificial Sequence
Description of Artificial Sequenceprobe for THO1 locus with 6x CATT
repeats 35 cccattggcc tgttcctccc ttatttccct cattcattca ttcattcatt
cattcacc 58 36 62 DNA Artificial Sequence Description of Artificial
Sequenceprobe for THO1 locus with 7x CATT repeats 36 cccattggcc
tgttcctccc ttatttccct cattcattca ttcattcatt cattcattca 60 cc 62 37
66 DNA Artificial Sequence Description of Artificial Sequenceprobe
for THO1 locus with 8x CATT repeats 37 cccattggcc tgttcctccc
ttatttccct cattcattca ttcattcatt cattcattca 60 ttcacc 66 38 70 DNA
Artificial Sequence Description of Artificial Sequenceprobe for
THO1 locus with 9x CATT repeats 38 cccattggcc tgttcctccc ttatttccct
cattcattca ttcattcatt cattcattca 60 ttcattcacc 70 39 58 DNA
Artificial Sequence Description of Artificial Sequenceprobe for THO
locus with 2 base pair mismatch 39 cccattggcc tgttcctccc ttatttccct
cattcattca ttcattcatt cattcagc 58 40 58 DNA Artificial Sequence
Description of Artificial Sequenceprobe to THO locus with A to C
mutation 3 from 3' terminus 40 cccattggcc tgttcctccc ttatttccct
cattcattca ttcattcatt cattcccc 58 41 58 DNA Artificial Sequence
Description of Artificial Sequenceprobe to THO locus with A to G
mutation 3 from 3' terminus 41 cccattggcc tgttcctccc ttatttccct
cattcattca ttcattcatt cattcgcc 58 42 58 DNA Artificial Sequence
Description of Artificial Sequenceprobe to THO locus with
additional base pair mismatches 4 from 3' terminus 42 cccattggcc
tgttcctccc ttatttccct cattcattca ttcattcatt cattgacc 58 43 58 DNA
Artificial Sequence Description of Artificial Sequenceprobe for THO
locus with additional base pair mismatches 4 from 3' terminus 43
cccattggcc tgttcctccc ttatttccct cattcattca ttcattcatt cattaacc 58
44 48 DNA Artificial Sequence Description of Artificial
SequenceTPOX allele 44 ggcacttagg gaaccctcac tgaatgaatg aatgaatgaa
tgaatatt 48 45 52 DNA Artificial Sequence Description of Artificial
SequenceTPOX allele 45 ggcacttagg gaaccctcac tgaatgaatg aatgaatgaa
tgaatgaata tt 52 46 56 DNA Artificial Sequence Description of
Artificial SequenceTPOX allele 46 ggcacttagg gaaccctcac tgaatgaatg
aatgaatgaa tgaatgaatg aatatt 56 47 60 DNA Artificial Sequence
Description of Artificial SequenceTPOX allele 47 ggcacttagg
gaaccctcac tgaatgaatg aatgaatgaa tgaatgaatg aatgaatatt 60 48 63 DNA
Unknown 48 gcacttaggg aaccctcact gaatgaatga atgaatgaat gaatgaatga
atgaatgaat 60 att 63 49 68 DNA Artificial Sequence Description of
Artificial SequenceTPOX allele 49 ggcacttagg gaaccctcac tgaatgaatg
aatgaatgaa tgaatgaatg aatgaatgaa 60 tgaatatt 68 50 72 DNA
Artificial Sequence Description of Artificial SequenceTPOX allele
50 ggcacttagg gaaccctcac tgaatgaatg aatgaatgaa tgaatgaatg
aatgaatgaa 60 tgaatgaata tt 72 51 76 DNA Artificial Sequence
Description of Artificial SequenceTPOX allele 51 ggcacttagg
gaaccctcac tgaatgaatg aatgaatgaa tgaatgaatg aatgaatgaa 60
tgaatgaatg aatatt 76 52 241 DNA Homo sapiens target allele 6 with 6
TGAA repeats 52 ggcacttagg gaaccctcac tgaatgaatg aatgaatgaa
tgaatgtttg ggcaaataaa 60 cgctgacaag gacagaaggg cctagcggga
agggaacagg agtaagacca gcgcacagcc 120 cgacttgtgt tcagaagacc
tgggattgga cctgaggatt caattttgga tgaatctctt 180 aattaacctg
tgtggttccc agttcctccc ctgagcgccc aggacagtag agtcaacctc 240 a 241 53
245 DNA Homo sapiens target allele 7 with 7 TGAA repeats 53
ggcacttagg gaaccctcac tgaatgaatg aatgaatgaa tgaatgaatg tttgggcaaa
60 taaacgctga caaggacaga agggcctagc gggaagggaa caggagtaag
accagcgcac 120 agcccgactt gtgttcagaa gacctgggat tggacctgag
gattcaattt tggatgaatc 180 tcttaattaa cctgtgtggt tcccagttcc
tcccctgagc gcccaggaca gtagagtcaa 240 cctca 245 54 249 DNA Homo
sapiens target allele 8 with 8 TGAA repeats 54 ggcacttagg
gaaccctcac tgaatgaatg aatgaatgaa tgaatgaatg aatgtttggg 60
caaataaacg ctgacaagga cagaagggcc tagcgggaag ggaacaggag taagaccagc
120 gcacagcccg acttgtgttc agaagacctg ggattggacc tgaggattca
attttggatg 180 aatctcttaa ttaacctgtg tggttcccag ttcctcccct
gagcgcccag gacagtagag 240 tcaacctca 249 55 253 DNA Homo sapiens
target allele 9 with 9 TGAA repeats 55 ggcacttagg gaaccctcac
tgaatgaatg aatgaatgaa tgaatgaatg aatgaatgtt 60 tgggcaaata
aacgctgaca aggacagaag ggcctagcgg gaagggaaca ggagtaagac 120
cagcgcacag cccgacttgt gttcagaaga cctgggattg gacctgagga ttcaattttg
180 gatgaatctc ttaattaacc tgtgtggttc ccagttcctc ccctgagcgc
ccaggacagt 240 agagtcaacc tca 253 56 257 DNA Homo sapiens target
allele 10 with 10 TGAA repeats 56 ggcacttagg gaaccctcac tgaatgaatg
aatgaatgaa tgaatgaatg aatgaatgaa 60 tgtttgggca aataaacgct
gacaaggaca gaagggccta gcgggaaggg aacaggagta 120 agaccagcgc
acagcccgac ttgtgttcag aagacctggg attggacctg aggattcaat 180
tttggatgaa tctcttaatt aacctgtgtg gttcccagtt cctcccctga gcgcccagga
240 cagtagagtc aacctca 257 57 261 DNA Homo sapiens target allele 11
with 11 TGAA repeats 57 ggcacttagg gaaccctcac tgaatgaatg aatgaatgaa
tgaatgaatg aatgaatgaa 60 tgaatgtttg ggcaaataaa cgctgacaag
gacagaaggg cctagcggga agggaacagg 120 agtaagacca gcgcacagcc
cgacttgtgt tcagaagacc tgggattgga cctgaggatt 180 caattttgga
tgaatctctt aattaacctg tgtggttccc agttcctccc ctgagcgccc 240
aggacagtag agtcaacctc a 261 58 265 DNA Homo sapiens target allele
12 with 12 TGAA repeats 58 ggcacttagg gaaccctcac tgaatgaatg
aatgaatgaa tgaatgaatg aatgaatgaa 60 tgaatgaatg tttgggcaaa
taaacgctga caaggacaga agggcctagc gggaagggaa 120 caggagtaag
accagcgcac agcccgactt gtgttcagaa gacctgggat tggacctgag 180
gattcaattt tggatgaatc tcttaattaa cctgtgtggt tcccagttcc tcccctgagc
240 gcccaggaca gtagagtcaa cctca 265 59 269 DNA Homo sapiens target
allele 13 with 13 TGAA repeats 59 ggcacttagg gaaccctcac tgaatgaatg
aatgaatgaa tgaatgaatg aatgaatgaa 60 tgaatgaatg aatgtttggg
caaataaacg ctgacaagga cagaagggcc tagcgggaag 120 ggaacaggag
taagaccagc gcacagcccg acttgtgttc agaagacctg ggattggacc 180
tgaggattca attttggatg aatctcttaa ttaacctgtg tggttcccag ttcctcccct
240 gagcgcccag gacagtagag tcaacctca 269 60 24 DNA Homo sapiens
probe to E. coli 60 cactttatgc ttccggctcg tatg 24 61 26 DNA Homo
sapiens probe to E. coli 61 gggataggtt acgttggtgt agatgg 26 62 20
DNA Escherichia coli common probe for E. coli lac 62 gttgggaagg
gcgatcggtg 20 63 21 DNA Artificial Sequence Description of
Artificial Sequenceprobe to E. coli lac 63 gggatgtgct gcaaggcgat t
21 64 20 DNA Artificial Sequence Description of Artificial
Sequenceprobe for lac deletion in E. coli 64 ggattcactg gccgtcgtgg
20 65 65 DNA Cytomegalovirus target for cytomegalovirus 65
cgtgtatgcc actttgatat tacacccatg aacgtgctca tcgacgtgaa cccgcacaac
60 gagct 65 66 65 DNA Cytomegalovirus target for cytomegalovirus 66
cgttgtgcgg gttcacgtcg atgagcacgt tcatgggtgt aatatcaaag tggcatacac
60 gagct 65 67 65 DNA Cytomegalovirus target for cytomegalovirus 67
cgtgtatgcc actttgatat tacacccgtg aacgtgctca tcgacgtgaa cccgcacaac
60 gagct 65 68 65 DNA Cytomegalovirus target for mutant
cytomegalovirus 68 cgttgtgcgg gttcacgtcg atgagcacgt tcacgggtgt
aatatcaaag tggcatacac 60 gagct 65 69 26 DNA Cytomegalovirus probe
for cytomegalovirus 69 tcacacagga aacagctatg accatg 26 70 24 DNA
Artificial Sequence Description of Artificial SequenceM13 forward
probe 70 gcaaggcgat taagttgggt aacg 24 71 21 DNA Cytomegalovirus
cytomegalovirus probe 71 cactttgata ttacacccat g 21 72 21 DNA
Cytomegalovirus probe for cytomegalovirus 72 cactttgata ttacacccgt
g 21 73 18 DNA Homo sapiens CAH oligo 6 for mutation site 1 73
cggagcctcc acctcccg 18 74 18 DNA Homo sapiens CAH oligo 2 for
mutation site 2 74 caccctccag cccccagc 18 75 18 DNA Homo sapiens
CAH oligo 1 for mutation site 1 75 cggagcctcc acctcctg 18 76 19 DNA
Homo sapiens CAH oligo 3 for mutation site 3 76 cctcacctgc
agcatcaac 19 77 18 DNA Homo sapiens CAH oligo 7 for mutation site 2
77 caccctccag cccccaac 18 78 19 DNA Homo sapiens CAH oligo 8 for
mutation site 3 78 cctcacctgc agcatcatc 19 79 15 DNA Homo sapiens
CAH oligo 4 for mutation site 4 79 cctggaaggg cactt 15 80 15 DNA
Homo sapiens CAH oligo site 9 for mutation site 4 80 cctggaaggg
cacgt 15 81 18 DNA Homo sapiens CAH oligo 5 for mutation site 5 81
gattcagcag cgactgta 18 82 18 DNA Homo sapiens CAH oligo 10 for
mutation site 5 82 gattcagcag cgactgca 18 83 18 DNA Homo sapiens
CAH oligo 11 for mutation site 6 83 cgaggtgctg cgcctgcg 18 84 18
DNA Homo sapiens CAH oligo 12 for mutation site 6 84 cgaggtgctg
cgcctgtg 18 85 20 DNA Homo sapiens CAH oligo 23 for mutation site 7
85 gggatcacat cgtggagatg 20 86 20 DNA Homo sapiens CAH oligo 24 for
mutation site 7 86 gggatcacaa cgaggagaag 20 87 15 DNA Artificial
Sequence Description of Artificial Sequenceprobe to Alu1 human gene
87 agaccccatc tctaa 15 88 17 DNA Artificial Sequence Description of
Artificial Sequenceprobe to Alu2 human gene 88 gcctgggtga cagagca
17 89 29 DNA Artificial Sequence Description of Artificial
Sequenceprobe which forms hairpin when allowed to self-anneal 89
atgaacgtac gtcggatgag cacgttcat 29 90 29 DNA Artificial Sequence
Description of Artificial Sequenceprobe which forms hairpin when
allowed to self-anneal 90 gtgaacgtac gtcggatgag cacgttcat 29 91 29
DNA Artificial Sequence Description of Artificial Sequenceprobe
which forms hairpin when allowed to self-anneal 91 ataaacgtac
gtcggatgag cacgttcat 29 92 24 DNA Artificial Sequence Description
of Artificial Sequenceprobe which forms hairpin when allowed to
self-anneal 92 ataaacgtac gtcggatgag cacg 24 93 68 DNA Artificial
Sequence Description of Artificial Sequenceprobe to Alu which forms
a hairpin 93 ctccagcctc ggtgacagag caagaccctg tctcaaaaaa aaagcctcgg
tgcagggtct 60 tgctctgt
68 94 69 DNA Artificial Sequence Description of Artificial
Sequenceprobe to Alu which forms a hairpin 94 ctccagcctg agcaacacag
caagaccctg tctcaaaaca aaacgcctga gccagggtct 60 tgctgtgtt 69 95 34
DNA Artificial Sequence Description of Artificial Sequenceprobe for
cytochrome B 95 aaactgcagc ccctcagaat gatatttgtc ctca 34 96 35 DNA
Artificial Sequence Description of Artificial Sequenceprobe for
cytochrome B 96 aaaaagcttc catccaacat ctcagcatga tgaaa 35 97 12 DNA
Homo sapiens Description of Artificial Sequencehuman cytochrome B
97 ccagacgcct ca 12 98 12 DNA Homo sapiens human cytochrome B 98
accttcacgc ca 12 99 11 DNA Unknown Description of Unknown
Organismcommon probe to cytochrome B 99 tgccgagacg t 11 100 12 DNA
chicken Description of Artificial Sequencechicken cytochrome B 100
gcagacacat cc 12 101 12 DNA chicken chicken cytochrome B 101
ggaatctcca cg 12 102 12 DNA cow bovine cytochrome B 102 acatacacgc
aa 12 103 12 DNA dog canine cytochrome B 103 atatgcacgc aa 12 104
22 DNA Artificial Sequence Description of Artificial Sequenceprobe
to prothrombin pcr productm, with phosphorothioate linkages between
the first five bases on the 5' end 104 atagcactgg gagcattgag gc 22
105 20 DNA Homo sapiens probe for human prothrombin gene 105
gcacagacgg ctgttctctt 20 106 13 DNA Homo sapiens probe for
Prothrombin mutant 106 gtgattctca gca 13 107 13 DNA Homo sapiens
probe for wild-type Prothrombin 107 gtgattctca gcg 13 108 22 DNA
Artificial Sequence Description of Artificial Sequenceamplification
primer 108 ggagctgcag atgctgacca ac 22 109 20 DNA Artificial
Sequence Description of Artificial Sequenceamplification primer 109
gctactggcc gctgaagggc 20 110 20 DNA Artificial Sequence Description
of Artificial Sequenceinterrogation primer 110 gctgaccatc
aataaggaag 20 111 62 DNA Artificial Sequence Description of
Artificial Sequence hypothetical example 111 cccggagaga cctccttaag
gggccatatt atttcgtcga ttccagtgtt ggccaaacgg 60 at 62 112 41 DNA
Artificial Sequence Description of Artificial Sequence hypothetical
example 112 ggggccatat tatttcgccg tttggccaac actggaatcg a 41 113 77
DNA Artificial Sequence Description of Artificial Sequence
hypothetical example 113 gggcctctct ggaggaattc cccggtataa
taaagcagct aaggtcacaa ccggtttgcc 60 gctttattat accgggg 77 114 16
DNA Artificial Sequence Description of Artificial Sequence
hypothetical example 114 cccggagaga cctcct 16 115 77 DNA Artificial
Sequence Description of Artificial Sequence hypothetical example
115 cccggagaga cctccttaag gggccatatt atttcgtcga ttccagtgtt
ggccaaacgg 60 cgaaataata tggcccc 77 116 35 DNA Escherichia coli 116
ttacccaact taatcagggg gatgtgctgc aaggc 35 117 38 DNA Escherichia
coli 117 cgttacccaa cttaatcgcc ttgcagcaca tccccctt 38 118 50 DNA
Escherichia coli 118 ttacccaact taatcagggg gatgtgctgc aaggcgatta
agttgggtaa 50 119 75 DNA Campylobacter jejuni probe to
Campylobacter jejuni 119 cttgaagcat agttcttgtt tttaaacttt
gtccatcttg agccgcttga gttgagttgc 60 cttagtttta atagt 75 120 70 DNA
Campylobacter jejuni probe to Campylobacter jejuni 120 actattaaaa
ctaaggcaac tcaagcggct caagatggac aaagtttaaa aacaagaact 60
atgcttcaag 70 121 30 DNA Campylobacter jejuni probe to
Campylobacter jejuni 121 agttcttgtt tttaaacttt gtccatcttg 30 122 30
DNA Campylobacter jejuni probe to Campylobacter jejuni 122
caagatggac aaagtttaaa aacaagaact 30 123 11 DNA Human
immunodeficiency virus probe for 'colirep' sequence from E. coli
123 agtgactggg g 11
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