U.S. patent application number 12/748329 was filed with the patent office on 2010-11-11 for methods, compositions, and kits for detecting allelic variants.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Caifu Chen, Ruoying Tan.
Application Number | 20100285478 12/748329 |
Document ID | / |
Family ID | 42781942 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100285478 |
Kind Code |
A1 |
Chen; Caifu ; et
al. |
November 11, 2010 |
Methods, Compositions, and Kits for Detecting Allelic Variants
Abstract
In some embodiments, the present inventions relates generally to
compositions, methods and kits for use in discriminating sequence
variation between different alleles. More specifically, in some
embodiments, the present invention provides for compositions,
methods and kits for quantitating rare (e.g., mutant) allelic
variants, such as SNPs, or nucleotide (NT) insertions or deletions,
in samples comprising abundant (e.g., wild type) allelic variants
with high specificity and selectivity. In particular, in some
embodiments, the invention relates to a highly selective method for
mutation detection referred to as competitive allele-specific
TaqMan PCR ("cast-PCR").
Inventors: |
Chen; Caifu; (Palo Alto,
CA) ; Tan; Ruoying; (Palo Alto, CA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
42781942 |
Appl. No.: |
12/748329 |
Filed: |
March 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12641321 |
Dec 17, 2009 |
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12748329 |
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61258582 |
Nov 5, 2009 |
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61253501 |
Oct 20, 2009 |
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61251623 |
Oct 14, 2009 |
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61186775 |
Jun 12, 2009 |
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61164230 |
Mar 27, 2009 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6883 20130101; C12Q 1/6858 20130101; C12Q 1/686 20130101;
C12Q 1/6858 20130101; C12Q 2561/113 20130101; C12Q 2561/101
20130101; C12Q 2537/161 20130101; C12Q 1/6858 20130101; C12Q
2525/186 20130101; C12Q 2565/107 20130101; C12Q 1/6858 20130101;
C12Q 2535/125 20130101; C12Q 2525/186 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 17, 2009 |
US |
PCT/US2009/006652 |
Claims
1. A method for detecting a first allelic variant of a target
sequence in a nucleic acid sample suspected of comprising at least
a second allelic variant of the target sequence, comprising: a)
forming a first reaction mixture by combining: i) the nucleic acid
sample; ii) a first allele-specific primer, wherein an
allele-specific nucleotide portion of the first allele-specific
primer is complementary to the first allelic variant of the target
sequence; iii) a first allele-specific blocker probe that is
complementary to a region of the target sequence comprising the
second allelic variant, wherein said region encompasses a position
corresponding to the binding position of the allele-specific
nucleotide portion of the first allele-specific primer, and wherein
the first allele-specific blacker probe comprises a minor groove
binder; iv) a first locus-specific primer that is complementary to
a region of the target sequence that is 3' from the first allelic
variant and on the opposite strand; and v) a first detector probe;
b) carrying out an amplification reaction on the first reaction
mixture using the first locus-specific primer and the first
allele-specific primer to form a first amplicon; and c) detecting
the first amplicon by detecting a change in a detectable property
of the first detector probe, thereby detecting the first allelic
variant of the target gene in the nucleic acid sample.
2. The method of claim 1, further comprising using the change in a
detectable property of the first detector probe to quantitate the
first allelic variant.
3. The method of claim 1, further comprising: d) forming a second
reaction mixture by combining: i) the nucleic acid sample; ii) a
second allele-specific primer, wherein an allele-specific
nucleotide portion of the second allele-specific primer is
complementary to the second allelic variant of the target sequence;
iii) a second allele-specific blocker probe that is complementary
to a region of the target sequence comprising the first allelic
variant, wherein said region encompasses a position corresponding
to the binding position of the allele-specific nucleotide portion
of the second allele-specific primer, and wherein the second
allele-specific blocker probe comprises a minor groove binder; iv)
a second locus-specific primer that is complementary to a region of
the target sequence that is 3' from the second allelic variant and
on the opposite strand; and v) a second detector probe; e) carrying
out an amplification reaction on the second reaction mixture using
the second allele-specific primer and the locus-specific primer, to
form a second amplicon; and f) detecting the second amplicon by
detecting a change in a detectable property of the detector probe,
thereby detecting the second allelic variant of the target gene in
the nucleic acid sample.
4. The method of claim 3, further comprising comparing the change
in a detectable property of the first detector probe in the first
reaction mixture to the change in a detectable property of the
second detector probe in the second reaction mixture.
5. The method of claim 1 or 3, wherein said first, second or first
and second allele-specific primer and/or said first, second, or
first and second allele-specific blocker probe comprises at least
one modified base.
6. The method of claim 5, wherein said modified base is an
8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A),
cytosine (C), guanine (G), or thymine (T).
7. The method of claim 5, wherein said modified base is a locked
nucleic acid (LNA) base.
8. The method of claim 5, wherein said modified base is a fdU or
iso dC base.
9. The method of claim 5, wherein said modified base is any
modified base that increases the Tm between matched and mismatched
target sequences or nucleotides.
10. The method of claim 5, wherein said modified base is located at
(a) the 3'-end, (b) the 5'-end, (e) at an internal position or at
any combination of (a), (b) or (c) within said allele-specific
primer and/or allele-specific blocker probe.
11. The method of claim 5, wherein the specificity of said
detecting is improved by the inclusion of said modified base in
said first, second or first and second allele-specific primer
and/or said first, second, or first and second allele-specific
blocker probe as compared to when it is not.
12. The method of claim 11, wherein said improvement is at least 2
fold.
13. The method of claim 1 or 3, wherein the specificity of said
detecting is improved by at least 2 fold as compared to the
specificity of detecting an allelic variant in a nucleic acid
sample using ASB-PCR methods.
14. The method of claim 1 or 3, wherein said carrying out an
amplification reaction comprises a 2-stage cycling protocol.
15. The method of claim 14, wherein the number of cycles in the
first stage of said 2-stage cycling protocol comprises fewer cycles
than the number of cycles used in the second stage.
16. The method of claim 14, wherein said number of cycles in the
first stage is about 90% fewer cycles than said number of cycles in
the second stage.
17. The method of claim 14, wherein said number of cycles in the
first stage is between 3-7 cycles and said number of cycles in the
second stage is between 42-48 cycles.
18. The method of claim 14, wherein the annealing/extension
temperature used during the first cycling stage of said 2-stage
cycling protocol is between 1-3.degree. C. lower than the
annealing/extension temperature used during the second stage.
19. The method of claim 14, wherein said annealing/extension
temperature used during the first cycling stage of said 2-stage
cycling protocol is between 56-59.degree. C. and said
annealing/extension temperature used during said second stage is
between 60-62.degree. C.
20. The method of claim 1, wherein said step (a) is preceded by a
pre-amplification step.
21. The method of claim 20, wherein said pre-amplification step
comprises a multiplex amplification reaction that uses at least two
complete sets of allele-specific primers and locus-specific
primers, wherein each set is suitable or operative for amplifying a
specific polynucleotide of interest.
22. The method of claim 21, wherein the products of said multiplex
amplification reaction are divided into secondary single-plex
amplification reactions, wherein each single-plex amplification
reaction contains at least one primer set previously used in said
multiplex reaction.
23. The method of claim 21, wherein said multiplex amplification
reaction further comprises a plurality of allele-specific blocker
probes.
24. The method of claim 21, wherein said multiplex amplification
reaction is carried out for a number of cycles suitable to keep the
reaction within the linear phase of amplification.
25. A reaction mixture comprising: a) a nucleic acid molecule; b)
an allele-specific primer, wherein an allele-specific nucleotide
portion of the allele-specific primer is complementary to a first
allelic variant of a target sequence; c) an allele-specific blocker
probe that is complementary to a region of the target sequence
comprising a second allelic variant, wherein said region
encompasses a position corresponding to the binding position of the
allele-specific nucleotide portion of the allele-specific primer,
and wherein the allele-specific blocker probe comprises a minor
groove binder; d) a locus-specific primer that is complementary to
a region of the target sequence that is 3' from the first allelic
variant and on the opposite strand; and e) a detector probe.
26. The reaction mixture of claim 25, wherein said allele-specific
primer and/or said allele-specific blocker probe comprises at least
one modified base.
27. The reaction mixture of claim 26, wherein said modified base is
an 8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A),
cytosine (C), guanine (G), or thymine (T).
28. The reaction mixture of claim 26, wherein said modified base is
a locked nucleic acid (LNA) base.
29. The reaction mixture of claim 26, wherein said modified base is
a fdU or iso dC base.
30. The reaction mixture of claim 26, wherein said modified base is
any modified base that increases the Tm between matched and
mismatched target sequences or nucleotides.
31. The reaction mixture of claim 26, wherein said modified base is
located at (a) the 3'-end, (b) the 5'-end, (c) at an internal
position or at any combination of (a), (b) or (c) within said
allele-specific primer and/or allele-specific blacker probe.
32. A composition comprising: a) a first allele-specific primer,
wherein an allele-specific nucleotide portion of the first
allele-specific primer is complementary to the first allelic
variant of a target sequence; b) a first allele-specific blocker
probe that is complementary to a region of the target sequence
comprising the second allelic variant, wherein said region
encompasses a position corresponding to the binding position of the
allele-specific nucleotide portion of the first allele-specific
primer, and wherein the first allele-specific blocker probe
comprises a minor groove binder;
33. The composition of claim 32, further comprising a
locus-specific primer that is complementary to a region of the
target sequence that is 3' from the first allelic variant and on
the opposite strand.
34. The composition of claim 32 or 33, wherein said first
allele-specific primer and/or said first allele-specific blocker
probe comprises at least one modified base.
35. The composition of claim 34, wherein said modified base is an
8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A),
cytosine (C), guanine (G), or thymine (T).
36. The composition of claim 34, wherein said modified base is a
locked nucleic acid (LNA) base.
37. The composition of claim 34, wherein said modified base is a
fdU or iso dC base.
38. The composition of claim 34, wherein said modified base is any
modified base that increases the Tin between matched and mismatched
target sequences or nucleotides.
39. The composition of claim 34, wherein said modified base is
located at (a) the 3'-end, (b) the 5'-end, (c) at an internal
position or at any combination of (a), (b) or (c) within said
allele-specific primer and/or allele-specific blocker probe.
40. A kit comprising, two or more containers comprising the
following components independently distributed in one of the two or
more containers: a) a first allele-specific primer, wherein an
allele-specific nucleotide portion of the first allele-specific
primer is complementary to the first allelic variant of a target
sequence; and b) a first allele-specific blocker probe that is
complementary to a region of the target sequence comprising the
second allelic variant, wherein said region encompasses a position
corresponding to the binding position of the allele-specific
nucleotide portion of the first allele-specific primer, and wherein
the first allele-specific blocker probe comprises a minor groove
binder.
41. The kit of claim 40, further comprising a locus-specific primer
that is complementary to a region of the target sequence that is 3'
from the first allelic variant and on the opposite strand.
42. The kit of claim 41, wherein said first allele-specific primer
and/or first allele-specific blocker probe comprises at least one
modified base.
43. The kit of claim 42, wherein said modified base is a
8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A),
cytosine (C), guanine (G), or thymine (T).
44. The kit of claim 42, wherein said modified base is a locked
nucleic acid (LNA) base.
45. The kit of claim 42, wherein said modified base is any modified
base that increases the Tin between matched and mismatched target
sequences and/or nucleotides.
46. The kit of claim 42, wherein said allele-specific blocker probe
comprises an MGB moiety at the 3'-end, the 5'-end and/or at an
internal position within said allele-specific blocker probe.
47. A method for detecting a first allelic variant in a target
sequence in a nucleic acid sample, comprising: a) forming a
reaction mixture comprising: i) a nucleic acid sample; ii) an
allele-specific primer, wherein an allele-specific nucleotide
portion of the allele-specific primer is complementary to the first
allelic variant of the target sequence; iii) an allele-specific
blocker probe that is complementary to a region of the target
sequence comprising a second allelic variant, wherein said region
encompasses a position corresponding to the binding position of the
allele-specific nucleotide portion of the allele-specific primer,
and wherein the allele-specific blocker probe comprises a blocking
moiety; iv) a locus-specific primer that is complementary to a
region of the target sequence that is 3' from the first allelic
variant and on the opposite strand; and v) a detector probe; b) PCR
amplifying the target sequence using a 2-stage cycling protocol,
comprising: i) a first amplification step comprising a first number
of cycles run at a first annealing/extension temperature; and ii) a
second amplification step comprising a second number of cycles run
at a second annealing/extension temperature, wherein the first
number of cycles is fewer than the second number of cycles and the
first annealing/extension temperature is lower than said second
annealing/extension temperature. c) detecting a change in a
detectable property of the detector probe in the amplified products
of the target sequence produced by step (b), thereby detecting the
first allelic variant of the target gene in the nucleic acid
sample.
48. The method of claim 47, wherein said number of cycles in the
first step is about 90% fewer cycles than said number of cycles in
the second step.
49. The method of claim 14, wherein said number of cycles in the
first step is between 3-7 cycles and said number of cycles in the
second step is between 42-48 cycles.
50. The method of claim 14, wherein the annealing/extension
temperature used during the first step of said 2-stage cycling
protocol is between 1-3.degree. C. lower than the
annealing/extension temperature used during the second step.
51. The method of claim 14, wherein said annealing/extension
temperature used during the first step of said 2-stage cycling
protocol is between 56-59.degree. C. and said annealing/extension
temperature used during said second step is between 60-62.degree.
C.
52. A method for detecting an allelic variant of a target sequence
in a nucleic acid sample, comprising: a) forming in a single vessel
a first reaction mixture comprising: i) a nucleic acid sample; and
ii) at least two sets of primers wherein each set comprises (a) a
first allele-specific primer and a second allele-specific primer,
wherein the allele-specific nucleotide portion of said first and
second allele-specific primers is complementary to a first allele
and a second allele, respectively, of a given SNP within a target
sequence; and (b) a locus-specific primer that is complementary to
a region of said target sequence that is 3' from the first and
second alleles and on the opposite strand; wherein the at least two
sets of primers is each specific for a different target sequence;
b) amplifying said different target sequences using a number of
cycles suitable to keep the reaction within a linear phase; c)
dividing the amplified products of step (b) into at least two
separate vessels; d) adding to each of the divided products of step
(c) i) at least one set of primers used in step (a), ii) an
allele-specific blocker probe that is complementary to a region of
said target sequence comprising a second allelic variant, wherein
said region encompasses a position corresponding to the binding
position of the allele-specific nucleotide portion of the first
allele-specific primer, and wherein the first allele-specific
blacker probe comprises a minor groove binder; and iv) a detector
probe to form a second and a third reaction mixture in the at least
two separate vessels; e) amplifying said target sequence in said
second and third reaction mixtures; and f) detecting a change in a
detectable property of the detector probe in each of the amplified
products of said target sequence produced by step (e), thereby
detecting the allelic variant of the target gene in the nucleic
acid sample.
53. The method of claim 52, wherein said first reaction mixture is
a multiplex reaction and said second and third reaction mixtures
are single-plea reactions.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 12/641,321, filed Dec. 17, 2009 and claims the
benefit of priority under 35 U.S.C. 119 to U.S. Provisional
Application Nos. 61/258,582, filed Nov. 5, 2009; 61/253,501, filed
Oct. 20, 2009; 61/251,623, filed Oct. 14, 2009; 61/186,775, filed
Jun. 12, 2009; and 61/164,230, filed Mar. 27, 2009, all of which
are incorporated herein by reference in their entireties.
BACKGROUND
[0002] Single nucleotide polymorphisms (SNPs) are the most common
type of genetic diversity in the human genome, occurring at a
frequency of about one SNP in 1,000 nucleotides or less in human
genomic DNA (Kwok, P-Y, Ann Rev Genom Hum Genet 2001, 2: 235-258).
SNPs have been implicated in genetic disorders, susceptibility to
different diseases, predisposition to adverse reactions to drugs,
and for use in forensic investigations. Thus, SNP (or rare
mutation) detection provides great potentials in diagnosing early
phase diseases, such as detecting circulating tumor cells in blood,
for prenatal diagnostics, as well as for detection of
disease-associated mutations in a mixed cell population.
[0003] Numerous approaches for SNP genotyping have been developed
based on methods involving hybridization, ligation, or DNA
polymerases (Chen, X., and Sullivan, P F, The Pharmacogeonomics
Journal 2003, 3, 77-96.). For example, allele-specific polymerase
chain reaction (AS-PCR) is a widely used strategy for detecting DNA
sequence variation (Wu D Y, Ugozzoli L, Pal B K, Wallace R B., Proc
Natl Acad Sci USA 1989; 86:2757-2760). AS-PCR, as its name implies,
is a PCR-based method whereby one or both primers are designed to
anneal at sites of sequence variations which allows for the ability
to differentiate among different alleles of the same gene. AS-PCR
exploits the fidelity of DNA polymerases, which extend primers with
a mismatched 3' base at much lower efficiency, from 100 to 100,000
fold less efficient, than that with a matched 3' base (Chen, X.,
and Sullivan, P F, The Pharmacogeonomics Journal 2003; 3:77-96).
The difficulty in extending mismatched primers results in
diminished PCR amplification that can be readily detected.
[0004] The specificity and selectivity of AS-PCR, however, is
largely dependent on the nature of exponential amplification of PCR
which makes the decay of allele discriminating power rapid. Even
though primers are designed to match a specific variant to
selectively amplify only that variant, in actuality significant
mismatched amplification often occurs. Moreover, the ability of
AS-PCR to differentiate between allelic variants can be influenced
by the type of mutation or the sequence surrounding the mutation or
SNP (Ayyadevara S, Thaden J J, Shmookler Reis R J., Anal Biochem
2000; 284:11-18), the amount of allelic variants present in the
sample, as well as the ratio between alternative alleles.
Collectively, these factors are often responsible for the frequent
appearance of false-positive results, leading many researchers to
attempt to increase the reliability of AS-PCR (Orou A, Fechner B,
Utermann G, Menzel H J., Hum Mutat 1995; 6:163-169) (Imyanitov E N,
Busboy K G, Suspitsin E N, Kuligina E S, Belogubova E V, Grigoriev
M Y, et al., Biotechniques 2002; 33:484-490) (McKinzie P B, Parsons
B L. Detection of rare K-ras codon 12 mutations using
allele-specific competitive blocker PCR. Mutat Res 2002;
517:209-220) (Latorra D, Campbell K, Wolter A, Hurley J M., Hum
Mutat 2003; 22:79-85).
[0005] In some cases, the selectivity of AS-PCR has been increased
anywhere from detection of 1 in 10 alleles to 1 in 100,000 alleles
by using SNP-based PCR primers containing locked nucleic acids
(LNAs) (Latorra, D., et al., Hum Mut 2003, 2:79-85; Nakiandwe, J.
et al., Plant Method 2007, 3:2) or modified bases (Koizumi, M. et
al. Anal Biochem. 2005, 340:287-294). However, these base "mimics"
or modifications increase the overall cost of analysis and often
require extensive optimization.
[0006] Another technology involving probe hybridization methods
used for discriminating allelic variations is TaqMan.RTM.
genotyping. However, like AS-PCR, selectivity using this method is
limited and not suitable for detecting rare (1 in .gtoreq.1,000)
alleles or mutations in a mixed sample.
SUMMARY
[0007] In some embodiments, the present inventions relates
generally to compositions, methods and kits for use in
discriminating sequence variation between different alleles. More
specifically, in some embodiments, the present invention provides
for compositions, methods and kits for quantitating rare (e.g.,
mutant) allelic variants, such as SNPs, or nucleotide (NT)
insertions or deletions, in samples comprising abundant (e.g., wild
type) allelic variants with high specificity. In particular, in
some embodiments, the invention relates to a highly selective
method for mutation detection referred to as competitive
allele-specific TaqMan PCR ("cast-PCR").
[0008] In one aspect, the present invention provides compositions
for use in identifying and/or quantitating allelic variants in
nucleic acid samples. Some of these compositions can comprise: (a)
an allele-specific primer; (b) an allele-specific blocker probe;
(c) a detector probe; and/or (d) a locus-specific primer.
[0009] In some embodiments of the compositions, the allele-specific
primer comprises a target-specific portion and an allele-specific
nucleotide portion. In some embodiments, the allele-specific primer
may further comprise a tail. In some exemplary embodiments, the
tail is located at the 5' end of the allele-specific primer. In
other embodiments, the tail of the allele-specific primer has
repeated guanine and cytosine residues ("GC-rich"). In some
embodiments, the melting temperature ("Tm") of the entire
allele-specific primer ranges from about 50.degree. C. to
67.degree. C. In some embodiments, the allele-specific primer
concentration is between about 20-900 nM.
[0010] In some embodiments of the compositions, the allele-specific
nucleotide portion of the allele-specific primer is located at the
3' terminus. In some embodiments, the selection of the
allele-specific nucleotide portion of the allele-specific primer
involves the use of a highly discriminating base (e.g., for
detection of A/A, A/G, G/A, G/G, A/C, or C/A alleles). In some
embodiments, for example when the allele to be detected involves
A/G or C/T SNPs, A or G is used as the 3' allele-specific
nucleotide portion of the allele-specific primer (e.g., if A or T
is the target allele), or C or T is used as the 3' allele-specific
nucleotide portion of the allele-specific primer (e.g., if C or G
is the target allele). In other embodiments, A is used as the
discriminating base at the 3' end of the allele-specific primer
when detecting and/or quantifying A/T SNPs. In other embodiments, G
is used as the discriminating base at the 3' end of the
allele-specific primer when detecting and/or quantifying C/G
SNPs.
[0011] In some embodiments of the compositions, the allele-specific
blocker probe comprises a non-extendable blocker moiety at the 3'
terminus. In some exemplary embodiments, the non-extendable blocker
moiety is a minor groove binder (MGB). In some embodiments, the
target allele position is located about 6-10, such as about 6,
about 7, about 8, about 9, or about 10 nucleotides away from the
non-extendable blacker moiety of the allele-specific blocker probe.
In some embodiments, the allele-specific blocker probe comprises an
MGB moiety at the 5' terminus. In some exemplary embodiments, the
allele-specific blocker probe is not cleaved during PCR
amplification. In some embodiments, the Tm of the allele-specific
blocker probe ranges from about 58.degree. C. to 66.degree. C.
[0012] In some embodiments of the compositions, the allele-specific
blocker probe and/or allele-specific primer comprise at least one
modified base. In some embodiments, the modified base(s) may
increase the difference in the Tm between matched and mismatched
target sequences and/or decrease mismatch priming efficiency,
thereby improving not only assay specificity bust also selectivity.
Such modified base(s) may include, for example, 8-Aza-7-deaza-dA
(ppA), 8-Aza-7-deaza-dG (ppG), 2'-Deoxypseudoisocytidine (iso dC),
5-fluoro-2'-deoxyuridine (fdU), locked nucleic acid (LNA), or
2'-O,4'-C-ethylene bridged nucleic acid (ENA) bases (also see, for
example, FIG. 4B). In some embodiments the modified base is located
at (a) the 3'-end, (b) the 5'-end, (c) at an internal position or
at any combination of (a), (b) or (c) within the allele-specific
blocker probe and/or the allele-specific primer.
[0013] In some embodiments of the compositions, the detector probe
is a sequence-based or locus-specific detector probe. In other
embodiments the detector probe is a 5' nuclease probe. In some
exemplary embodiments, the detector probe can comprises an MGB
moiety, a reporter moiety (e.g., FAM.TM., TET.TM., JOE.TM.,
VTC.TM., or SYBR.RTM. Green), a quencher moiety (e.g., Black Hole
Quencher.TM. or TAMRA.TM.;), and/or a passive reference (e.g.,
ROX.TM.). In some exemplary embodiments, the detector probe is
designed according to the methods and principles described in U.S.
Pat. No. 6,727,356 (the disclosure of which is incorporated herein
by reference in its entirety). In some exemplary embodiments, the
detector probe is a TaqMan.RTM. probe (Applied Biosystems, Foster
City).
[0014] In some embodiments of the compositions, the composition can
further comprise a polymerase; deoxyribonucleotide triphosphates
(dNTPs); other reagents and/or buffers suitable for amplification;
and/or a template sequence or nucleic acid sample. In some
embodiments, the polymerase can be a DNA polymerase. In some other
embodiments, the polymerase can be thermostable, such as Tag DNA
polymerase. In other embodiments, the template sequence or nucleic
acid sample can be DNA, such as genomic DNA (gDNA) or complementary
DNA (cDNA). In other embodiments the template sequence or nucleic
acid sample can be RNA, such as messenger RNA (mRNA).
[0015] In another aspect, the present disclosure provides methods
for amplifying an allele-specific sequence. Some of these methods
can include one or more of the following: (a) hybridizing an
allele-specific primer to a first nucleic acid molecule comprising
a first allele (allele-1); (b) hybridizing an allele-specific
blocker probe to a second nucleic acid molecule comprising a second
allele (allele-2), wherein allele-2 corresponds to the same loci as
allele-1; (c) hybridizing a detector probe to the first nucleic
acid molecule; (d) hybridizing a locus-specific primer to the
extension product of the allele-specific primer; and (e) PCR
amplifying the first nucleic acid molecule comprising allele-1.
[0016] In another aspect, the present invention provides methods
for detecting and/or quantitating an allelic variant in a pooled or
mixed sample comprising other alleles. Some of these methods can
include one or more of the following: (a) in a first reaction
mixture hybridizing a first allele-specific primer to a first
nucleic acid molecule comprising a first allele (allele-1) and in a
second reaction mixture hybridizing a second allele-specific primer
to a first nucleic acid molecule comprising a second allele
(allele-2), wherein allele-2 corresponds to the same locus as
allele-1; (b) in the first reaction mixture hybridizing a first
allele-specific blocker probe to a second nucleic acid molecule
comprising allele-2 and in the second reaction mixture hybridizing
a second allele-specific blocker probe to a second nucleic acid
molecule comprising allele-1; (c) in the first reaction mixture,
hybridizing a first detector probe to the first nucleic acid
molecule and in the second reaction mixture and hybridizing a
second detector probe to the first nucleic acid molecule; (d) in
the first reaction mixture hybridizing a first locus-specific
primer to the extension product of the first allele-specific primer
and in the second reaction mixture hybridizing a second
locus-specific primer to the extension product of the second
allele-specific primer; and (e) PCR amplifying the first nucleic
acid molecule to form a first set or sample of amplicons and PCR
amplifying the second nucleic acid molecule to form a second set or
sample of amplicons; and (f) comparing the first set of amplicons
to the second set of amplicons to quantitate allele-1 in the sample
comprising allele-2 and/or allele-2 in the sample comprising
allele-1.
[0017] In some embodiments of the methods, the first and/or second
allele-specific primer comprises a target-specific portion and an
allele-specific nucleotide portion. In some embodiments, the first
and/or second allele-specific primer may further comprise a tail.
In some embodiments, the Tm of the entire first and/or second
allele-specific primer ranges from about 50.degree. C. to
67.degree. C. In some embodiments the first and/or second
allele-specific primer concentration is between about 20-900
nM.
[0018] In some embodiments of the methods, the target-specific
portion of the first allele-specific primer and the target-specific
portion of the second allele-specific primer comprise the same
sequence. In other embodiments, the target-specific portion of the
first allele-specific primer and the target-specific portion of the
second allele-specific primer are the same sequence.
[0019] In some embodiments of the methods, the tail is located at
the 5'-end of the first and/or second allele-specific primer. In
some embodiments, the 5' tail of the first allele-specific primer
and the 5' tail of the second allele-specific primer comprise the
same sequence. In other embodiments, the 5' tail of the first
allele-specific primer and the 5' tail of the second
allele-specific primer are the same sequence. In other embodiments,
the tail of the first and/or second allele-specific primer is
GC-rich.
[0020] In some embodiments of the methods, the allele-specific
nucleotide portion of the first allele-specific primer is specific
to a first allele (allele-1) of a SNP and the allele-specific
nucleotide portion of the second allele-specific primer is specific
to a second allele (allele-2) of the same SNP. In some embodiments
of the methods, the allele-specific nucleotide portion of the first
and/or second allele-specific primer is located at the 3'-terminus.
In some embodiments, the selection of the allele-specific
nucleotide portion of the first and/or second allele-specific
primer involves the use of a highly discriminating base (e.g., for
detection of A/A, A/G, G/A, G/G, A/C, or C/A alleles). In some
embodiments, for example when the allele to be detected involves
A/G or C/T SNPs, A or G is used as the 3' allele-specific
nucleotide portion of the first and/or second allele-specific
primer (e.g., if A or T is the major allele), or C or T is used as
the 3' allele-specific nucleotide portion of the first and/or
second allele-specific primer (e.g., if C or G is the major
allele). In other embodiments, A is used as the discriminating base
at the 3' end of the first and/or second allele-specific primer
when detecting and/or quantifying A/T SNPs. In other embodiments, G
is used as the discriminating base at the 3' end of the first
and/or second allele-specific primer when detecting and/or
quantifying C/G SNPs.
[0021] In some embodiments of the methods, the first and/or second
allele-specific blocker probe comprises a non-extendable blocker
moiety at the 3' terminus. In some exemplary embodiments, the
non-extendable blocker moiety is an MGB. In some embodiments, the
target allele position is located about 6-10, such as about 6,
about 7, about 8, about 9, or about 10 nucleotides away from the
non-extendable blocker moiety of the first and/or second
allele-specific blocker probe. In some embodiments, the first
and/or second allele-specific blocker probe comprises an MGB moiety
at the 5'-terminus. In other embodiments, the first and/or second
allele-specific blocker probe is not cleaved during PCR
amplification. In some embodiments, the Tm of the first and/or
second allele-specific blocker probe ranges from about 58.degree.
C. to 66.degree. C.
[0022] In some embodiments of the methods, the first and/or second
allele-specific blocker probe and/or the first and/or second
allele-specific primer comprises at least one modified base. In
some embodiments, the modified base(s) may increase the difference
in the Tm between matched and mismatched target sequences and/or
decrease mismatch priming efficiency, thereby improving not only
assay specificity, but also selectivity. Such modified base(s) may
include, for example, 8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG
(ppG), 2'-Deoxypseudoisocytidine (iso dC), 5-fluoro-2'-deoxyuridine
(fdU), locked nucleic acid (LNA), or 2'-O,4'-C-ethylene bridged
nucleic acid (ENA) bases (see also, for example, FIG. 4B). In some
embodiments the modified base is located at (a) the 3'-end, (b) the
5'-end, (c) at an internal position or at any combination of (a),
(b) or (c) within said first and/or second allele-specific blocker
probe and/or the first and/or second allele-specific primer.
[0023] In some embodiments of the methods, the specificity of
allelic discrimination is improved by the inclusion of a modified
base in the first and/or second allele-specific primer and/or
first, and/or second allele-specific blocker probe as compared to
the use of a non-modified allelic-specific primer or blocker probe.
In some embodiments, the improvement in specificity is at least 2
fold.
[0024] In some embodiments of the methods, the specificity of
allelic discrimination is at least 2 fold (e.g., 2 fold, 3 fold, 4,
fold, 5 fold, and so on) better than the specificity of allelic
discrimination using Allele-Specific PCR with a Blocking reagent
(ASB-PCR) methods.
[0025] In some embodiments, the methods further comprise a 2-stage
cycling protocol. In some embodiments, the number of cycles in the
first stage of the 2-stage cycling protocol comprises fewer cycles
than the number of cycles used in the second stage. In other
embodiments, the number of cycles in the first stage is about 90%
fewer cycles than the number of cycles in the second stage. In yet
other embodiments, the number of cycles in the first stage is
between 3-7 cycles and the number of cycles in the second stage is
between 42-48 cycles.
[0026] In some embodiments, the annealing/extension temperature
used during the first cycling stage of the 2-stage cycling protocol
is between 1-3.degree. C. lower than the annealing/extension
temperature used during the second stage. In preferred embodiments,
the annealing/extension temperature used during the first cycling
stage of the 2-stage cycling protocol is between 56-59.degree. C.
and the annealing/extension temperature used during the second
stage is between 60-62.degree. C.
[0027] In some embodiments, the methods further comprise a
pre-amplification step. In preferred embodiments, the
pre-amplification step comprises a multiplex amplification reaction
that uses at least two complete sets of allele-specific primers and
locus-specific primers, wherein each set is suitable or operative
for amplifying a specific polynucleotide of interest. In other
embodiments, the products of the multiplex amplification reaction
are divided into secondary single-plex amplification reactions,
such as a cast-PCR reaction, wherein each single-plex reaction
contains at least one primer set previously used in the multiplex
reaction. In other embodiments, the multiplex amplification
reaction further comprises a plurality of allele-specific blocker
probes. In some embodiments, the multiplex amplification reaction
is carried out for a number of cycles suitable to keep the reaction
within the linear phase of amplification.
[0028] In some embodiments of the methods, the first and/or second
detector probes are the same. In some embodiments, the first and/or
second detector probes are different. In some embodiments, the
first and/or second detector probe is a sequence-based or
locus-specific detector probe. In other embodiments the first
and/or second detector probe is a 5' nuclease probe. In some
exemplary embodiments, the first and/or second detector probes
comprises an MGB moiety, a reporter moiety (e.g., FAM.TM., TET.TM.,
JOE.TM., VIC.TM., or SYBR.RTM. Green), a quencher moiety (e.g.,
Black Hole Quencher.TM. or TAMRA.TM.;), and/or a passive reference
(e.g., ROX.TM.). In some exemplary embodiments, the first and/or
second detector probe is designed according to the methods and
principles described in U.S. Pat. No. 6,727,356 (the disclosure of
which is incorporated herein by reference in its entirety). In some
exemplary embodiments, the detector probe is a TaqMan.RTM.
probe.
[0029] In some embodiments of the methods, the first locus-specific
primer and the second locus-specific primer comprise the same
sequence. In some embodiments the first locus-specific primer and
the second locus-specific primer are the same sequence.
[0030] In some embodiments of the methods, the first and/or second
reaction mixtures can further comprises a polymerase; dNTPs; other
reagents and/or buffers suitable for PCR amplification; and/or a
template sequence or nucleic acid sample. In some embodiments, the
polymerase can be a DNA polymerase. In some embodiments, the
polymerase can be thermostable, such as Taq DNA polymerase. In some
embodiments, the template sequence or nucleic acid sample can be
DNA, such as gDNA or cDNA. In other embodiments the template
sequence or nucleic acid sample can be RNA, such as mRNA.
[0031] In some embodiments of the methods, the first
allele-specific blocker probe binds to the same strand or sequence
as the second allele-specific primer, while the second
allele-specific blocker probe binds to the same strand or sequence
as the first allele-specific primer. In some embodiments, the first
and/or second allele-specific blocker probes are used to reduce the
amount of background signal generated from either the second allele
and/or the first allele, respectively. In some embodiments, first
and/or second allele-specific blocker probes are non-extendable and
preferentially anneal to either the second allele or the first
allele, respectively, thereby blocking the annealing of, for
example, the extendable first allele-specific primer to the second
allele and/or the extendable second allele-specific primer to first
allele.
[0032] In some exemplary embodiments, the first allele is a rare
(e.g., minor) or mutant allele. In other exemplary embodiments the
second allele is an abundant (e.g., major) or wild type allele.
[0033] In another aspect, the present invention provides kits for
quantitating a first allelic variant in a sample comprising a
second allelic variant involving: (a) a first allele-specific
primer; (b) a second allele-specific primer; (e), a first
locus-specific primer; (d) a second locus-specific primer; (e) a
first allele-specific blocker probe; (f) a second allele-specific
blocker probe; and (g) a first locus-specific detector probe and
(h) a second locus-specific detector probe.
[0034] In some embodiments of the kits, the first and/or second
allele-specific primer comprises a target-specific portion and an
allele-specific nucleotide portion. In some embodiments, the first
and/or second allele-specific primer may further comprise a tail.
In some embodiments, the Tm of the entire first and/or second
allele-specific primer ranges from about 50.degree. C. to
67.degree. C. In some embodiments the first and/or second
allele-specific primer concentrations are between about 20-900
nM.
[0035] In some embodiments of the kits, the target-specific portion
of the first allele-specific primer and the target-specific portion
of the second allele-specific primer comprise the same sequence. In
other embodiments, the target-specific portion of the first
allele-specific primer and the target-specific portion of the
second allele-specific primer are the same sequence.
[0036] in some embodiments of the kits, the tail is located at the
5' end of the first and/or second allele-specific primer. In some
embodiments, the 5' tail of the first allele-specific primer and
the 5' tail of the second allele-specific primer comprise the same
sequence. In other embodiments, the 5' tail of the first
allele-specific primer and the 5' tail of the second
allele-specific primer are the same sequence. In other embodiments,
the tail of the first and/or second allele-specific primer is GC
rich.
[0037] In some embodiments of the kits, the allele-specific
nucleotide portion of the first allele-specific primer is specific
to a first allele (allele-1) of a SNP and the allele-specific
nucleotide portion of the second allele-specific primer is specific
to a second allele (allele-2) of the same SNP. In some embodiments
of the disclosed methods, the allele-specific nucleotide portion of
the first and/or second allele-specific primer is located at the 3'
terminus. In some embodiments, the selection of the allele-specific
nucleotide portion of the first and/or second allele-specific
primer involves the use of a highly discriminating base (e.g., for
detection of A/A, A/G, G/A, G/G, A/C, or C/A alleles) (FIG. 2). In
some embodiments, for example when the allele to be detected
involves A/G or C/T SNPs, A or G is used as the 3' allele-specific
nucleotide portion of the first and/or second allele-specific
primer (e.g., if A or T is the major allele), or C or T is used as
the 3' allele-specific nucleotide portion of the first and/or
second allele-specific primer (e.g., if C or G is the major
allele). In other embodiments, A is used as the discriminating base
at the 3' end of the first and/or second allele-specific primer
when detecting and/or quantifying A/T SNPs. In other embodiments, G
is used as the discriminating base at the 3' end of the first
and/or second allele-specific primer when detecting and/or
quantifying C/G SNPs.
[0038] In some embodiments of the kits, the first and/or second
allele-specific blocker probe comprises a non-extendable blocker
moiety at the 3' terminus. In some exemplary embodiments, the
non-extendable blocker moiety is an MGB. In some embodiments, the
target allele position is located about 6-10, such as about 6,
about 7, about 8, about 9, or about 10 nucleotides away from the
non-extendable blocker moiety of the first and/or second
allele-specific blocker probe. In some embodiments, the first
and/or second allele-specific blocker probe comprises an MGB moiety
at the 5' terminus. In other embodiments, the first and/or second
allele-specific blocker probe is not cleaved during PCR
amplification. In some embodiments, the Tm of the first and/or
second allele-specific blocker probe ranges from about 58.degree.
C. to 66.degree. C.
[0039] In some embodiments of the kits, the allele-specific blocker
probe and/or the first and/or second allele-specific primer
comprises at least one modified base. In some embodiments, the
modified base(s) may increase the difference in the Tm between
matched and mismatched target sequences and/or decrease mismatch
priming efficiency, thereby improving not only assay specificity
bust also selectivity. Such modified base(s) may include, for
example, 8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG (ppG),
2'-Deoxypseudoisocytidine (iso dC), 5-fluoro-2'-deoxyuridine (fdU),
locked nucleic acid (LNA), or 2'-O,4'-C-ethylene bridged nucleic
acid (ENA) bases (see also, for example, FIG. 4B). In some
embodiments the modified base is located at (a) the 3'-end, (b) the
5'-end, (c) at an internal position or at any combination of (a),
(b) or (c) within said first and/or second allele-specific blocker
probe and/or the first and/or second allele-specific primer.
[0040] In some embodiments of the kits, the first and/or second
detector probes are the same. In some embodiments of the disclosed
kits the first and/or second detector probes are different. In some
embodiments of the disclosed kits, the first and/or second detector
probes are sequence-based or locus-specific detector probes. In
other embodiments the first and/or second detector probe are 5'
nuclease probes. In some exemplary embodiments, the first and/or
second detector probes comprise an MGB moiety, a reporter moiety
(e.g., FAM.TM., TET.TM., JOE.TM., VIC.TM., or SYBR.RTM. Green), a
quencher moiety (e.g., Black Hole Quencher.TM. or TAMRA.TM.;),
and/or a passive reference (e.g., ROX.TM.). In some exemplary
embodiments, the first and/or second detector probe are designed
according to the methods and principles described in U.S. Pat. No.
6,727,356 (the disclosure of which is incorporated herein by
reference in its entirety). In some exemplary embodiments, the
detector probe is a TagMan.RTM. probe.
[0041] In some embodiments of the kits, the first locus-specific
primer and the second locus-specific primer comprise the same
sequence. In some embodiments the first locus-specific primer and
the second locus-specific primer are the same sequence.
[0042] In some embodiments of the kits, the first and/or second
reaction mixture can further comprise a polymerase; dNTPs; other
reagents and/or buffers suitable for PCR amplification; and/or a
template sequence or nucleic acid sample. In some embodiments, the
polymerase can be a DNA polymerase. In some other embodiments, the
polymerase can be thermostable, such as Tag DNA polymerase.
[0043] In some embodiments, the compositions, methods and kits of
the present invention provide high allelic discrimination
specificity and selectivity. In some embodiments, the quantitative
determination of specificity and/or selectivity comprises a
comparison of Ct values between a first set of amplicons and a
second set of amplicons. In some embodiments, selectivity is at a
level whereby a single copy of a given allele in about 1 million
copies of another allele or alleles can be detected.
[0044] The foregoing has described various embodiments of the
invention that provide improved detection and discrimination of
allelic variants using one or more of the following: (a) tailed
allele-specific primers; (b) low allele-specific primer
concentration; (c) allele-specific primers designed to have lower
Tms; (d) allele-specific primers designed to target discriminating
bases; (e) allele-specific blocker probes containing MGB, designed
to prevent amplification from alternative, and potentially more
abundant, allelic variants in a sample; and (f) allele-specific
blocker probes and/or allele-specific primers designed to comprise
modified bases in order to increase the delta Tm between matched
and mismatched target sequences.
[0045] While particular embodiments employing several of the above
improvements have been discussed herein, it will be apparent to the
skilled artisan that depending on the nature of the sample to be
examined, various combinations of the above improvements can be
combined to arrive at a favorable result. Thus, for example,
non-MGB blocker probes can be used with an embodiment that include
methods employing allele-specific primers containing modified bases
to increase delta Tm; such primers can also be designed to target
discriminating bases; and the primers can be used at low primer
concentrations. Accordingly, alternative embodiments based upon the
present disclosure can be used to achieve a suitable level of
allelic detection.
[0046] The present disclosure provides the advantage that any of
the combinations of listed improvements could be utilized by a
skilled artisan in a particular situation. For example, the current
invention can include a method or reaction mixture that employs
improvements a, c, d and f; improvements b, c, and e; or
improvements
[0047] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed.
[0048] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
exemplary embodiments of the disclosure and together with the
description, serve to explain certain teachings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] The skilled artisan will understand that the drawings
described below are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0050] FIG. 1 depicts a schematic of an illustrative embodiment of
cast-PCR. In some embodiments, components of cast-PCR include the
following: one locus-specific TaqMan probe (LST); two MGB blockers:
one allele-1-specific MGB blocker (MGB1) and one allele-2-specific
MGB blocker (MGB2); 3 PCR primers: one locus-specific PCR primer
(LSP); one allele-1-specific primer (ASP1) and one
allele-2-specific primer (ASP2).
[0051] FIG. 2 depicts a schematic of an illustrative embodiment of
cast-PCR using allele-specific blocker probes comprising highly
discriminating bases for detecting rare allelic variants. Highly
discriminating bases may include, for example, A/A, A/G, G/A, G/G,
A/C, C/A. The least discriminating bases may include, for example,
C/C, T/C, G/T, T/G, C/T. In some embodiments, for example, for
detection of A-G or C-T SNPs, A & G are used as the
discriminating base if A//T is allelic variant (e.g., mutant
allele); or C & T are used as the discriminating base if C//G
allelic variant (e.g., mutant allele).
[0052] FIG. 3 depicts a schematic of an illustrative embodiment of
cast-PCR using an allele-specific blocker probe with an MGB moiety
at the 5' end. In some embodiments the blocker moiety at the 3'-end
of the probe may include, for example, NH.sub.2, biotin, MGB,
PO.sub.4, and PEG.
[0053] FIG. 4A depicts a schematic of an illustrative embodiment of
cast-PCR using modified bases in an MGB blocker probe or
allele-specific primer. (G* represents ppG.)
[0054] FIG. 4B depicts some examples of modified bases of an MGB
blocker probe or allele-specific primer.
[0055] FIG. 5 depicts the TaqMan-like sensitivity and dynamic range
of one exemplary embodiment of cast-PCR.
[0056] FIG. 6 depicts the sequence of KRAS mutations at codons 12
and 13 that are detectable using cast-PCR methods. KRAS mutations
at codons 12 and 13 are associated with resistance to cetuxima or
panitumumab in metastatic colorectal cancer (Di Nicolantonio F., et
al., J Clin Oncol. 2008; 26:5705-12.)
[0057] FIG. 7 depicts the specificity of KRAS mutation detection
using cast-PCR assays in one exemplary embodiment.
[0058] FIG. 8 depicts one exemplary embodiment using cast-PCR
methods to detect a single copy of mutant DNA in 10.sup.6 copies of
wild-type DNA.
[0059] FIG. 9 depicts detection of the relative copy number of
mutant samples (KRAS-G12A) spiked in wild type samples using
cast-PCR methods.
[0060] FIG. 10 depicts a number of different tumor markers (SNPs)
detected in tumor samples using one exemplary embodiment of
cast-PCR.
[0061] FIG. 11A-E shows a list of exemplary primers and probes used
in cast-PCR assays. Nucleotides shown in lower case are the tailed
portion of the primers. The nucleotide-portion of allele-specific
primers (ASP) is at the 3'-most terminus of each primer and are
indicated in bold. The allele positions of the blocker probes (MGB)
are located at various internal positions relative to the blocker
moieties, in some cases, are indicated in bold.
[0062] FIG. 12 depicts the specificity of allelic discrimination
for samples that were pre-amplified prior to analysis by
cast-PCR.
[0063] FIG. 13 depicts the specificity of allelic discrimination
for cast-PCR assays performed using tailed versus non-tailed
allele-specific primers.
[0064] FIG. 14 depicts the specificity of allelic discrimination
for samples analyzed by cast-PCR versus samples analyzed by ASB-PCR
methods.
[0065] FIG. 15 depicts the specificity of allelic discrimination
for cast-PCR assays performed using MGB blocker probes or phosphate
blocker probes.
[0066] FIG. 16 depicts the specificity of allelic discrimination
for cast-PCR assays performed using LNA-modified allele-specific
primers.
[0067] FIG. 17 compares the specificity of allelic discrimination
for cast-PCR assays performed using various chemically-modified
allele-specific primers.
[0068] FIG. 18A-B shows a list of exemplary allele-specific primers
and probes used in pre-amplification and cast-PCR assays.
[0069] FIG. 19A-D shows a list of exemplary primers and probes used
in cast-PCR assays using either tailed (ASP +tail) or non-tailed
(ASP -tail) allele-specific primers. (The tailed portion of the ASP
+tail primers are indicated in lower case lettering.)
[0070] FIG. 20A-C shows a list of exemplary primers and probes used
in ASB-PCR. The blocker probes used in ASB-PCR comprise a phosphate
group at the 3'-end of the blocker probes (PHOS).
[0071] FIG. 21A-C shows a list of exemplary primers and probes used
in cast-PCR assays performed using LNA-modified allele-specific
primers. In this exemplary embodiment, the LNA modifications of the
ASP are at the 3'-ends. ("+" indicates the LNA modified nucleotide
and are notated in parentheses.)
[0072] FIG. 22 shows a list of exemplary primers and probes used in
cast-PCR assays performed using other chemically modified
allele-specific primers. In this exemplary embodiment, the chemical
modifications (e.g., ppA, ppG, fdU, and iso dC) of the ASP are at
the 3'-ends. (The chemically modified nucleotides are shown in
parentheses.)
DETAILED DESCRIPTION
I. Introduction
[0073] The selective amplification of an allele of interest is
often complicated by factors including the mispriming and extension
of a mismatched allele-specific primer on an alternative allele.
Such mispriming and extension can be especially problematic in the
detection of rare alleles present in a sample populated by an
excess of another allelic variant. When in sufficient excess, the
mispriming and extension of the other allelic variant may obscure
the detection of the allele of interest. When using PCR-based
methods, the discrimination of a particular allele in a sample
containing alternative allelic variants relies on the selective
amplification of an allele of interest, while minimizing or
preventing amplification of other alleles present in the
sample.
[0074] A number of factors have been identified, which alone or in
combination, contribute to the enhanced discriminating power of
allele-specific PCR. As disclosed herein, a factor which provides a
greater .DELTA.Ct value between a mismatched and matched
allele-specific primer is indicative of greater discriminating
power between allelic variants. Such factors found to improve
discrimination of allelic variants using the present methods
include, for example, the use of one or more of the following: (a)
tailed allele-specific primers; (b) low allele-specific primer
concentration; (c) allele-specific primers designed to have lower
Tms; (d) allele-specific primers designed to target discriminating
bases; (e) allele-specific blocker probes designed to prevent
amplification from alternative, and potentially more abundant,
allelic variants in a sample; and (f) allele-specific blocker
probes and/or allele-specific primers designed to comprise modified
bases in order to increase the delta Tm between matched and
mismatched target sequences.
[0075] The above-mentioned factors, especially when used in
combination, can influence the ability of allele-specific PCR to
discriminate between different alleles present in a sample. Thus,
the present disclosure relates generally to novel amplification
methods referred to as cast-PCR, which utilizes a combination of
factors referred to above to improve discrimination of allelic
variants during PCR by increasing .DELTA.Ct values. In some
embodiments, the present methods can involve high levels of
selectivity, wherein one mutant molecule in a background of at
least 1,000 to 1,000,000, such as about 1000-10,000, about 10,000
to 100,000, about 20,000 to 250,000, about 30,000 to 300,000, about
40,000 to 400,000, about 50,000 to 500,000, about 75,000 to 750,000
or about 100,000 to 1,000,000 wild type molecules, or any
fractional ranges in between can be detected. In some embodiments,
the comparison of a first set of amplicons and a second set of
amplicons involving the disclosed methods provides improvements in
specificity from 1,000.times. to 100,000,000.times. fold
difference, such as about 1000-10,000.times., about 10,000 to
100,000.times., about 20,000 to 250,000.times., about 30,000 to
300,000.times., about 40,000 to 400,000.times., about 50,000 to
500,000.times., about 75,000 to 750,000.times., about 100,000 to
1,000,000.times., about 2,000,000 to 25,000,000.times., about
3,000,000 to 30,000,000.times., about 4,000,000.times. to
40,000,000.times., about 5,000,000 to 50,000,000.times., about
7,5000,000 to 75,000,000.times. or about 10,000,000 to
100,000,000.times. fold difference, or any fractional ranges in
between.
II. Definitions
[0076] For the purposes of interpreting this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
In the event that any definition set forth below conflicts with the
usage of that word in any other document, including any document
incorporated herein by reference, the definition set forth below
shall always control for purposes of interpreting this
specification and its associated claims unless a contrary meaning
is clearly intended.
[0077] As used herein, the term "allele" refers generally to
alternative DNA sequences at the same physical locus on a segment
of DNA, such as, for example, on homologous chromosomes. An allele
can refer to DNA sequences which differ between the same physical
locus found on homologous chromosomes within a single cell or
organism or which differ at the same physical locus in multiple
cells or organisms ("allelelic variant"). In some instances, an
allele can correspond to a single nucleotide difference at a
particular physical locus. In other embodiments and allele can
correspond to nucleotide (single or multiple) insertion or
deletion.
[0078] As used herein, the term "allele-specific primer" refers to
an oligonucleotide sequence that hybridizes to a sequence
comprising an allele of interest, and which when used in PCR can be
extended to effectuate first strand cDNA synthesis. Allele-specific
primers are specific for a particular allele of a given target DNA
or loci and can be designed to detect a difference of as little as
one nucleotide in the target sequence. Allele-specific primers may
comprise an allele-specific nucleotide portion, a target-specific
portion, and/or a tail.
[0079] As used herein, the terms "allele-specific nucleotide
portion" or "allele-specific target nucleotide" refers to a
nucleotide or nucleotides in an allele-specific primer that can
selectively hybridize and be extended from one allele (for example,
a minor or mutant allele) at a given locus to the exclusion of the
other (for example, the corresponding major or wild type allele) at
the same locus.
[0080] As used herein, the term "target-specific portion" refers to
the region of an allele-specific primer that hybridizes to a target
polynucleotide sequence. In some embodiments, the target-specific
portion of the allele-specific primer is the priming segment that
is complementary to the target sequence at a priming region 5' of
the allelic variant to be detected. The target-specific portion of
the allele-specific primer may comprise the allele-specific
nucleotide portion. In other instances, the target-specific portion
of the allele-specific primer is adjacent to the 3' allele-specific
nucleotide portion.
[0081] As used herein, the terms "tail" or "5'-tail" refers to the
non-3' end of a primer. This region typically will, although does
not have to contain a sequence that is not complementary to the
target polynucleotide sequence to be analyzed. The 5' tail can be
any of about 2-30, 2-5, 4-6, 5-8, 6-12, 7-15, 10-20, 15-25 or 20-30
nucleotides, or any range in between, in length.
[0082] As used herein, the term "allele-specific blocker probe"
(also referred to herein as "blocker probe," "blocker,") refers to
an oligonucleotide sequence that binds to a strand of DNA
comprising a particular allelic variant which is located on the
same, opposite or complementary strand as that bound by an
allelic-specific primer, and reduces or prevents amplification of
that particular allelic variant. As discussed in greater detail
herein, allele-specific blocker probes generally comprise
modifications, e.g., at the 3'-OH of the ribose ring, which prevent
primer extension by a polymerase. The allele-specific blocker probe
can be designed to anneal to the same or opposing strand of what
the allele-specific primer anneals to and can be modified with a
blocking group (e.g., a "non-extendable blocker moiety") at its 3'
terminal end. Thus, a blocker probe can be designed, for example,
so as to tightly bind to a wild type allele (e.g., abundant allelic
variant) in order to suppress amplification of the wild type allele
while amplification is allowed to occur on the same or opposing
strand comprising a mutant allele (e.g., rare allelic variant) by
extension of an allele-specific primer. In illustrative examples,
the allele-specific blocker probes do not include a label, such as
a fluorescent, radioactive, or chemiluminescent label
[0083] As used herein, the term "non-extendable blocker moiety"
refers generally to a modification on an oligonucleotide sequence
such as a probe and/or primer which renders it incapable of
extension by a polymerase, for example, when hybridized to its
complementary sequence in a PCR reaction. Common examples of
blocker moieties include modifications of the ribose ring 3'-OH of
the oligonucleotide, which prevents addition of further bases to
the '3-end of the oligonucleotide sequence a polymerase. Such 3'-OH
modifications are well known in the art. (See, e.g., Josefsen, M.,
et al., Molecular and Cellular Probes, 23 (2009):201-223; McKinzie,
P. et al., Mutagenesis. 2006, 21(6):391-7; Parsons, B. et al.,
Methods Mol Biol. 2005, 291:235-45; Parsons, B. et al., Nucleic
Acids Res. 1992, 25:20(10):2493-6; and Morlan, J. et al., PLoS One
2009, 4 (2): e4584, the disclosures of which are incorporated
herein by reference in their entireties.)
[0084] As used herein, the terms "MGB," "MGB group," "MGB
compound," or "MBG moiety" refers to a minor groove binder. When
conjugated to the 3' end of an oligonucleotide, an MGB group can
function as a non-extendable blocker moiety.
[0085] An MGB is a molecule that binds within the minor groove of
double stranded DNA. Although a general chemical formula for all
known MGB compounds cannot be provided because such compounds have
widely varying chemical structures, compounds which are capable of
binding in the minor groove of DNA, generally speaking, have a
crescent shape three dimensional structure. Most MGB moieties have
a strong preference for A-T (adenine and thymine) rich regions of
the B form of double stranded DNA. Nevertheless, MGB compounds
which would show preference to C-G (cytosine and guanine) rich
regions are also theoretically possible. Therefore,
oligonucleotides comprising a radical or moiety derived from minor
groove binder molecules having preference for C-G regions are also
within the scope of the present invention.
[0086] Some MGBs are capable of binding within the minor groove of
double stranded. DNA with an association constant of
10.sup.3M.sup.-1 or greater. This type of binding can be detected
by well established spectrophotometric methods such as ultraviolet
(UV) and nuclear magnetic resonance (NMR) spectroscopy and also by
gel electrophoresis. Shifts in UV spectra upon binding of a minor
groove binder molecule and NMR spectroscopy utilizing the "Nuclear
Overhauser" (NOSEY) effect are particularly well known and useful
techniques for this purpose. Gel electrophoresis detects binding of
an MGB to double stranded DNA or fragment thereof, because upon
such binding the mobility of the double stranded DNA changes.
[0087] A variety of suitable minor groove binders have been
described in the literature. See, for example, Kutyavin, et al.
U.S. Pat. No. 5,801,155; Wemmer, D. E., and Dervan P. B., Current
Opinion in Structural Biology, 7:355-361 (1997); Walker, W. L.,
Kopka, J. L. and Goodsell, D. S., Biopolymers, 44:323-334 (1997);
Zimmer, C.& Wahnert, U. Frog. Biophys. Molec. Bio. 47:31-112
(1986) and Reddy, B. S. P., Dondhi, S. M., and Lown, J. W.,
Pharmacol. Therap., 84:1-111 (1999) (the disclosures of which are
herein incorporated by reference in their entireties). A preferred
MGB in accordance with the present disclosure is DPI.sub.3.
Synthesis methods and/or sources for such MGBs are also well known
in the art. (See, e.g., U.S. Pat. Nos. 5,801,155; 6,492,346;
6,084,102; and 6,727,356, the disclosures of which are incorporated
herein by reference in their entireties.)
[0088] As used herein, the term "MGB blocker probe," "MBG blocker,"
or "MGB probe" is an oligonucleotide sequence and/or probe further
attached to a minor groove binder moiety at its 3' and/or 5' end.
Oligonucleotides conjugated to MGB moieties form extremely stable
duplexes with single-stranded and double-stranded DNA targets, thus
allowing shorter probes to be used for hybridization based assays.
In comparison to unmodified DNA, MGB probes have higher melting
temperatures (Tin) and increased specificity, especially when a
mismatch is near the MGB region of the hybridized duplex. (See,
e.g., Kutyavin, I. V., et al., Nucleic Acids Research, 2000, Vol.
28, No. 2: 655-661).
[0089] As used herein, the term "modified base" refers generally to
any modification of a base or the chemical linkage of a base in a
nucleic acid that differs in structure from that found in a
naturally occurring nucleic acid. Such modifications can include
changes in the chemical structures of bases or in the chemical
linkage of a base in a nucleic acid, or in the backbone structure
of the nucleic acid. (See, e.g., Latorra, D. et al., Hum Mut 2003,
2:79-85. Nakiandwe, J. et al., Plant Method 2007, 3:2.)
[0090] As used herein, the term "detector probe" refers to any of a
variety of signaling molecules indicative of amplification. For
example, SYBR.RTM. Green and other DNA-binding dyes are detector
probes. Some detector probes can be sequence-based (also referred
to herein as "locus-specific detector probe"), for example 5'
nuclease probes. Various detector probes are known in the art, for
example (TaqMan.RTM. probes described herein (See also U.S. Pat.
No. 5,538,848) various stem-loop molecular beacons (See, e.g., U.S.
Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, Nature
Biotechnology 1996, 14:303-308), stemless or linear beacons (See,
e.g., WO 99/21881), PNA Molecular Beacons.TM. (See, e.g., U.S. Pat.
Nos. 6,355,421 and 6,593,091), linear PNA beacons (See, e.g.,
Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (See, e.g.,
U.S. Pat. No. 6,150,097), Sunrise.RTM./Amplifluor.RTM. probes (U.S.
Pat. No. 6,548,250), stem-loop and duplex Scorpion.TM. probes
(Salinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat.
No. 6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo
knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No.
6,383,752), MGB Eclipse.TM. probe (Epoch Biosciences), hairpin
probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA.)
light-up probes, self-assembled nanoparticle probes, and
ferrocene-modified probes described, for example, in U.S. Pat. No.
6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et
al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000,
Molecular Cell Probes. 14:321-328; Svanvik et al., 2000, Anal
Biochem. 281:26-35; Wolffs et al., 2001, Biotechniques 766:769-771;
Tsourkas et al., 2002, Nucleic Acids Research. 30:4208-4215;
Riccelli et al., 2002, Nucleic Acids Research 30:4088-4093; Zhang
et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am.
Chem. Soc. 124:9606-9612; Braude et al., 2002, Trends Biotechnol.
20:249-56; Huang et al., 2002, Chem Res. Toxicol. 15:118-126; and
Yu et al., 2001, J. Am. Chem. Soc 14:11155-11161. Detector probes
can comprise reporter dyes such as, for example,
6-carboxyfluorescein (6-FAM) or tetrachlorofluorescin (TET).
Detector probes can also comprise quencher moieties such as
tetramethylrhodamine (TAMRA), Black Hole Quenchers (Biosearch),
Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and
Dabcel sulfonate/carboxylate Quenchers (Epoch). Detector probes can
also comprise two probes, wherein for example a fluor is on one
probe, and a quencher on the other, wherein hybridization of the
two probes together on a target quenches the signal, or wherein
hybridization on a target alters the signal signature via a change
in fluorescence. Detector probes can also comprise sulfonate
derivatives of fluorescein dyes with SO.sub.3 instead of the
carboxylate group, phosphoramidite forms of fluorescein,
phosphoramidite forms of CY5 (available, for example, from Amersham
Biosciences-GE Healthcare).
[0091] As used herein, the term "locus-specific primer" refers to
an oligonucleotide sequence that hybridizes to products derived
from the extension of a first primer (such as an allele-specific
primer) in a PCR reaction, and which can effectuate second strand
cDNA synthesis of said product. Accordingly, in some embodiments,
the allele-specific primer serves as a forward PCR primer and the
locus-specific primer serves as a reverse PCR primer, or vice
versa. In some preferred embodiments, locus-specific primers are
present at a higher concentration as compared to the
allele-specific primers.
[0092] As used herein, the term "rare allelic variant" refers to a
target polynucleotide present at a lower level in a sample as
compared to an alternative allelic variant. The rare allelic
variant may also be referred to as a "minor allelic variant" and/or
a "mutant allelic variant." For instance, the rare allelic variant
may be found at a frequency less than 1/10, 1/100, 1/1,000,
1/10,000, 1/100,000, 1/1,000,000, 1/10,000,000, 1/100,000,000 or
1/1,000,000,000 compared to another allelic variant for a given.
SNP or gene. Alternatively, the rare allelic variant can be, for
example, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75,
100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500, 10,000, 25,000,
50,000, 75,000, 100,000, 250,000, 500,000, 750,000, or 1,000,000
copies per 1, 10, 100, 1,000 micro liters of a sample or a reaction
volume.
[0093] As used herein, the terms "abundant allelic variant" may
refer to a target polynucleotide present at a higher level in a
sample as compared to an alternative allelic variant. The abundant
allelic variant may also be referred to as a "major allelic
variant" and/or a "wild type allelic variant." For instance, the
abundant allelic variant may be found at a frequency greater than
10.times., 100.times., 1,000.times., 10,000.times., 100,000.times.,
1,000,000.times., 10,000,000.times., 100,000,000.times. or
1,000,000,000.times. compared to another allelic variant for a
given SNP or gene. Alternatively, the abundant allelic variant can
be, for example, greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 50, 75, 100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500, 10,000,
25,000, 50,000, 75,000, 100,000, 250,000, 500,000, 750,000,
1,000,000 copies per 1, 10, 100, 1,000 micro liters of a sample or
a reaction volume.
[0094] As used herein, the terms "first" and "second" are used to
distinguish the components of a first reaction (e.g., a "first"
reaction; a "first" allele-specific primer) and a second reaction
(e.g., a "second" reaction; a "second" allele-specific primer). By
convention, as used herein the first reaction amplifies a first
(for example, a rare) allelic variant and the second reaction
amplifies a second (for example, an abundant) allelic variant or
vice versa.
[0095] As used herein, both "first allelic variant" and "second
allelic variant" can pertain to alleles of a given locus from the
same organism. For example, as might be the case in human samples
(e.g., cells) comprising wild type alleles, some of which have been
mutated to form a minor or rare allele. The first and second
allelic variants of the present teachings can also refer to alleles
from different organisms. For example, the first allele can be an
allele of a genetically modified organism, and the second allele
can be the corresponding allele of a wild type organism. The first
allelic variants and second allelic variants of the present
teachings can be contained on gDNA, as well as mRNA and cDNA, and
generally any target nucleic acids that exhibit sequence
variability due to, for example, SNP or nucleotide(s) insertion
and/or deletion mutations.
[0096] As used herein, the term "thermostable" or "thermostable
polymerase" refers to an enzyme that is heat stable or heat
resistant and catalyzes polymerization of deoxyribonucleotides to
form primer extension products that are complementary to a nucleic
acid strand. Thermostable DNA polymerases useful herein are not
irreversibly inactivated when subjected to elevated temperatures
for the time necessary to effect destabilization of single-stranded
nucleic acids or denaturation of double-stranded nucleic acids
during PCR amplification. Irreversible denaturation of the enzyme
refers to substantial loss of enzyme activity. Preferably a
thermostable DNA polymerase will not irreversibly denature at about
90.degree.-100.degree. C. under conditions such as is typically
required for PCR amplification.
[0097] As used herein, the term "PCR amplifying" or "PCR
amplification" refers generally to cycling polymerase-mediated
exponential amplification of nucleic acids employing primers that
hybridize to complementary strands, as described for example in
Innis et al., PCR Protocols: A Guide to Methods and Applications,
Academic Press (1990). Devices have been developed that can perform
thermal cycling reactions with compositions containing fluorescent
indicators which are able to emit a light beam of a specified
wavelength, read the intensity of the fluorescent dye, and display
the intensity of fluorescence after each cycle. Devices comprising
a thermal cycler, light beam emitter, and a fluorescent signal
detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907;
6,015,674; 6,174,670; and 6,814,934 and include, but are not
limited to, the ABI Prism.RTM. 7700 Sequence Detection System
(Applied Biosystems, Foster City, Calif.), the ABI GeneAmp.RTM.
5700 Sequence Detection System (Applied Biosystems, Foster City,
Calif.), the ABI GeneAmp.RTM. 7300 Sequence Detection System
(Applied Biosystems, Foster City, Calif.), the ABI GeneAmp.RTM.
7500 Sequence Detection System (Applied Biosystems, Foster City,
Calif.), the StepOne.TM. Real-Time PCR System (Applied Biosystems,
Foster City, Calif.) and the ABI GeneAmp.RTM. 7900 Sequence
Detection System (Applied. Biosystems, Foster City, Calif.).
[0098] As used herein, the term "pre-amplification" or
"pre-amplify" refers to a process wherein a plurality of primer
pairs are included in a multiplexed PCR amplification reaction, and
the multiplexed amplification reaction undergoes a limited number
of cycles so that the PCR-based pre-amplification reaction ends
prior to the PCR plateau and/or reagent depletion. The term
"PCR-based pre-amplification" can be considered to indicate that a
secondary amplification reaction is subsequently performed,
typically of lower plexy level than the PCR-based pre-amplification
reaction. This secondary amplification reaction, typically a
plurality of separate secondary amplification reactions, can employ
primer pairs encoded by the primers used in the multiplexed
PCR-based pre-amplification reaction. However, each secondary
amplification reaction typically comprises a single or a few primer
pairs. Further examples of PCR-based pre-amplification approaches
can be found, for example, in U.S. Pat. No. 6,605,451, and U.S.
patent application Ser. No. 10/723,520, the disclosures of which
are herein incorporated by reference in their entireties.
[0099] As used herein, the term "Tm'" or "melting temperature" of
an oligonucleotide refers to the temperature (in degrees Celsius)
at which 50% of the molecules in a population of a single-stranded
oligonucleotide are hybridized to their complementary sequence and
50% of the molecules in the population are not-hybridized to said
complementary sequence. The Tm of a primer or probe can be
determined empirically by means of a melting curve. In some cases
it can also be calculated using formulas well know in the art (See,
e.g., Maniatis, T., et al., Molecular cloning: a laboratory
manual/Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.:
1982).
[0100] As used herein, the term "sensitivity" refers to the minimum
amount (number of copies or mass) of a template that can be
detected by a given assay. As used herein, the term "specificity"
refers to the ability of an assay to distinguish between
amplification from a matched template versus a mismatched template.
Frequently, specificity is expressed as
.DELTA.C.sub.t=Ct.sub.mismatch-Ct.sub.match. An improvement in
specificity or "specificity improvement" or "fold difference" is
expressed herein as
2.sup.(.DELTA.Ct.sup.--.sup.condition1-(.DELTA.Ct.sup.--.sup.condition2).
The term "selectivity" refers to the extent to which an AS-PCR
assay can be used to determine minor (often mutant) alleles in
mixtures without interferences from major (often wild type)
alleles. Selectivity is often expressed as a ratio or percentage.
For example, an assay that can detect 1 mutant template in the
presence of 100 wild type templates is said to have a selectivity
of 1:100 or 1%. As used herein, assay selectivity can also be
calculated as 1/2.sup..DELTA.Ct or as a percentage using
(1/2.sup..DELTA.Ct.times.100).
[0101] As used herein, the term "Ct" or "Ct value" refers to
threshold cycle and signifies the cycle of a PCR amplification
assay in which signal from a reporter that is indicative of
amplicon generation (e.g., fluorescence) first becomes detectable
above a background level. In some embodiments, the threshold cycle
or "Ct" is the cycle number at which PCR amplification becomes
exponential.
[0102] As used herein, the term "delta Ct" or ".DELTA.Ct" refers to
the difference in the numerical cycle number at which the signal
passes the fixed threshold between two different samples or
reactions. In some embodiments delta Ct is the difference in
numerical cycle number at which exponential amplification is
reached between two different samples or reactions. The delta Ct
can be used to identify the specificity between a matched primer to
the corresponding target nucleic acid sequence and a mismatched
primer to the same corresponding target nucleic acid sequence.
[0103] In some embodiments, the calculation of the delta Ct value
between a mismatched primer and a matched primer is used as one
measure of the discriminating power of allele-specific PCR. In
general, any factor which increases the difference between the Ct
value for an amplification reaction using a primer that is matched
to a target sequence (e.g., a sequence comprising an allelic
variant of interest) and that of a mismatched primer will result in
greater allele discrimination power.
[0104] According to various embodiments, a Ct value may be
determined using a derivative of a PCR curve. For example, a first,
second, or nth order derivative method may be performed on a PCR
curve in order to determine a Ct value. In various embodiments, a
characteristic of a derivative may be used in the determination of
a Ct value. Such characteristics may include, but are not limited
by, a positive inflection of a second derivative, a negative
inflection of a second derivative, a zero crossing of the second
derivative, or a positive inflection of a first derivative. In
various embodiments, a Ct value may be determined using a
thresholding and baselining method. For example, an upper bound to
an exponential phase of a PCR curve may be established using a
derivative method, while a baseline for a PCR curve may be
determined to establish a lower bound to an exponential phase of a
PCR curve. From the upper and lower bound of a PCR curve, a
threshold value may be established from which a Ct value is
determined. Other methods for the determination of a Ct value known
in the art, for example, but not limited by, various embodiments of
a fit point method, and various embodiments of a sigmoidal method
(See, e.g., U.S. Pat. Nos. 6,303,305; 6,503,720; 6,783,934,
7,228,237 and U.S. Application No. 2004/0096819; the disclosures of
which are herein incorporated by reference in their
entireties).
III. Compositions, Methods and Kits
[0105] In one aspect, the present invention provides compositions
for use in identifying and/or quantitating an allelic variant in a
nucleic acid sample. Some of these compositions can comprise: (a)
an allele-specific primer; (b) an allele-specific blocker probe;
(c) a detector probe; and (d) a locus-specific primer, or any
combinations thereof. In some embodiments of the compositions, the
compositions may further comprise a polymerase, dNTPs, reagents
and/or buffers suitable for PCR amplification, and/or a template
sequence or nucleic acid sample. In some embodiments, the
polymerase can be thermostable.
[0106] In another aspect, the invention provides compositions
comprising: (i) a first allele-specific primer, wherein an
allele-specific nucleotide portion of the first allele-specific
primer is complementary to the first allelic variant of a target
sequence; and (ii) a first allele-specific blocker probe that is
complementary to a region of the target sequence comprising the
second allelic variant, wherein said region encompasses a position
corresponding to the binding position of the allele-specific
nucleotide portion of the first allele-specific primer, and wherein
the first allele-specific blocker probe comprises a minor groove
binder.
[0107] In some illustrative embodiments, the compositions can
further include a locus-specific primer that is complementary to a
region of the target sequence that is 3' from the first allelic
variant and on the opposite strand.
[0108] In further embodiments, the compositions can further include
a detector probe.
[0109] In other embodiments, compositions for pre-amplification are
further provided as described in further detail below.
[0110] In another aspect, the present invention provides methods
for amplifying an allele-specific sequence. Some of these methods
can include: (a) hybridizing an allele-specific primer to a first
nucleic acid molecule comprising a target allele; (b) hybridizing
an allele-specific blocker probe to a second nucleic acid molecule
comprising an alternative allele wherein the alternative allele
corresponds to the same loci as the target allele; (c) hybridizing
a locus-specific detector probe to the first nucleic acid molecule;
(d) hybridizing a locus-specific primer to the extension product of
the allele-specific primer and (e) PCR amplifying the target
allele.
[0111] In another aspect, the present invention provides methods
for detecting and/or quantitating an allelic variant in a mixed
sample. Some of these methods can involve: (a) in a first reaction
mixture hybridizing a first allele-specific primer to a first
nucleic acid molecule comprising a first allele (allele-1) and in a
second reaction mixture hybridizing a second allele-specific primer
to a first nucleic acid molecule comprising a second allele
(allele-2), wherein the allele-2 corresponds to the same loci as
allele-1; (b) in the first reaction mixture hybridizing a first
allele-specific blocker probe to a second nucleic acid molecule
comprising allele-2 and in the second reaction mixture hybridizing
a second allele-specific blocker probe to a second nucleic acid
molecule comprising allele-1; (c) in the first reaction mixture,
hybridizing a first detector probe to the first nucleic acid
molecule and in the second reaction mixture, hybridizing a second
detector probe to the first nucleic acid molecule; (d) in the first
reaction mixture hybridizing a first locus-specific primer to the
extension product of the first allele-specific primer and in the
second reaction mixture hybridizing a second locus-specific primer
to the extension product of the second allele-specific primer; and
(e) PCR amplifying the first nucleic acid molecule to form a first
set or sample of amplicons and PCR amplifying the second nucleic
acid molecule to form a second set or sample of amplicons; and (f)
comparing the first set of amplicons to the second set of amplicons
to quantitate allele-1 in the sample comprising allele-2 and/or
allele-2 in the sample comprising allele-1.
[0112] In yet another aspect, the present invention provides
methods for detecting and/or quantitating allelic variants. Some of
these methods can comprise: (a) PCR amplifying a first allelic
variant in a first reaction comprising (i) a low-concentration
first allele-specific primer, (ii) a first locus-specific primer,
and (iii) a first blocker probe to form first amplicons; (b) PCR
amplifying a second allelic variant in a second reaction comprising
(i) a low-concentration second allele-specific primer, (ii) a
second locus-specific primer, and (iii) a second blocker probe to
form second amplicons; and (d) comparing the first amplicons to the
second amplicons to quantitate the first allelic variant in the
sample comprising second allelic variants.
[0113] In yet another aspect, the present invention provides
methods for detecting a first allelic variant of a target sequence
in a nucleic acid sample suspected of comprising at least a second
allelic variant of the target sequence. Methods of this aspect
include forming a first reaction mixture by combining the
following: (i) a nucleic acid sample; (ii) a first allele-specific
primer, wherein an allele-specific nucleotide portion of the first
allele-specific primer is complementary to the first allelic
variant of the target sequence; (iii) a first allele-specific
blocker probe that is complementary to a region of the target
sequence comprising the second allelic variant, wherein said region
encompasses a position corresponding to the binding position of the
allele-specific nucleotide portion of the first allele-specific
primer, and wherein the first allele-specific blacker probe
comprises a minor groove binder; (iv) a first locus-specific primer
that is complementary to a region of the target sequence that is 3'
from the first allelic variant and on the opposite strand; and (v)
a first detector probe.
[0114] Next an amplification reaction, typically a PCR
amplification reaction, is carried out on the first reaction
mixture using the first locus-specific primer and the first
allele-specific primer to form a first amplicon. Then, the first
amplicon is detected by a change in a detectable property of the
first detector probe upon binding to the amplicon, thereby
detecting the first allelic variant of the target gene in the
nucleic acid sample. The detector probe in some illustrative
embodiments is a 5' nuclease probe. The detectable property in
certain illustrative embodiments is fluorescence.
[0115] In some embodiments, the 3' nucleotide position of the 5'
target region of the first allele-specific primer is an
allele-specific nucleotide position. In certain other illustrative
embodiments, including those embodiments where the 3' nucleotide
position of the 5' target region of the first allele-specific
primer is an allele-specific nucleotide position, the blocking
region of the allele-specific primer encompasses the
allele-specific nucleotide position. Furthermore, in illustrative
embodiments, the first allele-specific blacker probe includes a
minor groove binder. Furthermore, the allele-specific blocker probe
in certain illustrative embodiments does not have a label, for
example a fluorescent label, or a quencher.
[0116] In certain illustrative embodiments, the quantity of the
first allelic variant is determined by evaluating the change in a
detectable property of the first detector probe.
[0117] In certain illustrative embodiments, the method further
includes forming a second reaction mixture by combining (i) the
nucleic acid sample; (ii) a second allele-specific primer, wherein
an allele-specific nucleotide portion of the second allele-specific
primer is complementary to the second allelic variant of the target
sequence; (iii) a second allele-specific blocker probe that is
complementary to a region of the target sequence comprising the
first allelic variant, wherein said region encompasses a position
corresponding to the binding position of the allele-specific
nucleotide portion of the second allele-specific primer, and
wherein the second allele-specific blocker probe comprises a minor
groove binder; (iv) a second locus-specific primer that is
complementary to a region of the target sequence that is 3' from
the second allelic variant and on the opposite strand; and (v) a
second detector probe. Next, an amplification reaction is carried
out on the second reaction mixture using the second allele-specific
primer and the locus-specific primer, to form a second amplicon.
Then the second amplicon is detected by a change in a detectable
property of the detector probe.
[0118] In certain embodiments, the method further includes
comparing the change in a detectable property of the first detector
probe in the first reaction mixture to the change in a detectable
property of the second detector probe in the second reaction
mixture.
[0119] In some preferred embodiments, the methods may include a
2-stage cycling protocol. In some embodiments, the methods for
detecting an allelic variant in a target sequence in a nucleic acid
sample comprises forming a reaction mixture comprising: [0120] i) a
nucleic acid sample; [0121] ii) an allele-specific primer, wherein
an allele-specific nucleotide portion of the allele-specific primer
is complementary to the first allelic variant of the target
sequence; [0122] iii) an allele-specific blocker probe that is
complementary to a region of the target sequence comprising a
second allelic variant, wherein said region encompasses a position
corresponding to the binding position of the allele-specific
nucleotide portion of the allele-specific primer, and wherein the
allele-specific blocker probe comprises a blocking moiety; [0123]
iv) a locus-specific primer that is complementary to a region of
the target sequence that is 3' from the first allelic variant and
on the opposite strand; and [0124] v) a detector probe; [0125] b)
PCR amplifying the target sequence using a 2-stage cycling
protocol, comprising: [0126] i) a first amplification step
comprising a first number of cycles run at a first
annealing/extension temperature; and [0127] ii) a second
amplification step comprising a second number of cycles run at a
second annealing/extension temperature, [0128] wherein the first
number of cycles is fewer than the second number of cycles and the
first annealing/extension temperature is lower than said second
annealing/extension temperature; and [0129] c) detecting a change
in a detectable property of the detector probe in the amplified
products of the target sequence produced by step (b).
[0130] In some embodiments, the cycling protocol comprises a first
stage of amplification that employs an initial number of cycles at
a lower annealing/extension temperature, followed by a second stage
of amplification that employs a number of cycles at a higher
annealing/extension temperature. Due to the lower Tm of cast-PCR
allele-specific primers (e.g., 53-56.degree. C.), PCR may not be
optimal at standard annealing/extension conditions (e.g.,
60-64.degree. C.). Consequently, lower annealing/extension
temperatures may be used during the initial cycling stage which
improve cast-PCR efficiency significantly.
[0131] In some embodiments, the number of cycles used in the first
stage of the cast-PCR cycling protocol is fewer than the number of
cycles used in the second stage. In some embodiments of the
cast-PCR methods, the number of cycles used in the first stage of
the cycling protocol is about 2%-20%, 4%-18%, 6%-16%, 8%-14%,
10%-12%, or any percent in between, of the total number of cycles
used in the second stage. In some embodiments, the first stage
employs between 1 to 10 cycles, 2 to 8 cycles, 3 to 7 cycles, or 4
to 6 cycles, and all number of cycles in between, e.g., 2, 3, 4, 5,
6, or 7 cycles.
[0132] In some embodiments, the number of cycles used in the second
stage of the cast-PCR cycling protocol is greater than the number
of cycles used in the second stage. In some embodiments of the
cast-PCR methods, the number of cycles used in the second stage of
the cycling protocol is 5 times, 6 times, 8 times, 10 times, 12
times, 18 times, 25 times, or 30 times the number of cycles used in
the first stage. In some embodiments, the second stage employs
between 30 to 50 cycles, 35 to 48 cycles, 40 to 46 cycles, or any
number of cycles in between, e.g., 42, 43, 44, 45, or 46
cycles.
[0133] In some embodiments, the lower annealing/extension
temperature used during the first cycling stage is about 1.degree.
C., about 2.degree. C., about 3.degree. C., about 4.degree. C., or
about 5.degree. C. lower than the annealing/extension temperature
used during the second cycling stage. In some preferred
embodiments, the annealing/extension temperature of the first stage
is between 50.degree. C. to 60.degree. C., 52.degree. C. to
58.degree. C., or 54.degree. C. to 56.degree. C., e.g., 53.degree.
C., 54.degree. C., 55.degree. C. or 55.degree. C. In some preferred
embodiments the annealing/extension temperature of the second stage
is between 56.degree. C. to 66.degree. C., 58.degree. C. to
64.degree. C., or 60.degree. C. to 62.degree. C., e.g., 58.degree.
C., 60.degree. C., 62.degree. C. or 64.degree. C.
[0134] There are several major advantages of this 2-stage PCR
cycling protocol used in cast-PCR that make it better than
conventional AS-PCR methods. First, it improves the detection
sensitivity by lowering the Ct value for matched targets or
alleles. Next, it improves the specificity of cast-PCR by
increasing the .DELTA.Ct between Ct values of matched and
mismatched sequences. Finally, it can improve the uniformity of
cast-PCR by making it similarly efficient across various
assays.
[0135] In some embodiments, the 2-stage cycling protocol improves
cast-PCR efficiency (e.g. specificity and sensitivity). In some
embodiments, the specificity is increased by 2-8.times.,
3-7.times., or 4-6.times., or any number of times in between (e.g.,
3.times., 4.times., 5.times., 6.times., or 7.times.), more than the
specificity of cast-PCR reactions run without the 2-stage cycling
protocol. In other embodiments, the sensitivity is increased by
1-3.times., (e.g., 1.times., 2.times., or 3.times.) more than the
sensitivity of cast-PCR reactions run without the 2-stage cycling
protocol. In some embodiments, the difference in .DELTA.Ct between
assays run using cast-PCR with the 2-stage cycling protocol
compared to cast-PCR assays run without the 2-stage cycling
protocol is a .DELTA.Ct of 1, 2 or 3.
[0136] In yet another aspect, the present invention provides a
reaction mixture that includes the following (i) nucleic acid
molecule; (ii) an allele-specific primer, wherein an
allele-specific nucleotide portion of the allele-specific primer is
complementary to a first allelic variant of a target sequence;
(iii) an allele-specific blocker probe that is complementary to a
region of the target sequence comprising a second allelic variant,
wherein said region encompasses a position corresponding to the
binding position of the allele-specific nucleotide portion of the
allele-specific primer, and wherein the allele-specific blocker
probe comprises a minor groove binder; (iv) a locus-specific primer
that is complementary to a region of the target sequence that is 3'
from the first allelic variant and on the opposite strand; and (v)
a detector probe.
[0137] In certain embodiments, the methods of the invention are
used to detect a first allelic variant that is present at a
frequency less than 1/10, 1/100, 1/1,000, 1/10,000, 1/100,000,
1/1,000,000, 1/10,000,000, 1/100,000,000 or 1/1,000,000,000, and
any fractional ranges in between, of a second allelic variant for a
given SNP or gene. In other embodiments, the methods are used to
detect a first allelic variant that is present in less than 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 250, 500, 750,
1,000, 2,500, 5,000, 7,500, 10,000, 25,000, 50,000, 75,000,
100,000, 250,000, 500,000, 750,000, 1,000,000 copies per 1, 10,
100, 1,000 micro liters, and any fractional ranges in between, of a
sample or a reaction volume.
[0138] In some embodiments, the first allelic variant is a mutant.
In some embodiments the second allelic variant is wild type. In
some embodiments, the present methods can involve detecting one
mutant molecule in a background of at least 1,000 to 1,000,000,
such as about 1000 to 10,000, about 10,000 to 100,000, or about
100,000 to 1,000,000 wild type molecules, or any fractional ranges
in between. In some embodiments, the methods can provide high
sensitivity and the efficiency at least comparable to that of
TaqMan.RTM.-based assays.
[0139] In some embodiments, the comparison of the first amplicons
and the second amplicons involving the disclosed methods provides
improvements in specificity from 1,000.times. to 1,000,000.times.
fold difference, such as about 1000 to 10,000.times., about 10,000
to 100,000.times., or about 100,000 to 1,000,000.times. fold
difference, or any fractional ranges in between. In some
embodiments, the size of the amplicons range from about 60-120
nucleotides long.
[0140] In some embodiments, the methods can further include
additional steps used for pre-amplification of cast-PCR reactions
as described in further detail below.
[0141] In another aspect, the present invention provides kits for
quantitating a first allelic variant in a sample comprising an
alternative second allelic variants that include: (a) a first
allele-specific primer; (b) a second allele-specific primer; (c) a
first locus-specific primer; (d) a second locus-specific primer;
(e) a first allele-specific blocker probe; (f) a second
allele-specific blacker probe; and (g) a polymerase. In some
embodiments of the disclosed kits, the kit further comprises a
first locus-specific detector probe and a second locus-specific
detector probe.
[0142] In another aspect, the present invention provides kits that
include two or more containers comprising the following components
independently distributed in one of the two or more containers: (i)
a first allele-specific primer, wherein an allele-specific
nucleotide portion of the first allele-specific primer is
complementary to the first allelic variant of a target sequence;
and (ii) a first allele-specific blocker probe that is
complementary to a region of the target sequence comprising the
second allelic variant, wherein said region encompasses a position
corresponding to the binding position of the allele-specific
nucleotide portion of the first allele-specific primer, and wherein
the first allele-specific blocker probe comprises a minor groove
binder.
[0143] In some illustrative embodiments, the kits can further
include a locus-specific primer that is complementary to a region
of the target sequence that is 3' from the first allelic variant
and on the opposite strand.
[0144] In other embodiments, the kits can further include a
detector probe.
[0145] In some embodiments, the kits can further include additional
components used for pre-amplification as described in further
detail below.
[0146] In some embodiments, the compositions, methods, and/or kits
can be used in detecting circulating cells in diagnosis. In one
embodiment, the compositions, methods, and/or kits can be used to
detect tumor cells in blood for early cancer diagnosis. In some
embodiments, the compositions, methods, and/or kits can be used for
cancer or disease-associated genetic variation or somatic mutation
detection and validation. In some embodiments, the compositions,
methods, and/or kits can be used for genotyping tera-, tri- and
di-allelic SNPs. In other embodiments, the compositions, methods,
and/or kits can be used for identifying single or multiple
nucleotide insertion or deletion mutations. In some embodiments,
the compositions, methods, and/or kits can be used for DNA typing
from mixed DNA samples for QC and human identification assays, cell
line QC for cell contaminations, allelic gene expression analysis,
virus typing/rare pathogen detection, mutation detection from
pooled samples, detection of circulating tumor cells in blood,
and/or prenatal diagnostics.
[0147] In some embodiments, the compositions, methods, and/or kits
are compatible with various instruments such as, for example, SDS
instruments from Applied. Biosystems (Foster City, Calif.).
[0148] Allele-Specific Primers
[0149] Allele-specific primers (ASPS) designed with low Tms exhibit
increased discrimination of allelic variants. In some embodiments,
the allele-specific primers are short oligomers ranging from about
15-30, such as about 16-28, about 17-26, about 18-24, or about
20-22, or any range in between, nucleotides in length. In some
embodiments, the Tm of the allele-specific primers range from about
50.degree. C. to 70.degree. C., such as about 52.degree. C. to
68.degree. C., about 54.degree. C. to 66.degree. C., about
56.degree. C. to 64.degree. C., about 58.degree. C. to 62.degree.
C., or any temperature in between (e.g., 53.degree. C., 54.degree.
C., 55.degree. C., 56.degree. C.). In other embodiments, the Tm of
the allele-specific primers is about 3.degree. C. to 6.degree. C.
higher than the anneal/extend temperature of the PCR cycling
conditions employed during amplification.
[0150] Low allele-specific primer concentration can also improve
selectivity. Reduction in concentration of allele-specific primers
below 900 nM can increase the delta Ct between matched and
mismatched sequences. In some embodiments of the disclosed
compositions, the concentration of allele-specific primers ranges
from about 20 nM to 900 nM, such as about 50 nM to 700 nM, about
100 nM to 500 nM, about 200 nM to 300 nM, about 400 nM to 500 nM,
or any range in between. In some exemplary embodiments, the
concentration of the allele-specific primers is between about 200
nM to 400 nM.
[0151] In some embodiments, allele-specific primers can comprise an
allele-specific nucleotide portion that is specific to the target
allele of interest. The allele-specific nucleotide portion of an
allele-specific primer is complementary to one allele of a gene,
but not another allele of the gene. In other words, the
allele-specific nucleotide portion binds to one or more variable
nucleotide positions of a gene that is nucleotide positions that
are known to include different nucleotides for different allelic
variants of a gene. The allele-specific nucleotide portion is at
least one nucleotide in length. In exemplary embodiments, the
allele-specific nucleotide portion is one nucleotide in length. In
some embodiments, the allele-specific nucleotide portion of an
allele-specific primer is located at the 3' terminus of the
allele-specific primer. In other embodiments, the allele-specific
nucleotide portion is located about 1-2, 3-4, 5-6, 7-8, 9-11,
12-15, or 16-20 nucleotides in from the 3' most-end of the
allele-specific primer.
[0152] Allele-specific primers designed to target discriminating
bases can also improve discrimination of allelic variants. In some
embodiments, the nucleotide of the allele-specific nucleotide
portion targets a highly discriminating base (e.g., for detection
of A/A, A/G, G/A, G/G, A/C, or C/A alleles). Less discriminating
bases, for example, may involve detection of C/C, T/C, G/T, T/G,
C/T alleles. In some embodiments, for example when the allele to be
detected involves A/G or C/T SNPs, A or G may be used as the 3'
allele-specific nucleotide portion of the allele-specific primer
(e.g., if A or T is the major allele), or C or T may be used as the
3' allele-specific nucleotide portion of the allele-specific primer
(e.g., if C or G is the major allele). In other embodiments, A may
be used as the nucleotide-specific portion at the 3' end of the
allele specific primer (e.g., the allele-specific nucleotide
portion) when detecting and/or quantifying A/T SNPs. In other
embodiments, G may be used as the nucleotide-specific portion at
the 3' end of the allele specific primer when detecting and/or
quantifying C/G SNPs.
[0153] In some embodiments, the allele-specific primer can comprise
a target-specific portion that is specific to the polynucleotide
sequence (or locus) of interest. In some embodiments the
target-specific portion is about 75-85%, 85-95%, 95-99% or 100%
complementary to the target polynucleotide sequence of interest. In
some embodiments, the target-specific portion of the
allele-specific primer can comprise the allele-specific nucleotide
portion. In other embodiments, the target-specific portion is
located 5' to the allele-specific nucleotide portion. The
target-specific portion can be about 4-30, about 5-25, about 6-20,
about 7-15, or about 8-10 nucleotides in length. In some
embodiments, the Tm of the target specific portion is about
5.degree. C. below the anneal/extend temperature used for PCR
cycling. In some embodiments, the Tm of the target specific portion
of the allele-specific primer ranges from about 51.degree. C. to
60.degree. C., about 52.degree. C. to 59.degree. C., about
53.degree. C. to 58.degree. C., about 54.degree. C. to 57.degree.
C., about 55.degree. C. to 56.degree. C., or about 50.degree. C. to
about 60.degree. C.
[0154] In some embodiments of the disclosed methods and kits, the
target-specific portion of the first allele-specific primer and the
target-specific portion of the second allele-specific primer
comprise the same sequence. In other embodiments, the
target-specific portion of the first allele-specific primer and the
target-specific portion of the second allele-specific primer are
the same sequence.
[0155] In some embodiments, the allele-specific primer comprises a
tail. Allele-specific primers comprising tails, enable the overall
length of the primer to be reduced, thereby lowering the Tm without
significant impact on assay sensitivity.
[0156] In some exemplary embodiments, the tail is on the 5'
terminus of the allele-specific primer. In some embodiments, the
tail is located 5' of the target-specific portion and/or
allele-specific nucleotide portion of the allele-specific primer.
In some embodiments, the tail is about 65-75%, about 75-85%, about
85-95%, about 95-99% or about 100% non-complementary to the target
polynucleotide sequence of interest. In some embodiments the tail
can be about 2-40, such as about 4-30, about 5-25, about 6-20,
about 7-15, or about 8-10 nucleotides in length. In some
embodiments the tail is GC-rich. For example, in some embodiments
the tail sequence is comprised of about 50-100%, about 60-100%,
about 70-100%, about 80-100%, about 90-100% or about 95-100% G
and/or C nucleotides.
[0157] The tail of the allele-specific primer may be configured in
a number of different ways, including, but not limited to a
configuration whereby the tail region is available after primer
extension to hybridize to a complementary sequence (if present) in
a primer extension product. Thus, for example, the tail of the
allele-specific primer can hybridize to the complementary sequence
in an extension product resulting from extension of a
locus-specific primer.
[0158] In some embodiments of the disclosed methods and kits, the
tail of the first allele-specific primer and the tail of the second
allele-specific primer comprise the same sequence. In other
embodiments, the 5' tail of the first allele-specific primer and
the 5' tail of the second allele-specific primer are the same
sequence.
[0159] Allele-Specific Blocker Probes
[0160] Allele-specific blocker probes (or ASBs) (herein sometimes
referred to as "blocker probes") may be designed as short oligomers
that are single-stranded and have a length of 100 nucleotides or
less, more preferably 50 nucleotides or less, still more preferably
30 nucleotides or less and most preferably 20 nucleotides or less
with a lower limit being approximately 5 nucleotides.
[0161] In some embodiments, the Tm of the blocker probes range from
58.degree. C. to 70.degree. C., 61.degree. C. to 69.degree. C.,
62.degree. C. to 68.degree. C., 63.degree. C. to 67.degree. C.,
64.degree. C. to 66.degree. C., or about 60.degree. C. to about
63.degree. C., or any range in between. In yet other embodiments,
the Tm of the allele-specific blocker probes is about 3.degree. C.
to 6.degree. C. higher than the anneal/extend temperature in the
PCR cycling conditions employed during amplification.
[0162] In some embodiments, the blocker probes are not cleaved
during PCR amplification. In some embodiments, the blocker probes
comprise a non-extendable blocker moiety at their 3'-ends. In some
embodiments, the blocker probes can further comprise other moieties
(including, but not limited to additional non-extendable blocker
moieties, quencher moieties, fluorescent moieties, etc) at their
3'-end, 5'-end, and/or any internal position in between. In some
embodiments, the allele position is located about 5-15, such as
about 5-11, about 6-10, about 7-9, about 7-12, or about 9-11, such
as about 6, about 7, about 8, about 9, about 10, or about 11
nucleotides away from the non-extendable blocker moiety of the
allele-specific blocker probes when hybridized to their target
sequences. In some embodiments, the non-extendable blocker moiety
can be, but is not limited to, an amine (NH.sub.2), biotin, PEG,
DPI.sub.3, or PO.sub.4. In some preferred embodiments, the blocker
moiety is a minor groove binder (MGB) moiety. (The
oligonucleotide-MGB conjugates of the present invention are
hereinafter sometimes referred to as "MGB blocker probes" or "MGB
blockers.")
[0163] As disclosed herein, the use of MGB moieties in
allele-specific blocker probes can increase the specificity of
allele-specific PCR. One possibility for this effect is that, due
to their strong affinity to hybridize and strongly bind to
complementary sequences of single or double stranded nucleic acids,
MGBs can lower the Tm of linked oligonucleotides (See, for example,
Kutyavin, I., et al., Nucleic Acids Res., 2000, Vol. 28, No. 2:
655-661). Oligonucleotides comprising MGB moieties have strict
geometric requirements since the linker between the oligonucleotide
and the MGB moiety must be flexible enough to allow positioning of
the MGB in the minor groove after DNA duplex formation. Thus, MGB
blocker probes can provide larger Tm differences between matched
versus mismatched alleles as compared to conventional DNA blocker
probes.
[0164] In general, MGB moieties are molecules that bind within the
minor groove of double stranded. DNA. Although a generic chemical
formula for all known MGB compounds cannot be provided because such
compounds have widely varying chemical structures, compounds which
are capable of binding in the minor groove of DNA, generally
speaking, have a crescent shape three dimensional structure. Most
MGB moieties have a strong preference for A-T (adenine and thymine)
rich regions of the B form of double stranded DNA. Nevertheless,
MGB compounds which would show preference to C-G (cytosine and
guanine) rich regions are also theoretically possible. Therefore,
oligonucleotides comprising a radical or moiety derived from minor
groove binder molecules having preference for C-G regions are also
within the scope of the present invention.
[0165] Some MGBs are capable of binding within the minor groove of
double stranded DNA with an association constant of
10.sup.3M.sup.-1 or greater. This type of binding can be detected
by well established spectrophotometric methods such as ultraviolet
(UV) and nuclear magnetic resonance (NMR) spectroscopy and also by
gel electrophoresis. Shifts in UV spectra upon binding of a minor
groove binder molecule and NMR spectroscopy utilizing the "Nuclear
Overhauser" (NOSEY) effect are particularly well known and useful
techniques for this purpose. Gel electrophoresis detects binding of
an MGB to double stranded DNA or fragment thereof, because upon
such binding the mobility of the double stranded DNA changes.
[0166] A variety of suitable minor groove binders have been
described in the literature. See, for example, Kutyavin, et al.
U.S. Pat. No. 5,801,155; Wemmer, D. E., and Dervan P. B., Current
Opinion in Structural Biology, 7:355-361 (1997); Walker, W. L.,
Kopka, J. L. and Goodsell, D. S., Biopolymers, 44:323-334 (1997);
Zimmer, C & Wahnert, U. Prog. Biophys. Molec. Bio. 47:31-112
(1986) and Reddy, B. S. P., Dondhi, S. M., and Lown, J. W.,
Pharmacol. Therap., 84:1-111 (1999). In one group of embodiments,
the MGB is selected from the group consisting of CC1065 analogs,
lexitropsins, distamycin, netropsin, berenil, duocarmycin,
pentamidine, 4,6-diamino-2-phenylindole and
pyrrolo[2,1-c][1,4]benzodiazepines. A preferred MGB in accordance
with the present disclosure is DPI.sub.3 (see U.S. Pat. No.
6,727,356, the disclosure of which is incorporated herein by
reference in its entirety).
[0167] Suitable methods for attaching MGBs through linkers to
oligonucleotides or probes and have been described in, for example,
U.S. Pat. Nos. 5,512,677; 5,419,966; 5,696,251; 5,585,481;
5,942,610; 5,736,626; 5,801,155 and 6,727,356. (The disclosures of
each of which are incorporated herein by reference in their
entireties.) For example, MGB-oligonucleotide conjugates can be
synthesized using automated oligonucleotide synthesis methods from
solid supports having cleavable linkers. In other examples, MGB
probes can be prepared from an MGB modified solid support
substantially in accordance with the procedure of Lukhtanov et al.
Bioconjugate Chern., 7: 564-567 (1996). (The disclosure of which is
also incorporated herein by reference in its entirety.) According
to these methods, one or more MGB moieties can be attached at the
5'-end, the 3'-end and/or at any internal portion of the
oligonucleotide.
[0168] The location of an MGB moiety within an MGB-oligonucleotide
conjugate can affect the discriminatory properties of such a
conjugate. An unpaired region within a duplex will likely result in
changes in the shape of the minor groove in the vicinity of the
mismatched base(s). Since MGBs fit best within the minor groove of
a perfectly-matched DNA duplex, mismatches resulting in shape
changes in the minor groove would reduce binding strength of an MGB
to a region containing a mismatch. Hence, the ability of an MGB to
stabilize such a hybrid would be decreased, thereby increasing the
ability of an MGB-oligonucleotide conjugate to discriminate a
mismatch from a perfectly-matched duplex. On the other hand, if a
mismatch lies outside of the region complementary to an
MGB-oligonucleotide conjugate, discriminatory ability for
unconjugated and MGB-conjugated oligonucleotides of equal length is
expected to be approximately the same. Since the ability of an
oligonucleotide probe to discriminate single base pair mismatches
depends on its length, shorter oligonucleotides are more effective
in discriminating mismatches. The first advantage of the use of
MGB-oligonucleotides conjugates in this context lies in the fact
that much shorter oligonucleotides compared to those used in the
art (i.e., 20-mers or shorter), having greater discriminatory
powers, can be used, due to the pronounced stabilizing effect of
MGB conjugation. Consequently, larger delta Tms of allele-specific
blocker probes can improve AS-PCR assay specificity and
selectivity.
[0169] Blocker probes having MGB at the 5' termini may have
additional advantages over other blocker probes having a blocker
moiety (e.g., MGB, PO.sub.4, NH.sub.2, PEG, or biotin) only at the
3' terminus. This is at least because blocker probes having MGB at
the 5' terminus (in addition to a blocking moiety at the 3'-end
that prevents extension) will not be cleaved during PCR
amplification. Thus, the probe concentration can be maintained at a
constant level throughout PCR, which may help maintain the
effectiveness of blocking non-specific priming, thereby increasing
cast-PCR assay specificity and selectivity (FIG. 3).
[0170] In some embodiments, as depicted in FIG. 4A, the
allele-specific primer and/or the allele-specific blocker probe can
comprise one or more modified nucleobases or nucleosidic bases
different from the naturally occurring bases (i.e., adenine,
cytosine, guanine, thymine and uracil). In some embodiments, the
modified bases are still able to effectively hybridize to nucleic
acid units that contain adenine, guanine, cytosine, uracil or
thymine moieties. In some embodiments, the modified base(s) may
increase the difference in the Tm between matched and mismatched
target sequences and/or decrease mismatch priming efficiency,
thereby improving not only assay specificity, bust also
selectivity.
[0171] Modified bases are considered to be those that differ from
the naturally-occurring bases by addition or deletion of one or
more functional groups, differences in the heterocyclic ring
structure (i.e., substitution of carbon for a heteroatom, or vice
versa), and/or attachment of one or more linker arm structures to
the base. In some embodiments, all tautomeric forms of naturally
occurring bases, modified bases and base analogues may also be
included in the oligonucleotide primers and probes of the
invention.
[0172] Some examples of modified base(s) may include, for example,
the general class of base analogues 7-deazapurines and their
derivatives and pyrazolopyrimidines and their derivatives
(described in PCT WO 90/14353; and U.S. application Ser. No.
09/054,630, the disclosures of each of which are incorporated
herein by reference in their entireties). Examples of base
analogues of this type include, for example, the guanine analogue
6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (ppG), the adenine
analogue 4-amino-1H-pyrazolo[3,4-d]pyrimidine (ppA), and the
xanthine analogue 1H-pyrazolo[4,4-d]pyrimidin-4(5H)-6(7H)-dione
(ppX). These base analogues, when present in an oligonucleotide of
some embodiments of this invention, strengthen hybridization and
can improve mismatch discrimination.
[0173] Additionally, in some embodiments, modified sugars or sugar
analogues can be present in one or more of the nucleotide subunits
of an oligonucleotide conjugate in accordance with the invention.
Sugar modifications include, but are not limited to, attachment of
substituents to the 2', 3' and/or 4' carbon atom of the sugar,
different epimeric forms of the sugar, differences in the .alpha.-
or .beta.-configuration of the glycosidic bond, and other anomeric
changes. Sugar moieties include, but are not limited to, pentose,
deoxypentose, hexose, deoxyhexose, ribose, deoxyribose, glucose,
arabinose, pentofuranose, xylose, lyxose, and cyclopentyl.
[0174] Locked nucleic acid (LNA)-type modifications, for example,
typically involve alterations to the pentose sugar of ribo- and
deoxyribonucleotides that constrains, or "locks," the sugar in the
N-type conformation seen in A-form DNA. In some embodiments, this
lock can be achieved via a 2'-O, 4'-C methylene linkage in
1,2:5,6-di-O-isopropylene-.alpha.-D-allofuranose. In other
embodiments, this alteration then serves as the foundation for
synthesizing locked nucleotide phosphoramidite monomers. (See, for
example, Wengel J., Ace. Chem. Res., 32:301-310 (1998), U.S. Pat.
No. 7,060,809; Obika, et al., Tetrahedron Lett 39: 5401-5405
(1998); Singh, et al., Chem Commun 4:455-456 (1998); Koshkin, et
al., Tetrahedron 54: 3607-3630 (1998), the disclosures of each of
which are incorporated herein by reference in their
entireties.)
[0175] In some preferred embodiments, the modified bases include
8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG (ppG),
2'-Deoxypseudoisocytidine (iso dC), 5-fluoro-2'-deoxyuridine (fdU),
locked nucleic acid (LNA), or 2'-O,4'-C-ethylene bridged nucleic
acid (ENA) bases. Other examples of modified bases that can be used
in the invention are depicted in FIG. 4B and described in U.S. Pat.
No. 7,517,978 (the disclosure of which is incorporated herein by
reference in its entirety).
[0176] Many modified bases, including for example, LNA, ppA, ppG,
5-Fluoro-dU (fdU), are commercially available and can be used in
oligonucleotide synthesis methods well known in the art. In some
embodiments, synthesis of modified primers and probes can be
carried out using standard chemical means also well known in the
art. For example, in certain embodiments, the modified moiety or
base can be introduced by use of a (a) modified nucleoside as a DNA
synthesis support, (b) modified nucleoside as a phosphoramidite,
(c) reagent during DNA synthesis (e.g., benzylamine treatment of a
convertible amidite when incorporated into a DNA sequence), or (d)
by post-synthetic modification.
[0177] In some embodiments, the primers or probes are synthesized
so that the modified bases are positioned at the 3' end. In some
embodiments, the modified base are located between, 1-6
nucleotides, e.g., 2, 3, 4 or 5 nucleotides away from the 3'-end of
the allele-specific primer or blocker probe. In some preferred
embodiments, the primers or probes are synthesized so that the
modified bases are positioned at the 3'-most end of the
allele-specific primer or blocker probe.
[0178] Modified internucleotide linkages can also be present in
oligonucleotide conjugates of the invention. Such modified linkages
include, but are not limited to, peptide, phosphate,
phosphodiester, phosphodiester, alkylphosphate, alkanephosphonate,
thiophosphate, phosphorothioate, phosphorodithioate,
methylphosphonate, phosphoramidate, substituted phosphoramidate and
the like. Several further modifications of bases, sugars and/or
internucleotide linkages, that are compatible with their use in
oligonucleotides serving as probes and/or primers, will be apparent
to those of skill in the art.
[0179] In addition, in some embodiments, the nucleotide units which
are incorporated into the oligonucleotides of the allele-specific
primers and/or blocker probes of the present invention may have a
cross-linking function (an alkylating agent) covalently bound to
one or more of the bases, through a linking arm.
[0180] In some embodiments of the methods and kits, the first
allele-specific blocker probe binds to the same strand or sequence
as the first allele-specific primer, while the second
allele-specific blocker probe binds to the opposite strand and/or
complementary sequence as the first allele-specific primer.
[0181] Detector Probes
[0182] In some embodiments, detector probe is designed as short
oligomers ranging from about 15-30 nucleotides, such as about 16,
about 18, about 22, about 24, about 30, or any number in between.
In some embodiments, the Tm of the detector probe ranges from about
60.degree. C. to 70.degree. C., about 61.degree. C. to 69.degree.
C., about 62.degree. C. to 68.degree. C., about 63.degree. C. to
67.degree. C., or about 64.degree. C. to 66.degree. C., or any
temperature in between.
[0183] In some embodiments, the detector probe is a locus-specific
detector probes (LST). In other embodiments the detector probe is a
5' nuclease probe. In some exemplary embodiments, the detector
probe can comprises an MGB moiety, a reporter moiety (e.g.,
FAM.TM., TET.TM., JOE.TM., VIC.TM., or SYBR.RTM. Green), a quencher
moiety (e.g., Black Hole Quencher.TM. or TAMRA.TM.;), and/or a
passive reference (e.g., ROX.TM.). In some exemplary embodiments,
the detector probe is designed according to the methods and
principles described in U.S. Pat. No. 6,727,356 (the disclosure of
which is incorporated herein by reference in its entirety). In some
exemplary embodiments, the detector probe is a TaqMan.RTM. probe
(Applied Biosystems, Foster City). In exemplary embodiments, the
locus-specific detector probe can be designed according to the
principles and methods described in U.S. Pat. No. 6,727,356 (the
disclosure of which is incorporated herein by reference in its
entirety). For example, fluorogenic probes can be prepared with a
quencher at the 3' terminus of a single DNA strand and a
fluorophore at the 5' terminus. In such an example, the 5'-nuclease
activity of a Tag DNA polymerase can cleave the DNA strand, thereby
separating the fluorophore from the quencher and releasing the
fluorescent signal. In some embodiments, the detector probes are
hybridized to the template strands during primer extension step of
PCR amplification (e.g., at 60-65.degree. C.). In yet other
embodiments, an MGB is covalently attached to the quencher moiety
of the locus-specific detector probes (e.g., through a linker).
[0184] In some embodiments of the disclosed methods and kits, the
first and second detector probes are the same and/or comprise the
same sequence or are the same sequence.
[0185] Locus-Specific Primers
[0186] In some embodiments, locus-specific primer (LSP) is designed
as a short oligomer ranging from about 15-30 nucleotides, such as
about 16, about 18, about 22, about 24, about 30, or any number in
between. In some embodiments, the Tm of the locus-specific primer
ranges from about 60.degree. C. to 70.degree. C., about 61.degree.
C. to 69.degree. C., about 62.degree. C. to 68.degree. C., about
63.degree. C. to 67.degree. C., or about 64.degree. C. to
66.degree. C., or any range in between.
[0187] In some other embodiments of the disclosed methods and kits,
the first locus-specific detector probe and/or second
locus-specific detector probes comprise the same sequence or are
the same sequence.
[0188] Additional Components
[0189] Polymerase enzymes suitable for the practice of the present
invention are well known in the art and can be derived from a
number of sources. Thermostable polymerases may be obtained, for
example, from a variety of thermophilic bacteria that are
commercially available (for example, from American Type Culture
Collection, Rockville, Md.) using methods that are well-known to
one of ordinary skill in the art (See, e.g., U.S. Pat. No.
6,245,533). Bacterial cells may be grown according to standard
microbiological techniques, using culture media and incubation
conditions suitable for growing active cultures of the particular
species that are well-known to one of ordinary skill in the art
(See, e.g., Brock, T. D., and Freeze, H., J. Bacterial.
98(1):289-297 (1969); Oshima, T., and Imahori, K, Int. J. Syst.
Bacteriol. 24(1):102-112 (1974)). Suitable for use as sources of
thermostable polymerases are the thermophilic bacteria Thermus
aquaticus, Thermus thermophilus, Thermococcus litoralis, Pyrococcus
furiosus, Pyrococcus woosii and other species of the Pyrococcus
genus, Bacillus stearothermophilus, Sulfolobus acidocaldarius,
Thermoplasma acidophilum, Thermus flavus, Thermus ruber, Thermus
brockianus, Thermotoga neapolitana, Thermotoga maritima and other
species of the Thermotoga genus, and Methanobacterium
thermoautotrophicum, and mutants of each of these species.
Preferable thermostable polymerases can include, but are not
limited to, Tag DNA polymerase, The DNA polymerase, Tma DNA
polymerase, or mutants, derivatives or fragments thereof.
[0190] Various Sources and/or Preparation Methods of Nucleic
Acids
[0191] Sources of nucleic acid samples in the disclosed
compositions, methods and/or kits include, but are not limited to,
human cells such as circulating blood, buccal epithelial cells,
cultured cells and tumor cells. Also other mammalian tissue, blood
and cultured cells are suitable sources of template nucleic acids.
In addition, viruses, bacteriophage, bacteria, fungi and other
micro-organisms can be the source of nucleic acid for analysis. The
DNA may be genomic or it may be cloned in plasmids, bacteriophage,
bacterial artificial chromosomes (BACs), yeast artificial
chromosomes (YACs) or other vectors. RNA may be isolated directly
from the relevant cells or it may be produced by in vitro priming
from a suitable RNA promoter or by in vitro transcription. The
present invention may be used for the detection of variation in
genomic DNA whether human, animal or other. It finds particular use
in the analysis of inherited or acquired diseases or disorders. A
particular use is in the detection of inherited diseases.
[0192] In some embodiments, template sequence or nucleic acid
sample can be gDNA. In other embodiments, the template sequence or
nucleic acid sample can be cDNA. In yet other embodiments, as in
the case of simultaneous analysis of gene expression by RT-PCR, the
template sequence or nucleic acid sample can be RNA. The DNA or RNA
template sequence or nucleic acid sample can be extracted from any
type of tissue including, for example, formalin-fixed
paraffin-embedded tumor specimens.
[0193] Pre-Amplification
[0194] In another aspect, additional compositions, methods and kits
are provided for "boosting" or pre-amplifying cast-PCR
amplification reactions. In some embodiments, pre-amplification of
cast-PCR reactions is employed for limited quantity specimens
having very low nucleic acid copy number.
[0195] In some embodiments, said "boosting" compositions, methods
and kits involve a two-step amplification process comprising a
first pre-amplification reaction (see, for example, U.S. Pat. Nos.
6,605,451 and 7,087,414 and U.S. Published Application No.
2004/0175733, the disclosures of which are herein incorporated by
reference in their entireties), followed by a second amplification
(i.e., cast-PCR) reaction.
[0196] In some embodiments, pre-amplification composition used in
cast-PCR pre-amplification reactions comprise: [0197] i) a nucleic
acid sample; and [0198] ii) at least two sets of primers wherein
each set comprises (a) a first allele-specific primer and a second
allele-specific primer, wherein the allele-specific nucleotide
portion of said first and second allele-specific primers is
complementary to a first allele and a second allele, respectively,
of a given SNP within a target sequence; and (b) a locus-specific
primer that is complementary to a region of said target sequence
that is 3' from the first and second alleles and on the opposite
strand; wherein the at least two sets of primers is each specific
for a different target sequence.
[0199] In some embodiments, the cast-PCR pre-amplification methods
comprise: [0200] a) forming in a single vessel a first reaction
mixture comprising: [0201] i) a nucleic acid sample; and [0202] ii)
at least two sets of primers wherein each set comprises (a) a first
allele-specific primer and a second allele-specific primer, wherein
the allele-specific nucleotide portion of said first and second
allele-specific primers is complementary to a first allele and a
second allele, respectively, of a given SNP within a target
sequence; and (b) a locus-specific primer that is complementary to
a region of said target sequence that is 3' from the first and
second alleles and on the opposite strand; wherein the at least two
sets of primers is each specific for a different target sequence;
[0203] b) amplifying said different target sequences using a number
of cycles suitable to keep the reaction within a linear phase;
[0204] c) dividing the amplified products of step (b) into at least
two separate vessels; [0205] d) adding to each of the divided
products of step (c) [0206] i) at least one set of primers used in
step (a), [0207] ii) an allele-specific blocker probe that is
complementary to a region of said target sequence comprising a
second allelic variant, wherein said region encompasses a position
corresponding to the binding position of the allele-specific
nucleotide portion of the first allele-specific primer, and wherein
the first allele-specific blocker probe comprises a minor groove
binder; and [0208] iv) a detector probe [0209] to form a second and
a third reaction mixture in the at least two separate vessels;
[0210] e) amplifying said target sequence in said second and third
reaction mixtures; and [0211] f) detecting a change in a detectable
property of the detector probe in each of the amplified products of
said target sequence produced by step (e), thereby detecting the
allelic variant of the target gene in the nucleic acid sample.
[0212] In some embodiments, the first reaction is a multiplex
reaction and said second reaction is a single-plex reaction. In
some preferred embodiments, the multiplex reaction involves the use
of at least two complete sets of primers (e.g., a set comprises one
forward allele-1-specific primer, one forward allele-2-specific
primer and one reverse locus-specific primer), each set of which is
suitable or operative for amplifying a specific polynucleotide of
interest. In other embodiments, the resultant multiplex products
acquired in the first step are divided into optimized secondary
single-plex cast-PCR amplification reactions, each containing at
least one primer set previously used in the first multiplexing step
and then PCR amplified using the cast-PCR methods described
herein.
[0213] In other preferred embodiments, the first multiplex reaction
is a cast-PCR amplification reaction (although other well known
amplification methods such as, but not limited to PCR, RT-PCR,
NASBA, SDA, TMA, CRCA, Ligase Chain Reaction, etc. can be used). In
certain embodiments, the first multiplex reaction comprises a
plurality of allele-specific primers, and locus-specific primers,
each group of which is specific for a particular allele of interest
and designed according to the cast-PCR methods described herein.
Unlike single-plex cast-PCR reactions that generate a single
amplified sequence, multiplex cast-PCR amplification reactions, by
virtue of utilizing a plurality of different primer sets, can
permit the simultaneous amplification of a plurality of different
sequences of interest in a single reaction. Because a plurality of
different sequences is amplified simultaneously in a single
reaction, the multiplex amplifications can effectively increase the
concentration or quantity of a sample available for downstream
cast-PCR assays. Thus, in some preferred embodiments, significantly
more analyses or assays can be performed with a pre-amplified
cast-PCR sample than could have been performed with the original
sample.
[0214] The number of different amplification primer pairs utilized
in the multiplex amplification is not critical and can range from
as few as two, to as many as tens, hundreds, thousands, or even
more. Thus, depending upon the particular conditions, the multiplex
amplifications permit the simultaneous amplification of from as few
as two to as many as tens, hundreds, thousands, or even more
polynucleotide sequences of interest.
[0215] The number of amplification cycles performed with a
multiplex amplification may depend upon, among other factors, the
degree of amplification desired. The degree of amplification
desired, in turn, may depend upon such factors as the amount of
polynucleotide sample to be amplified or the number of alleles or
mutations to be detected using subsequent cast-PCR assays.
[0216] In preferred embodiments, it may be desirable to keep the
multiplex amplification from progressing beyond the exponential
phase or the linear phase. Indeed, in some embodiments, it may be
desirable to carry out the multiplex amplification for a number of
cycles suitable to keep the reaction within the exponential or
linear phase. Utilization of a truncated multiplex amplification
round can result in a sample having a boosted product copy number
of about 100-1000 fold increase.
[0217] In many embodiments, pre-amplification permits the ability
to perform cast-PCR assays or analyses that require more sample, or
a higher concentration of sample, than was originally available.
For example, after a 10.times., 100.times., 1000.times.,
10,000.times., and so on, multiplex amplification, subsequent
cast-PCR single-plex assays can then be performed using,
respectively, a 10.times., 100.times., 1000.times., 10,000.times.,
and so on, less sample volume. In some embodiments, this allows
each single-plex cast-PCR reaction to be optimized for maximum
sensitivity and requires only one method of detection for each
allele analyzed. This can be a significant benefit to cast-PCR
analysis since, in some embodiments, it allows for the use of
off-the-shelf commercially available cast-PCR reagents and kits to
be pooled together and used in a multiplex amplification reaction
without extensive effort toward or constraints against redesigning
and/or re-optimizing cast-PCR assays for any given target sequence.
Moreover, in some embodiments, the ability to carry out a multiplex
amplification with reagents and kits already optimized for cast-PCR
analysis permits the creation of multiplex amplification reactions
that are ideally correlated or matched with subsequent single-plex
cast-PCR assays.
[0218] In one aspect, kits are provided that include at least one
separate container comprising the following components
independently distributed in at least one of the separate
containers: (i) a first allele-specific primer, wherein an
allele-specific nucleotide portion of the first allele-specific
primer is complementary to the first allelic variant of a target
sequence; (ii) a second allele-specific primer wherein an
allele-specific nucleotide portion of the second allele-specific
primer is complementary to the second allelic variant of a target
sequence; and (iii) a common locus-specific primer.
[0219] The following examples are intended to illustrate but not
limit the invention.
EXAMPLES
I. General Cast-PCR Assay Design
[0220] The general schema for the cast-PCR assays used in the
following examples is illustrated in FIG. 1. For each SNP that was
analyzed, allele-specific primers (ASPs) were designed to target a
first allele (i.e. allele-1) and a second allele (i.e. allele-2).
The cast-PCR assay reaction mixture for allele-1 analysis included
a 5'-tailed allele-1-specific primer (ASP1), one MGB allele-2
blocker probe (MGB2), one common locus-specific TaqMan probe (LST)
and one common locus-specific primer (LSP). The cast-PCR assay
reaction mixture for analysis of allele-2 included a 5'-tailed
allele-2-specific primer (ASP2), one MGB allele-1 blacker probe
(MGB1), one common locus-specific TaqMan probe (LST) and one common
locus-specific primer (LSP).
II. Reaction Conditions
[0221] Each assay reaction mixture (10 .mu.l total) contained
1.times. TaqMan Genotyping Master Mixture (Applied Biosystems,
Foster City, Calif.; P/N 437135), 0.5 ng/.mu.L genomic DNA or 1
million copies of plasmid DNA (or as indicated otherwise), 300 nM
(unless specified otherwise) tailed-, or in some cases untailed-,
allele-specific primer (ASP1 for detection of allele-1 or ASP2 for
detection of allele-2), 200 nM TaqMan probe (LST), 900 nM
locus-specific primer (LSP), 150 nM allele-specific MGB blocker
probe (MGB1 for detection of allele-2 or MGB2 for detection of
allele-1). The reactions were incubated in a 384-well plate at
95.degree. C. for 10 minutes, then for 5 cycles at 95.degree. C.
for 15 seconds and 58.degree. C. for 1 minute, then by 45 cycles at
95.degree. C. for 15 seconds and 60.degree. C. for 1 minute. All
reactions were run in duplicate or higher replication in an ABI
PRISM 7900HT.RTM. Sequence Detection System, according to the
manufacturer's instructions.
[0222] The 2-stage cycling protocol used in the following examples
for cast-PCR amplification reactions is different from conventional
allele-specific PCR (AS-PCR). The 2-stage cycling protocol
comprises an initial 5 cycles at a lower annealing/extension
temperature (e.g., 58.degree. C.), followed by 45 standard cycles
at a higher annealing/extension temperature (e.g., 60.degree. C.).
Due to the lower Tm of cast-PCR allele-specific primers (e.g.,
53-56.degree. C.), PCR is not optimal at standard
annealing/extension conditions (e.g., 60.degree. C.). Consequently,
lower annealing/extension temperatures used during the initial 5
cycles increases overall cast-PCR efficiency.
III. Nucleic Acid Samples
[0223] Plasmids containing specific SNP sequences were designed and
ordered from BlueHeron (Bothell, Wash.). (See Table 1 for a list of
plasmids comprising SNPs used in some of the following examples.)
The plasmids were quantified using TaqMan RNase P Assay (Applied
Biosystems, Foster City, Calif.; P/N 4316838) according to the
manufacturer's instructions and were used as templates (See Table
1, RNase P Control) to validate sensitivity, linear dynamic range,
specificity, and selectivity of the given assays.
[0224] Genomic DNAs were purchased from Coriell Institute for
Medical Research (Camden, N.J.; NA17203, NA17129, NA17201). The
genotypes of target SNPs were validated with TaqMan SNP Genotyping
Assays (Applied Biosystems, Foster City, Calif.; P/N 4332856)
according to the manufacturer's instructions.
IV. Modified Oligonucleotides
[0225] Modified bases were purchased from Berry and Associates
(ppA: P/N BA 0239; ppG: P/N BA 0242; fdU: P/N BA 0246; and iso dC:
P/N BA 0236) or Exicon (LNA-T Amidite: P/N EQ-0064; LNA-mC Amidite:
P/N EQ-0066; LNA-G Amidite: P/N EQ-0082; and LNA-A Amidite: P/N
EQ-0063). Oligonucleotides comprising the modified nucleotides at
their 3' ends were synthesized according to the manufacturer's
instructions.
TABLE-US-00001 TABLE 1 Plasmid SNP Sequences (target alleles are
indicated in brackets). SNP ID Sequence CV11201742
GCTCTGCTTCATTCCTGTCTGAAGAAGGGCAGATAGTTTGGCTGCTCCTGTG[C/T]TGTCAC-
CTGCAATTCTCC
CTTATCAGGGCCATTGGCCTCTCCCTTCTCTCTGTGAGGGATATTTTCTCTGACTTGTCAATCCACATCTTCC
CV11349123
GGCTTGCAATGGCTCCAACCGGAAGGGCGGTGCTCGAGCTGTGGTGCGTGC[C/T]GCTAAGT-
TGTGCGTTCCA GGGTGCACTCGC CV1207700
GCAACTATACCCTTGATGGATGGAGATTTA[C/T]GCAATGTGTTTTACTGGGTAGAGTGACAG-
ACCTT CV25594064
CCTGAACTTATTTGGCAAGAGCGATGAGTACTCTTAAAATTACTATCTGGAAATTATATTATT-
TAGAATCTGCC AATTACCTAGATCCCCCCT[C/G]AACAATTGTTTCACCAAGGAACTTCCTGAA
CV25639181
GAATTGGTTGTCTCCTTATGGGAACTGGAAGTATTTTGACA[G/T]CTTTACCACATTTCTTC-
ATGGGATAGTAA
GTGTTAAACAGCTCTGAGCCATTTATTATCAGCTACTTGTAAATTAGCAGTAGAATTTTATTTTTATACTTGT-
AA GTGGGCAGTTACCTTTTGAGAGGAATACCTATAG RNaseP Control
GCGGAGGGAAGCTCATCAGTGGGGCCACGAGCTGAGTGCGTCCTGTCACTCCACTCCCATGTCCCTTGGGAAG
GTCTGAGACTAGGG BRAF-1799TA
TACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG
[T/A]GAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAA-
TT GAGGCTATTTTTCCACTGATTAAATTTTTGGCCCTGAGATGCTGCTGAGTT CTNNB1-121AG
TGCTAATACTGTTTCGTATTTATAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGG
CTGTTAGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACT[A/G]CCACAGCTC-
CT TCTCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTC
CTNNB1-134-CT
TTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTAC
CTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTT[C/T]TCTGAGTGGTAAAGGCAATCCTGAG-
G AAGAGGATGTGGATACCTCCCAAGTCCTGTATGAGTGGGAA EGFR-2369CT
GTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCAG-
C
TCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAA-
C TGGTGTGTGCAGATCGCAAAGGTAATCAGGGAAGGGA EGFR-2573TG
GCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACC
GCAGCATGTCAAGATCACAGATTTTGGGC[T/G]GGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGC
AGAAGGAGGCAAAGTAAGGAGGTG KRAS-176CG
CAGGATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGAC-
ACAG[C/G]AG
GTCAAGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCAT
AAATAATACTAAATCATTTGAAGATATTC KRAS-183AC
ACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTC-
A[A/C]GAGG
AGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATAC
TAAATCATTTGAAGATATTCACCATTATAGGTGGGTTTAAATTGAATATAATAAGCTGACATTAA
KRAS-34GA
TATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCT-
GCTGAAAATGA
CTGAATATAAACTTGTGGTAGTTGGAGCT[G/A]GTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTC-
A GAATCATTTTGTGGACGAATATGA KRAS-35GA
TATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCT-
GCTGAAAATGA
CTGAATATAAACTTGTGGTAGTTGGAGCTG[G/A]TGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTC-
A GAATCATTTTGTGGACGAATATGATC KRAS-38GA
CATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGG-
TG[G/A]CGTA
GGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAA
ATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTT-
CA TGAGTACTTAT NRAS-181CA
ATTCTTACAGAAAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAG-
CTGGA[C/A]A
AGAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAAT
AATAGCAAGTCATTTGCGGATATTAACCTCTACAGGTACTAGGAGCATTATTTTCTCTGAAAGGATG
NRAS-183AT
TTACAGAAAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGG-
ACA[A/T]GAA
GAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAATAATA
GCAAGTCATTTGCGGATATTAACCTCTACAGGTACTAGGAGCATTATTTTCTCTGAAAGGATG
NRAS-35GA
TGGTTTCCAACAGGTTCTTGCTGGTGTGAAATGACTGAGTACAAACTGGTGGTGGTTGGAGCAG-
[G/A]TGGTG
TTGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATCCCACCATAGAGGT
GAGGCCCAGTGGTAGCCCG NRAS-38GA
TTTCCAACAGGTTCTTGCTGGTGTGAAATGACTGAGTACAAACTGGTGGTGGTTGGAGCAGGTG-
[G/A]TGTTG
GGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATCCCACCATAGAGGTGAG
GCCCAGTGGTAGCCC TP53-524GA
GGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGG-
C[G/A]CTGC
CCCCACCATGAGCGCTGCTCAGATAGCGATGGTGAGCAGCTGGGGCTGGAGAGACGACAGGGCTGGTTGCCCA
GGGTCCCCAGGCCTCTGATTCCTCACTGATTGCTCTTAGGTCTGGCC TP53-637CT
CCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAAC-
ACTTTT[C/T]
GACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTCTGGTTTGCAACTGGGGTCTCTGGGAGGAGGGGTTA
AGGGTGGTTGTCAGTGGCCCTC TP53-721TG
CTTGGGCCTGTGTTATCTCCTAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGT-
GTAACAGT[T/G]
CCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCA
CTTGCCACCCTGCACACTGGCCTGCTGTGCCCCAGCCTC TP53-733GA
TAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGC-
[G/A]GCATGA
ACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCACTTGCCACCCTGCACACTG
GCCTGCTGTGCCCCAGCCTC TP53-742CT
CTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAAC[-
C/T]GGAGGCC
CATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCACTTGCCACCCTGCACACTGGCCTGCTGTG-
C CCCAGCCTCTGCTTGCCTC TP53-743GA
TGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACC[-
G/A]GAGGCCC
ATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCACTTGCCACCCTGCACACTGGCCTGCTGTGC-
C CCAGCCTCTGCTTGCCTC TP53-817CT
CCTCTTGCTTCTCTTTTCCTATCCTGAGTAGTGGTAATCTACTGGGACGGAACAGCTTTGAGG-
TG[C/T]GTGTTT
GTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGC
TGCCCCCAGGGAGCACTAAGCGAGGTAAGCAA
[0226] Data Analysis:
[0227] An automatic baseline and manual threshold of 0.2 were used
to calculate the threshold cycle (C.sub.t) which is defined as the
fractional cycle number at which the fluorescence passes the fixed
threshold. PCR reactions were run for a total of 50 cycles. For
cast-PCR reactions, there was a pre-run of five cycles at a lower
annealing/extension temperature followed by an additional 45 cycles
at a higher annealing/extension temperature. The .DELTA.Ct between
amplification reactions for matched vs. mismatched sequences is
defined as the specificity of cast-PCR
(.DELTA.Ct=Ct.sub.mismatch-Ct.sub.match). The larger the .DELTA.Ct
between mismatched and matched targets, the better assay
specificity. The 2.sup..DELTA.Ct value was used to estimate the
power of discrimination (or selectivity) which is equal to
1/2.sup..DELTA.Ct or, in some cases, calculated as %
(1/2.sup..DELTA.Ct.times.100).
Example 1
Tailed Primers Improve Discrimination of Allelic Variants
[0228] The following example demonstrates that the application of
allele-specific primers comprising tails significantly improves the
discrimination of allelic variants.
[0229] In conventional AS-PCR, the discrimination of 3' nucleotide
mismatches is largely dependent on the sequence surrounding the SNP
and the nature of the allele. The .DELTA.Ct between the
amplification reactions for matched and mismatched primers varies.
To improve the discrimination between the amplification of matched
and mismatched sequences, allele-specific primers were designed to
comprise tails at their 5' termini and then tested for their
suitability in AS-PCR assays.
[0230] Assays were performed using the general experimental design
and reaction conditions indicated above (with the exception that no
blocker probes were included and either tailed or non-tailed
allele-specific primers were added), using 0.5 ng/uL genomic DNA
containing the hsv 11711720 SNP comprising one of three alleles (A,
C, or T) as the nucleic acid template (see Table 2A). The three
genotypes are indicated in Table 2B. Primers and probes were
designed according to the sequences shown in Table 3.
TABLE-US-00002 TABLE 2A Genomic DNA Sequence for hsv11711720 SNP
(target alleles are indicated in brackets).
AGAAAATAACTAAGGGAAGGAGGAAAGTGGGGAGGAAGGAAGAACAGTGT
GAAGACAATGGCCTGAAAACTGAAAAAGTCTGTTAAAGTTAATTATCAGT
TTTTGAGTCCAAGAACTGGCTTTGCTACTTTCTGTAAGTTTCTAATTTAC
TGAATAAGCATGAAAAAGATTGCTTTGAGGAATGGTTATAAACACATTCT
TAGAGCATAGTAAGCAGTAGGGAGTAACAAAATAACACTGATTAGAATAC
TTTACTCTACTTAATTAATCAATCATATTTAGTTTGACTCACCTTCCCAG
[A/C/T]ACCTTCTAGTTCTTTCTTATCTTTCAGTGCTTGTCCAGACAAC
ATTTTCATTTCAACAACTCCTGCTATTGCAATGATGGGTACAATTGCTAA
GAGTAACAGTGTTAGTTGCCAACCATAGATGAAGGATATAATTATTCCTG
TCCCAAGATTTGCTATATTCTGGGTAATTACAGCAAGCCTGGAACCTATA
GCCTGCAAAACAAAACAAATTAGAGAAATTTTAAAAATATTATCTTCACA
ACTCATGCTTCTATTTTCTGAAAACTCACCTTCATGAGACTATATTCATT ATTTTAT
TABLE-US-00003 TABLE 2B Genotypes of Genomic DNA Sequence for
hsv11711720 SNP Genomic DNA ID Genotype NA17203 AA NA17129 CC
NA17201 TT
TABLE-US-00004 TABLE 3 List and Sequences of Primers and Probes for
genomic DNA: conventional allele-specific primers ("ASP"); tailed
allele-specific primers ("tailASP"); locus-specific TaqMan probe
(LST); locus-specific primer (LSP). The nucleotides shown in lower
case are the tailed portion of the primers. The nucleotide-specific
portion of each allele-specific primer is at the 3'-most terminus
of each primer (indicated in bold). Primer/ Tm Probe ID Sequence
(5' to 3') (.degree. C.) 17129-ASP ATATTTAGTTTGACTCACCTTCCCAGC 63.2
17129- accACTCACCTTTCCCAGC 63.0 tailASP 17203-ASP
ATATTTAGTTTGACTCACCTTCCCAGA 62.0 17203- accACTCACCTTTCCCAGA 63.7
tailASP 17201-ASP ATATTTAGTTTGACTCACCTTCCCAGT 62.2 17201-
accACTCACCTTTCCCAGT 64.0 tailASP LST
(6-FAM)-TGGACAAGCACTGAAAGA-(MGB) 67.4 LSP
GCAGGAGTTGTTGAAATGAAAATGTTG 62.5
[0231] As shown in Table 4, when using non-tailed ASPs
("ASP-tail"), the discrimination of 3' nucleotide mismatch is
largely dependent on the nature of the allele, as a considerable
range of .DELTA.Ct values is observed depending on the identity of
the 3'-terminal base. The range of .DELTA.Ct values between matched
and mismatched nucleotides ("NT") were from -0.1 to 10. However,
with tailed ASPs (ASP+tail), the discrimination of 3' nucleotide
mismatch was significantly improved. In fact, as Table 4 shows, the
.DELTA.Ct value between matched and mismatched nucleotides was
consistently equal to or greater than 10 when tailed ASPs were
used. The Ct values for amplification of matched sequences using
tailed ASPs were comparable to those using conventional or
non-tailed ASPs. These results indicate that tailed ASP, can
improve the specificity of AS-PCR, but may not improve the
sensitivity of detection.
TABLE-US-00005 TABLE 4 Tailed allele-specific primers ("ASP")
significantly improve discrimination of allelic variants. The
specificity ("fold difference") was calculated based on the
difference between Ct values using tailed vs. untailed primers
(2.sup.(.DELTA.Ct(ASP+tail) - (.DELTA.Ct(ASP-tail)). The mismatched
nucleotides of the 3' allele-specific nucleotide portion of the
ASPs (+/- tail) and the target allele are also indicated ("NT
mismatch"). NT .DELTA.Ct Specificity Improvement mismatch (ASP -
tail) .DELTA.Ct (ASP + tail) (fold difference) C-A 0.9 11.5 1552.1
C-T 1.2 11.5 1278.3 A-C 10.0 11.9 3.7 A-G 9.8 11.9 4.3 T-G 2.3 11.5
588.1 T-C -0.1 11.5 3104.2 Average 4.0 11.6 1088.5
Example 2
Low Primer Concentrations Improve Discrimination of Allelic
Variants
[0232] Assays were performed using the general experimental design
and reaction conditions indicated above, in the presence of 1
million copies of plasmid DNA containing various SNP target
sequences (see Table 1) and 200 nM or 800 nM tailed ASP (as
indicated). Assay primers and probes were designed according to the
sequences shown in FIG. 11A.
[0233] The effect of tailed ASP concentration on discrimination of
allelic variants is summarized in Table 5. The .DELTA.Ct between
the amplification reactions for matched and mismatched primers
demonstrate that lower tailed ASP concentrations improve
discrimination of allelic variants.
TABLE-US-00006 TABLE 5 Assay Results Using Different Concentrations
of Tailed Allele-specific Primers .DELTA.Ct .DELTA.Ct Specificity
Plasmid (ASP @ (ASP @ Improvement SNP ID 800 nM) 200 nM) (fold
difference) CV11201742 14.1 15.2 2.14 CV11349123 8.2 10 3.48
CV1207700 5.2 6.6 2.64 CV25594064 20.1 19.1 0.5 CV25639181 11.9
12.9 2 Average 12.6 13.44 2.14
Example 3
Primers Designed with Reduced Tms Improves Discrimination of
Allelic Variants
[0234] Assays were performed using the general experimental design
and reaction conditions indicated above, in the presence of 1
million copies of plasmid DNA containing various SNP target
sequences (see Table 1) using tailed ASP with a higher Tm
(.about.57.degree. C.) or tailed ASP with a lower Tm
(.about.53.degree. C.). Assay primers and probes were designed
according to the sequences shown in FIG. 11B-E (see FIG. 11B for
higher Tm ASP and FIG. 11C or lower Tm ASP).
[0235] The effect of allele-specific primer Tm on discrimination of
allelic variants is summarized in Table 6. The .DELTA.Ct of
allele-specific primers with a lower Tm are significantly higher
than those of allele-specific primers with a higher Tm.
Allele-specific primers designed with reduced Tms improved
discrimination of allelic variants by as much as 118-fold in some
cases or an average of about 13-fold difference.
TABLE-US-00007 TABLE 6 .DELTA.Ct Values Using Tailed ASPs with
Lower Tm (~53.degree. C.) or with Higher Tm (~57.degree. C.)
Specificity Plasmid .DELTA.Ct (ASP w/ .DELTA.Ct (ASP w/ Improvement
SNP ID Tm ~57.degree. C.) Tm ~53.degree. C.) (fold difference)
BRAF-1799TA 12.2 19.1 118.9 CTNNB1-121AG 11.6 14.9 10.0 KRAS-176CG
18.8 22.5 13.1 NRAS-35GA 13.0 14.0 1 TP53-721TG 14.7 19.1 20.6
CTNNB1-134CT 8.6 14.1 44.8 EGFR-2369CT 9.7 10.7 2 KRAS-183AC 22.2
23.1 1.8 NRAS-38GA 14.0 14.3 1.2 TP53-733GA 13.6 13.5 1.0
EGFR-2573TG 16.7 20.2 10.9 KRAS-34GA 14 14.8 1.8 KRAS-38GA 11.2
14.4 8.9 NRAS-181CA 24.0 27.1 8.6 TP53-742CT 9.1 8.0 0.5 KRAS-35GA
11.5 15.1 12.3 NRAS-183AT 23.6 22.7 0.5 TP53-524GA 11.4 13.5 4.6
TP53-637CT 11.4 14.4 7.8 TP53-743GA 10.1 13.2 8.4 TP53-817CT 13.6
13.9 1.2 Average 14.1 16.3 13.3
Example 4
Use of Blocker Probes Improves Discrimination of Allelic
Variants
[0236] The following example illustrates that the use of MGB
blocker probes improves the discrimination between 3' nucleotide
mismatched and matched primers to target sequences in AS-PCR
reactions.
[0237] Assays were performed using the general cast-PCR schema and
reaction conditions indicated above, using 1 million copies of
plasmid DNA containing various SNP target sequences (see Table 1)
in the presence of MGB blocker probes or in the absence of MGB
blacker probes. Assay primers and probes were designed according to
the sequences shown in FIG. 11C-E.
[0238] To improve the selectivity of AS-PCR, blocker probes were
synthesized to comprise an MGB group at their 3' terminus. (See,
for example, Kutyavin, I. V., et al., Nucleic Acids Research, 2000,
Vol. 28, No. 2: 655-661, U.S. Pat. Nos. 5,512,677; 5,419,966;
5,696,251; 5,585,481; 5,942,610 and 5,736,626.)
[0239] The results of cast-PCR using MGB blocker probes are
summarized in Table 7. The .DELTA.Ct between cast-PCR with MGB
blocker probes is larger than that without MGB blocker probes. As
shown, MGB blocker probes improve the discrimination of allelic
variants.
TABLE-US-00008 TABLE 7 MGB Blocker Probes Improve Discrimination of
Allelic Variants Specificity .DELTA.Ct .DELTA.Ct (+MGB Improvement
SNP ID (no MGB blocker) blocker) (fold difference) BRAF-1799TA 11.4
14.9 11.5 CTNNB1-121AG 11.6 14.1 5.4 KRAS-176CG 17.8 20.9 9
NRAS-35GA 13.9 14.3 1.4 TP53-721TG 12.5 14.7 4.4 CTNNB1-134CT 6.7
10.2 11.6 EGFR-2369CT 7.7 10.1 5.3 KRAS-183AC 22.4 23 1.5 NRAS-38GA
14.5 14.6 1.1 TP53-733GA 13.2 14.4 2.3 EGFR-2573TG 18.2 21.8 11.6
KRAS-34GA 14.4 15.1 1.7 KRAS-38GA 11.9 15.1 1.7 NRAS-181CA 19.3
24.2 30.2 TP53-742CT 12.7 13.6 1.9 KRAS-35GA 11.0 13.7 6.5
NRAS-183AT 20.2 21.7 2.9 TP53-524GA 13.5 13.5 1 TP53-637CT 9.3 12.1
7.0 TP53-743GA 9.9 11.5 3.1 TP53-817CT 12.6 13.2 1.5 Average 13.6
15.5 6.0
Example 5
Primers Designed to Target Discriminating Bases Improves
Discrimination of Allelic Variants
[0240] Assays were performed using the general cast-PCR schema and
reaction conditions indicated above, in the presence of 1 million
copies of plasmid DNA containing SNP target sequences (see Table
1). Assay primers and probes were designed according to the
sequences shown in FIG. 11C-E.
[0241] According to the data summarized in Table 8, the
discrimination of cast-PCR was dependent on the nature of the
allele being analyzed. As Table 8 indicates, the .DELTA.Ct between
mismatched and matched sequences for allele-1 were different from
.DELTA.Ct between mismatched and matched sequences for allele-2.
However, both A and G bases, as compared to a T base, were highly
discriminating for allele-1 and allele-2 in all four SNPs
examined.
TABLE-US-00009 TABLE 8 Primers Designed to Target Discriminating
Bases Improve Discrimination of Allelic Variants ASP design SNP
allele-1 SNP allele-2 3' NT 3' NT .DELTA.Ct Specificity .DELTA.Ct
Specificity of of Allele (Ct_mismatch - (fold Allele (Ct_mismatch -
(fold SNP ID ASP1 ASP2 NT Ct_match) difference) NT Ct_match)
difference) KRAS-38GA G A C 13.4 10809 T 8.2 294 NRAS-181CA C A G
27.5 189812531 T 9.8 891 NRAS-183AT A T T 17.9 244589 A 23.4
11068835 TP53-742CT C T G 12.3 5043 A 8.3 315
Example 6
Determination of the Sensitivity and Dynamic Range for Cast-PCR
[0242] In this example, the sensitivity and dynamic range of
cast-PCR was determined by performing cast-PCR using various copy
numbers of a target plasmid.
[0243] Assays were performed using the general cast-PCR schema and
reaction conditions indicated above, using 1.times.10.sup.0 (1
copy) to 1.times.10.sup.7 copies of plasmid DNA containing the
NRAS-181CA SNP target sequence (see Table 1). Assay primers and
probes were designed according to the sequences shown in FIG.
11C-E.
[0244] As shown in FIG. 5, the use of tailed primers and
MGB-blocker probes does not adversely affect the sensitivity of
cast-PCR, as the sensitivity of cast-PCR is comparable to TaqMan
assays which do not utilize tailed primers or blocker probes.
Furthermore, FIG. 5 shows that the cast-PCR assay shows a linear
dynamic range over at least 7 logs.
Example 7
Determination of the Specificity of Cast-PCR
[0245] In this example, the specificity of cast-PCR was determined
by comparing the amplification of particular alleles of KRAS using
either matched or mismatched ASPs to a given allele in the presence
of their corresponding blocker probes.
[0246] Assays were performed using the general cast-PCR schema and
reaction conditions indicated above, using 1.times.10.sup.6 copies
of plasmid DNA containing either one of two alleles of the
KRAS-183AC SNP target sequence (see Table 1). Assay primers and
probes were designed according to the sequences shown in FIG.
11C-E.
[0247] The left panel of FIG. 7 shows the an amplification plot of
cast-PCR on allele-1 DNA using matched (A1) primers in the presence
of A2 blocker probes or mismatched (A2) primers in the presence of
A1 blocker probes. The right hand panel shows a similar experiment
in which cast-PCR was performed on allele-2 DNA. As indicated in
the data summary in FIG. 7, a robust .DELTA.Ct values of over 20
were observed for cast-PCR on both alleles of KRAS-183AC tested.
This corresponds to a specificity as determined by a calculation of
2.sup..DELTA.Ct of 9.times.10.sup.6, and 2.times.10.sup.6,
respectively, for allele-1 and allele-2. Furthermore, a calculation
of selectivity (1/2.sup..DELTA.Ct) indicates that values of
1/1.1.times.10.sup.7 and 1/5.0.times.10.sup.7 are observed for
allele-1 and allele-2, respectively.
Example 8
Cast-PCR is Able to Detect a Single Copy Mutant DNA in One Million
Copies of Wild Type DNA
[0248] In this example, the selectivity of cast-PCR, i.e., the
ability of cast-PCR to detect a rare mutant DNA in an excess of
wild type DNA, was determined.
[0249] Assays were performed using the general cast-PCR schema and
reaction conditions indicated above, using various copy numbers of
mutant KRAS-183AC plasmid DNA (1 copy to 1.times.10.sup.6 copies)
mixed with 1.times.10.sup.6 copies of wild type KRAS-183AC plasmid
DNA (see Table 1). Assay primers and probes were designed according
to the sequences shown in FIG. 11C-E, and cast-PCR reactions were
performed using wild type or mutant allele-specific primers and the
corresponding MGB blocker probes.
[0250] FIG. 8 shows that cast-PCR is able to detect as little as
one copy of a mutant DNA sequence, even when surrounded by a
million-fold excess of a wild type sequence.
Example 9
Selectivity of Cast-PCR in Discriminating Tumor Cell DNA from
Normal Cell DNA
[0251] In this example, the selectivity of cast-PCR was determined
by performing assays on samples in which various amounts of tumor
cell genomic DNA were mixed with or "spiked" into genomic DNA from
normal cells. DNA samples were extracted using QIAmp DNA Mini Prep
Kits (Qiagen). Wild type DNA was extracted from the SW48 cell line
and mutant DNA was extracted the H1573 cell line.
[0252] The mutant DNA contained the KRAS-G12A mutation (See FIG.
6). The percentage of tumor cell DNA in the spiked samples varied
from 0.5 to 100%. cast-PCR was used to detect the presence of tumor
cell DNA when present in these percentages.
[0253] Assays were performed using the general cast-PCR schema and
reaction conditions indicated above, using 30 ng of gDNA per
reaction. Assay primers and probes were designed according to the
sequences corresponding to KRAS-G12A SNP ID, as shown in FIG.
18A-B.
[0254] As shown in FIG. 9, tumor cell DNA, even when present only
at a level of 0.5% as compared to normal cell DNA, is easily
detected using cast-PCR.
Example 10
Use of Cast-PCR to Detect Tumor Cells in Tumor Samples
[0255] In this example, cast-PCR was used to detect and determine
the percentage of tumor cells in tumor samples. Various normal and
tumor samples were obtained and assayed by cast-PCR for the
presence of a number of SNPs associated with cancer as shown in
FIG. 10.
[0256] Assays were performed using the general cast-PCR schema and
reaction conditions indicated above, using 5 ng of gDNA or 1.5 ng
cDNA derived from either normal or tumor samples. Assay primers and
probes corresponding to the SNPs shown in FIG. 10 were designed
according to the sequences as shown in FIG. 11C-E.
[0257] The results shown in FIG. 10 indicate that cast-PCR has a
low false positive rate as indicated by the failure of cast-PCR to
detect the presence of mutant cells in normal samples. In contrast,
cast-PCR was able to provide a determination of the percentage of
tumor cells in various tumor samples that ranged from just under 2%
for a tumor sample containing the NRAS-183AT SNP to greater than
80% for a sample containing the CTNNB1-134CT SNP.
Example 11
Use of Pre-Amplification in Cast-PCR
[0258] The following example demonstrates that pre-amplification
combined with cast-PCR methods enables detection of multiple
alleles from a limited amount of genomic DNA template.
[0259] Prior to conducting cast-PCR amplification, multiplex
reactions were performed for 7 different KRAS mutations (see Table
9).
[0260] 10 .mu.l multiplex reactions were prepared in a single tube
by combining into one reaction 45 nM of each allele-specific primer
(including the allele-1-specific primer and the allele-2-specific
primer) and 45 nM of each locus-specific primer, as listed in FIG.
18A-B for each of the seven different KRAS SNPs, 0.1 ng/.mu.L
genomic DNA, and 1.times. Preamp Master Mix (Applied Biosystems,
Foster City, Calif.; P/N 437135). The 10 .mu.l pre-amplification
reactions were then incubated in an Applied Biosystems 9700
Thermocyler in a 96- or 384-well plate for 95.degree. C. for 10
minutes, followed by 10 cycles of 95.degree. C. for 15 seconds,
60.degree. C. for 4 minutes, and 99.9.degree. C. for 10 minutes,
and then held at 4.degree. C. Next, 190 .mu.l of 0.1.times.TE pH
8.0 was added to each 10 .mu.l pre-amplification reaction
(20.times. dilution). The diluted pre-amplification reaction
products were then directly used in subsequent cast-PCR reactions
or stored at -20.degree. C. for at least one week prior to use.
TABLE-US-00010 TABLE 9 KRAS SNP Sequences (target alleles are
indicated in brackets). SNP ID SNP Sequence KRAS-G12A_GC
TGACTGAATATAAACTTGTGGTAGTTGGAGCTG[G/C]TGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAA-
T
TCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTG-
C AGGACCATTCTTTGATACA KRAS-G12R_GC
TGACTGAATATAAACTTGTGGTAGTTGGAGCT[G/C]GTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAA-
T
TCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTG-
C AGGACCATTCTTTGATACA KRAS-G12D_GA
TGACTGAATATAAACTTGTGGTAGTTGGAGCTG[G/A]TGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAA-
T
TCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTG-
C AGGACCATTCTTTGATACA KRAS-G12S_GA
TGACTGAATATAAACTTGTGGTAGTTGGAGCT[G/A]GTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAA-
T
TCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTG-
C AGGACCATTCTTTGATACA KRAS-G13D_GA
TGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAAGAGTGCCTTGACGATACAGCTAA-
T
TCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTG-
C AGGACCATTCTTTGATACA KRAS-G12C_GT
GTGAGTTTGTATTAAAAGGTACTGGTGGAGTATNNGATAGTGTATTAACCTTATGTGTGACATGTTCTAATAT-
A
GTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCT[-
G/ T]GTGGCGTAGGCAAGAGT
[0261] Following pre-amplification, the diluted pre-amplification
products were aliquotted into single-plex cast-PCR reactions.
Individual assays were performed for each of the 7 different KRAS
mutations using the general experimental design and reaction
conditions indicated above (see section II of Examples). 10 .mu.L,
cast-PCR reactions were run for assays containing, as the nucleic
acid template, either 1 .mu.L 20.times. diluted pre-amplification
reaction product (as prepared above) or, as a comparison, 0.07 ng
genomic DNA. All assay primers and probes were designed according
to the sequences shown in FIG. 18A-B.
[0262] As shown in FIG. 12, for assays without pre-amplification
the average .DELTA.Ct of the 7 tested KRAS mutations was 12.0,
whereas for assays using pre-amplification the average .DELTA.Ct
was 17.0. Thus, the .DELTA..DELTA.Ct between the two gave an
improvement of about 5, which is about a 32 fold increase in
sensitivity, for the pre-amplified reactions over those without
pre-amplification.
[0263] In an ideal situation (where PCR efficiency=100%), the copy
number of the target gene increases about 1000 fold in 10 cycles.
Under these conditions, if the starting copy number in a
pre-amplification reaction is 0.1 ng/.mu.L (or approximately 33
copies/.mu.L), then after 10 cycles the copy number increases to
approximately 33,000 copies/.mu.L. In the example above, the copy
number of a 20 fold diluted pre-amplification product is estimated
to be approximately 1,650 copies/.mu.L. Therefore, after adding 1
.mu.L of 1,650 copies/.mu.L diluted pre-amplification product into
a cast-PCR reaction (final volume of 10 .mu.L), the concentration
of the cast-PCR products is approximately 165 copies/.mu.L. Based
on this estimation, 10 .mu.L of pre-amplification products from 1
ng/.mu.L genomic DNA can be diluted by as much as 200 fold, and
still provide up to 2000 .mu.L of nucleic acid template for use in
subsequent cast-PCR reactions.
Example 12
Effect of Tailed ASP on Cast-PCR Specificity
[0264] Assays were performed using the general cast-PCR schema and
reaction conditions indicated above (see section II of Examples),
using 1 million copies of plasmid DNA containing various SNP target
sequences (see Table 10). Assay primers and probes were designed
according to the sequences shown in FIG. 19A-D. For each SNP
analyzed, the blocker probes, locus-specific probes and
locus-specific primers were the same and only the allele-specific
primers varied (e.g., tailed or non-tailed).
TABLE-US-00011 TABLE 10 Plasmid SNP Sequences (target alleles are
indicated in brackets). SNP ID Sequence BRAF-1799TA_TP
ATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGAT
GGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTG CTNNB1-121AG_TP
GTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGAGAAGGAGCTGTGG[A/G]AGTGGCACCAG
AATGGATTNCAGAGTNCAGGTAAGACTGTTGCTGCCAGTGACTA CTNNB1-134CT_TP
CAGGACTTGGGAGGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGA[C/T]AAGGAGCTGTG
GTAGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTG EGFR-2369CT_TP
CCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCAGCTCATGCC
CTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATA EGFR-2573TG_TP
TTACTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCC[T/G]GCCCAAAATCT
GTGATCTTGACATGCTGCGGTGTTTTCACCAGTACGTTCCTGG KRAS-176CG_TP
AGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAG[C/G]AGGTCANGAG
GAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGC KRAS-183AC_TP
AAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCA[A/C]GAGGAGTACA
GTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTT KRAS-34GA_TP
CCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC[G/A]AGCTCCAACTAC
CACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAAA KRAS-35GA_TP
TCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCA[G/A]CAGCTCCAACTA
CCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAA KRAS-3GA_TP
ATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAA
GAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGA NRAS-181CA_TP
AACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGA[C/A]AAGAAGAGT
ACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCC NRAS-183AT_TP
CAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACA[A/T]GAAGAGTAC
AGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTC BRAF-1799TA_TP
ATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGAT
GGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTG CTNNB1-121AG_TP
GTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGAGAAGGAGCTGTGG[A/G]AGTGGCACCAG
AATGGATTNCAGAGTNCAGGTAAGACTGTTGCTGCCAGTGACTA CTNNB1-134CT_TP
CAGGACTTGGGAGGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGA[C/T]AAGGAGCTGTG
GTAGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTG EGFR-2369CT_TP
CCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCAGCTCATGCC
CTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATA EGFR-2573TG_TP
TTACTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCC[T/G]GCCCAAAATCT
GTGATCTTGACATGCTGCGGTGTTTTCACCAGTACGTTCCTGG KRAS-176CG_TP
AGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAG[C/G]AGGTCANGAG
GAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGC KRAS-183AC_TP
AAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCA[C/G]GAGGAGTACA
GTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTT KRAS-34GA_TP
CCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC[G/A]AGCTCCAACTAC
CACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAAA KRAS-35GA_TP
TCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCA[G/A]CAGCTCCAACTA
CCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAA KRAS-3GA_TP
ATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAA
GAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGA NRAS-181CA_TP
AACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGA[C/A]AAGAAGAGT
ACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCC NRAS-183AT_TP
CAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACA[A/T]GAAGAGTAC
AGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTC BRAF-1799TA_TP
ATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGAT
GGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTG CTNNB1-121AG_TP
GTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGAGAAGGAGCTGTGG[A/G]AGTGGCACCAG
AATGGATTNCAGAGTNCAGGTAAGACTGTTGCTGCCAGTGACTA CTNNB1-134CT_TP
CAGGACTTGGGAGGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGA[C/T]AAGGAGCTGTG
GTAGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTG EGFR-2369CT_TP
CCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCAGCTCATGCC
CTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATA EGFR-2573TG_TP
TTACTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCC[C/T]GCCCAAAATCT
GTGATCTTGACATGCTGCGGTGTTTTCACCAGTACGTTCCTGG KRAS-176CG_TP
AGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAG[T/G]AGGTCANGAG
GAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGC KRAS-183AC_TP
AAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCA[A/C]GAGGAGTACA
GTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTT KRAS-34GA_TP
CCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC[G/A]AGCTCCAACTAC
CACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAAA KRAS-35GA_TP
TCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCA[G/A]CAGCTCCAACTA
CCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAA KRAS-3GA_TP
ATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAA
GAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGA NRAS-181CA_TP
AACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGA[C/A]AAGAAGAGT
ACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCC NRAS-183AT_TP
CAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACA[A/T]GAAGAGTAC
AGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTC
[0265] The results of cast-PCR using non-tailed ASP and cast-PCR
using tailed-ASP are summarized in FIG. 13. In the cast-PCR
reactions having no tailed primers, the average .DELTA.Ct of the 12
tested mutations was 10.3, whereas in the cast-PCR reactions with
tailed primers the average .DELTA.Ct of 12 tested assays is 16.3.
Thus, the average A.DELTA.Ct between cast-PCR comprising tailed ASP
versus cast-PCR comprising non-tailed ASP was about 6.0, which is
about a 64 fold improvement in specificity for reactions comprising
ASP +tail primers.
Example 13
Comparison of Cast-PCR and ASB-PCR
[0266] Allelic discrimination for assays using cast-PCR methods was
compared to assays using other Allele-Specific PCR with a Blocking
reagent (ASB-PCR) methods (see, e.g., Morlan et al., 2009).
[0267] cast-PCR assays were performed using the general schema and
reaction conditions indicated above, using 1 million copies of
plasmid DNA containing various SNP target sequences (see Table 10).
Assays were performed using the general experimental design and
reaction conditions indicated above (see section II of Examples).
Assay primers and probes were designed according to the sequences
shown in FIG. 19B-D.
[0268] ASB-PCR assays were performed using 1 million copies of
plasmid DNA containing various SNP target sequences (see Table 10),
900 nM non-tailed allele-specific primers (Tm 58.about.62.degree.
C.), 3600 nM allele-specific phosphate blocker (Tm
58.about.62.degree. C.), 200 nM locus-specific TaqMan probe (Tm
70.about.74.degree. C.), and 900 nM locus-specific primers (Tm
60.about.63.degree. C.).
[0269] ASB-PCR assay primers and probes were designed according to
the sequences shown in FIG. 20A-C. The ASB-PCR reactions were
incubated in a 384-well plate at 95.degree. C. for 10 minutes,
followed by 50 cycles of at 92.degree. C. for 20 seconds,
60.degree. C. for 45 seconds. All reactions were run in 4
replications in an ABI PRISM 7900HT.RTM. Sequence Detection System,
according to the manufacturer's instructions.
[0270] The results for this example are summarized in FIG. 14. In
the ASB-PCR assays, the average .DELTA.Ct of 12 different mutations
was 14.1. In cast-PCR assays, the average .DELTA.Ct of the same 12
mutations was 16.3. The A.DELTA.Ct between ASB-PCR and cast-PCR was
2.2, which indicates that the specificity of cast-PCR was
approximately 4.6 fold higher than that of the ASB-PCR assay.
Example 14
Comparison of MGB and Phosphate Blocker Probes in Cast-PCR
[0271] The use of MGB blocker probes was compared to the use of
other types of blocker probes, such as PO.sub.4 blocker probes
(e.g., Morlan et al., 2009), in cast-PCR assays.
[0272] All assays were performed using the general cast-PCR schema
and reaction conditions indicated above (see Section II in
Examples), using 1 million copies of plasmid DNA containing various
SNP target sequences (see Table 10), except that reactions
contained either 150 nm allele-specific MGB blocker probes or 150
nm allele-specific 3'-phosphate blocker probes. Assay primers and
probes were designed according to the sequences shown in FIG. 19B-D
(for cast-PCR using MGB blocker probes) or FIG. 19B-C and FIG. 20C
(for cast-PCR using phosphate blocker probes; "PHOS1" to block
allele-1 and "PHOS2" to block allele-2).
[0273] The results of assays with phosphate blocker probes or with
MGB blocker probes are summarized in FIG. 15. In cast-PCR assays
performed using phosphate blocker probes the average .DELTA.Ct of
12 different mutations was 15.1. In comparison, the average
.DELTA.Ct for the same 12 mutations using cast-PCR assays performed
with MGB blocker probes was slightly higher and gave a .DELTA.Ct of
15.8.
Example 15
Improving the Specificity of Cast-PCR Using Lna Modified Asp
[0274] LNA-modified cast-PCR assays were performed using the
general experimental design and reaction conditions indicated above
(see Section H in Examples), using 0.5 ng/.mu.L of genomic DNA.
Assay primers and probes were designed according to the sequences
shown in FIGS. 21A-C. For each SNP analyzed, the blocker probes,
locus-specific probes and locus-specific primers were the same and
only the allele-specific primers varied (i.e., with or without an
LNA-modification at the 3' end.).
[0275] The effect of LNA modification of the ASP on the specificity
of cast-PCR is summarized in FIG. 16. For the 12 cast-PCR assays
performed using LNA-modified allele-specific primers, the average
.DELTA.Ct was 16.3. In comparison, the average .DELTA.Ct for the
same 12 mutations using cast-PCR assays performed allele-specific
primers having no modifications the .DELTA.Ct was noticeably higher
at 18.5. Based on the .DELTA..DELTA.Ct the assay specificity
increased by approximately 4 fold for those assays that used
LNA-modified allele-specific primers.
Example 16
Improving the Specificity of Cast-PCR Using Other Modified ASP
[0276] cast-PCR assays using other chemically-modified ASPs were
performed using the general experimental design and reaction
conditions indicated above conditions indicated above (see Section
II in Examples), performed in the presence of 1 million copies of
plasmid DNA containing various SNP target sequences (see Table 10).
Assay primers and probes were designed according to the sequences
shown in FIG. 22. For each SNP analyzed, the blocker probes,
locus-specific probes and locus-specific primers were the same and
only the allele-specific primers varied (i.e., with or without
chemical modifications, i.e., ppA, ppG, iso dC or fdU, at the 3'
end).
[0277] The results of cast-PCR assays using unmodified ASP and
cast-PCR assays with modified ASP are summarized in FIG. 17. As
shown, allele-specific primers having pyrophosphate modifications
(ppA or ppG) at their 3'-ends increased .DELTA.Ct by 2-3, which is
approximately a 4-6 fold increase in assay specificity.
[0278] It s noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the," include
plural referents unless expressly and unequivocally limited to one
referent. The use of "or" means "and/or" unless stated otherwise.
The use of "comprise," "comprises," "comprising," "include,"
"includes," and "including" are interchangeable and not intended to
be limiting. Furthermore, where the description of one or more
embodiments uses the term "comprising," those skilled in the art
would understand that, in some specific instances, the embodiment
or embodiments can be alternatively described using the language
"consisting essentially of" and/or "consisting of."
[0279] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including but not limited to patents, patent
applications, articles, books, and treatises are hereby expressly
incorporated by reference in their entirety for any purpose. In the
event that one or more of the incorporated documents defines a term
that contradicts that term's definition in this application, this
application controls.
[0280] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated by reference in their entirety. In the event that one
or more of the incorporated literature and similar materials
differs.
Sequence CWU 1
1
6351144DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1gctctgcttc attcctgtct gaagaagggc
agatagtttg gctgctcctg tgytgtcacc 60tgcaattctc ccttatcagg gccattggcc
tctcccttct ctctgtgagg gatattttct 120ctgacttgtc aatccacatc ttcc
144282DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2ggcttgcaat ggctccaacc ggaagggcgg
tgctcgagct gtggtgcgtg cygctaagtt 60gtgcgttcca gggtgcactc gc
82365DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3gcaactatac ccttgatgga tggagattta
ygcaatgtgt tttactgggt agagtgacag 60acctt 654124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
4cctgaactta tttggcaaga gcgatgagta ctcttaaaat tactatctgg aaattatatt
60atttagaatc tgccaattac ctagatcccc cctsaacaat tgtttcacca aggaacttcc
120tgaa 1245180DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 5gaattggttg tctccttatg ggaactggaa
gtattttgac akctttacca catttcttca 60tgggatagta agtgttaaac agctctgagc
catttattat cagctacttg taaattagca 120gtagaatttt atttttatac
ttgtaagtgg gcagttacct tttgagagga atacctatag 180687DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6gcggagggaa gctcatcagt ggggccacga gctgagtgcg
tcctgtcact ccactcccat 60gtcccttggg aaggtctgag actaggg
877192DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 7tactacacct cagatatatt tcttcatgaa
gacctcacag taaaaatagg tgattttggt 60ctagctacag wgaaatctcg atggagtggg
tcccatcagt ttgaacagtt gtctggatcc 120attttgtgga tggtaagaat
tgaggctatt tttccactga ttaaattttt ggccctgaga 180tgctgctgag tt
1928198DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 8tgctaatact gtttcgtatt tatagctgat
ttgatggagt tggacatggc catggaacca 60gacagaaaag cggctgttag tcactggcag
caacagtctt acctggactc tggaatccat 120tctggtgcca ctrccacagc
tccttctctg agtggtaaag gcaatcctga ggaagaggat 180gtggatacct cccaagtc
1989184DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 9tttgatggag ttggacatgg ccatggaacc
agacagaaaa gcggctgtta gtcactggca 60gcaacagtct tacctggact ctggaatcca
ttctggtgcc actaccacag ctccttytct 120gagtggtaaa ggcaatcctg
aggaagagga tgtggatacc tcccaagtcc tgtatgagtg 180ggaa
18410181DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 10gtggacaacc cccacgtgtg ccgcctgctg
ggcatctgcc tcacctccac cgtgcagctc 60atcaygcagc tcatgccctt cggctgcctc
ctggactatg tccgggaaca caaagacaat 120attggctccc agtacctgct
caactggtgt gtgcagatcg caaaggtaat cagggaaggg 180a
18111166DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 11gcatgaacta cttggaggac cgtcgcttgg
tgcaccgcga cctggcagcc aggaacgtac 60tggtgaaaac accgcagcat gtcaagatca
cagattttgg gckggccaaa ctgctgggtg 120cggaagagaa agaataccat
gcagaaggag gcaaagtaag gaggtg 16612172DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
12caggattcct acaggaagca agtagtaatt gatggagaaa cctgtctctt ggatattctc
60gacacagsag gtcaagagga gtacagtgca atgagggacc agtacatgag gactggggag
120ggctttcttt gtgtatttgc cataaataat actaaatcat ttgaagatat tc
17213207DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 13acaggaagca agtagtaatt gatggagaaa
cctgtctctt ggatattctc gacacagcag 60gtcamgagga gtacagtgca atgagggacc
agtacatgag gactggggag ggctttcttt 120gtgtatttgc cataaataat
actaaatcat ttgaagatat tcaccattat aggtgggttt 180aaattgaata
taataagctg acattaa 20714169DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 14tattaacctt
atgtgtgaca tgttctaata tagtcacatt ttcattattt ttattataag 60gcctgctgaa
aatgactgaa tataaacttg tggtagttgg agctrgtggc gtaggcaaga
120gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatga
16915171DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 15tattaacctt atgtgtgaca tgttctaata
tagtcacatt ttcattattt ttattataag 60gcctgctgaa aatgactgaa tataaacttg
tggtagttgg agctgrtggc gtaggcaaga 120gtgccttgac gatacagcta
attcagaatc attttgtgga cgaatatgat c 17116230DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
16cattattttt attataaggc ctgctgaaaa tgactgaata taaacttgtg gtagttggag
60ctggtgrcgt aggcaagagt gccttgacga tacagctaat tcagaatcat tttgtggacg
120aatatgatcc aacaatagag gtaaatcttg ttttaatatg catattactg
gtgcaggacc 180attctttgat acagataaag gtttctctga ccattttcat
gagtacttat 23017210DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 17attcttacag aaaacaagtg
gttatagatg gtgaaacctg tttgttggac atactggata 60cagctggama agaagagtac
agtgccatga gagaccaata catgaggaca ggcgaaggct 120tcctctgtgt
atttgccatc aataatagca agtcatttgc ggatattaac ctctacaggt
180actaggagca ttattttctc tgaaaggatg 21018206DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
18ttacagaaaa caagtggtta tagatggtga aacctgtttg ttggacatac tggatacagc
60tggacawgaa gagtacagtg ccatgagaga ccaatacatg aggacaggcg aaggcttcct
120ctgtgtattt gccatcaata atagcaagtc atttgcggat attaacctct
acaggtacta 180ggagcattat tttctctgaa aggatg 20619162DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
19tggtttccaa caggttcttg ctggtgtgaa atgactgagt acaaactggt ggtggttgga
60gcagrtggtg ttgggaaaag cgcactgaca atccagctaa tccagaacca ctttgtagat
120gaatatgatc ccaccataga ggtgaggccc agtggtagcc cg
16220158DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 20tttccaacag gttcttgctg gtgtgaaatg
actgagtaca aactggtggt ggttggagca 60ggtgrtgttg ggaaaagcgc actgacaatc
cagctaatcc agaaccactt tgtagatgaa 120tatgatccca ccatagaggt
gaggcccagt ggtagccc 15821189DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 21ggcacccgcg
tccgcgccat ggccatctac aagcagtcac agcacatgac ggaggttgtg 60aggcrctgcc
cccaccatga gcgctgctca gatagcgatg gtgagcagct ggggctggag
120agacgacagg gctggttgcc cagggtcccc aggcctctga ttcctcactg
attgctctta 180ggtctggcc 18922165DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 22cctcctcagc
atcttatccg agtggaagga aatttgcgtg tggagtattt ggatgacaga 60aacactttty
gacatagtgt ggtggtgccc tatgagccgc ctgaggtctg gtttgcaact
120ggggtctctg ggaggagggg ttaagggtgg ttgtcagtgg ccctc
16523183DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 23cttgggcctg tgttatctcc taggttggct
ctgactgtac caccatccac tacaactaca 60tgtgtaacag tkcctgcatg ggcggcatga
accggaggcc catcctcacc atcatcacac 120tggaagactc caggtcagga
gccacttgcc accctgcaca ctggcctgct gtgccccagc 180ctc
18324163DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 24taggttggct ctgactgtac caccatccac
tacaactaca tgtgtaacag ttcctgcatg 60ggcrgcatga accggaggcc catcctcacc
atcatcacac tggaagactc caggtcagga 120gccacttgcc accctgcaca
ctggcctgct gtgccccagc ctc 16325163DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 25ctgactgtac
caccatccac tacaactaca tgtgtaacag ttcctgcatg ggcggcatga 60acyggaggcc
catcctcacc atcatcacac tggaagactc caggtcagga gccacttgcc
120accctgcaca ctggcctgct gtgccccagc ctctgcttgc ctc
16326162DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 26tgactgtacc accatccact acaactacat
gtgtaacagt tcctgcatgg gcggcatgaa 60ccrgaggccc atcctcacca tcatcacact
ggaagactcc aggtcaggag ccacttgcca 120ccctgcacac tggcctgctg
tgccccagcc tctgcttgcc tc 16227176DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 27cctcttgctt
ctcttttcct atcctgagta gtggtaatct actgggacgg aacagctttg 60aggtgygtgt
ttgtgcctgt cctgggagag accggcgcac agaggaagag aatctccgca
120agaaagggga gcctcaccac gagctgcccc cagggagcac taagcgaggt aagcaa
17628601DNAHomo sapiens 28agaaaataac taagggaagg aggaaagtgg
ggaggaagga agaacagtgt gaagacaatg 60gcctgaaaac tgaaaaagtc tgttaaagtt
aattatcagt ttttgagtcc aagaactggc 120tttgctactt tctgtaagtt
tctaatttac tgaataagca tgaaaaagat tgctttgagg 180aatggttata
aacacattct tagagcatag taagcagtag ggagtaacaa aataacactg
240attagaatac tttactctac ttaattaatc aatcatattt agtttgactc
accttcccag 300haccttctag ttctttctta tctttcagtg cttgtccaga
caacattttc atttcaacaa 360ctcctgctat tgcaatgatg ggtacaattg
ctaagagtaa cagtgttagt tgccaaccat 420agatgaagga tataattatt
cctgtcccaa gatttgctat attctgggta attacagcaa 480gcctggaacc
tatagcctgc aaaacaaaac aaattagaga aattttaaaa atattatctt
540cacaactcat gcttctattt tctgaaaact caccttcatg agactatatt
cattatttta 600t 6012927DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 29atatttagtt tgactcacct
tcccagc 273019DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 30accactcacc tttcccagc
193127DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 31atatttagtt tgactcacct tcccaga
273219DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 32accactcacc tttcccaga 193327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
33atatttagtt tgactcacct tcccagt 273419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
34accactcacc tttcccagt 193518DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 35tggacaagca ctgaaaga
183627DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 36gcaggagttg ttgaaatgaa aatgttg
2737163DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 37tgactgaata taaacttgtg gtagttggag
ctgstggcgt aggcaagagt gccttgacga 60tacagctaat tcagaatcat tttgtggacg
aatatgatcc aacaatagag gtaaatcttg 120ttttaatatg catattactg
gtgcaggacc attctttgat aca 16338163DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 38tgactgaata
taaacttgtg gtagttggag ctsgtggcgt aggcaagagt gccttgacga 60tacagctaat
tcagaatcat tttgtggacg aatatgatcc aacaatagag gtaaatcttg
120ttttaatatg catattactg gtgcaggacc attctttgat aca
16339163DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 39tgactgaata taaacttgtg gtagttggag
ctgrtggcgt aggcaagagt gccttgacga 60tacagctaat tcagaatcat tttgtggacg
aatatgatcc aacaatagag gtaaatcttg 120ttttaatatg catattactg
gtgcaggacc attctttgat aca 16340163DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 40tgactgaata
taaacttgtg gtagttggag ctrgtggcgt aggcaagagt gccttgacga 60tacagctaat
tcagaatcat tttgtggacg aatatgatcc aacaatagag gtaaatcttg
120ttttaatatg catattactg gtgcaggacc attctttgat aca
16341163DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 41tgactgaata taaacttgtg gtagttggag
ctggtgrcgt aggcaagagt gccttgacga 60tacagctaat tcagaatcat tttgtggacg
aatatgatcc aacaatagag gtaaatcttg 120ttttaatatg catattactg
gtgcaggacc attctttgat aca 16342164DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 42gtgagtttgt
attaaaaggt actggtggag tatnngatag tgtattaacc ttatgtgtga 60catgttctaa
tatagtcaca ttttcattat ttttattata aggcctgctg aaaatgactg
120aatataaact tgtggtagtt ggagctkgtg gcgtaggcaa gagt
16443111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 43atatttcttc atgaagacct cacagtaaaa
ataggtgatt ttggtctagc tacagwgaaa 60tctcgatgga gtgggtccca tcagtttgaa
cagttgtctg gatccatttt g 11144111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 44gtatccacat
cctcttcctc aggattgcct ttaccactca gagaaggagc tgtggragtg 60gcaccagaat
ggattncaga gtncaggtaa gactgttgct gccagtgact a 11145111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
45caggacttgg gaggtatcca catcctcttc ctcaggattg cctttaccac tcagayaagg
60agctgtggta gtggcaccag aatggattnc agagtncagg taagactgtt g
11146111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 46cccacgtgtg ccgcctgctg ggcatctgcc
tcacctccac cgtgcagctc atcaygcagc 60tcatgccctt cggctgcctc ctggactatg
tccgggaaca caaagacaat a 11147111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 47ttactttgcc
tccttctgca tggtattctt tctcttccgc acccagcagt ttggcckgcc 60caaaatctgt
gatcttgaca tgctgcggtg ttttcaccag tacgttcctg g 11148111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
48aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga cacagsaggt
60cangaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg c
11149111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 49aagtagtaat tgatggagaa acctgtctct
tggatattct cgacacagca ggtcamgagg 60agtacagtgc aatgagggac cagtacatga
ggactgggga gggctttctt t 11150111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 50ccacaaaatg
attctgaatt agctgtatcg tcaaggcact cttgcctacg ccacragctc 60caactaccac
aagtttatat tcagtcattt tcagcaggcc ttataataaa a 11151111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
51tccacaaaat gattctgaat tagctgtatc gtcaaggcac tcttgcctac gccarcagct
60ccaactacca caagtttata ttcagtcatt ttcagcaggc cttataataa a
11152111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 52attataaggc ctgctgaaaa tgactgaata
taaacttgtg gtagttggag ctggtgrcgt 60aggcaagagt gccttgacga tacagctaat
tcagaatcat tttgtggacg a 11153111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 53aacaagtggt
tatagatggt gaaacctgtt tgttggacat actggataca gctggamaag 60aagagtacag
tgccatgaga gaccaataca tgaggacagg cgaaggcttc c 11154111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
54caagtggtta tagatggtga aacctgtttg ttggacatac tggatacagc tggacawgaa
60gagtacagtg ccatgagaga ccaatacatg aggacaggcg aaggcttcct c
11155111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 55atatttcttc atgaagacct cacagtaaaa
ataggtgatt ttggtctagc tacagwgaaa 60tctcgatgga gtgggtccca tcagtttgaa
cagttgtctg gatccatttt g 11156111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 56gtatccacat
cctcttcctc aggattgcct ttaccactca gagaaggagc tgtggragtg 60gcaccagaat
ggattncaga gtncaggtaa gactgttgct gccagtgact a 11157111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
57caggacttgg gaggtatcca catcctcttc ctcaggattg cctttaccac tcagayaagg
60agctgtggta gtggcaccag aatggattnc agagtncagg taagactgtt g
11158111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 58cccacgtgtg ccgcctgctg ggcatctgcc
tcacctccac cgtgcagctc atcaygcagc 60tcatgccctt cggctgcctc ctggactatg
tccgggaaca caaagacaat a 11159111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 59ttactttgcc
tccttctgca tggtattctt tctcttccgc acccagcagt ttggcckgcc 60caaaatctgt
gatcttgaca tgctgcggtg ttttcaccag tacgttcctg g 11160111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
60aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga cacagsaggt
60cangaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg c
11161111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 61aagtagtaat tgatggagaa acctgtctct
tggatattct cgacacagca ggtcasgagg 60agtacagtgc aatgagggac cagtacatga
ggactgggga gggctttctt t 11162111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide
62ccacaaaatg attctgaatt agctgtatcg tcaaggcact cttgcctacg ccacragctc
60caactaccac aagtttatat tcagtcattt tcagcaggcc ttataataaa a
11163111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 63tccacaaaat gattctgaat tagctgtatc
gtcaaggcac tcttgcctac gccarcagct 60ccaactacca caagtttata ttcagtcatt
ttcagcaggc cttataataa a 11164111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 64attataaggc
ctgctgaaaa tgactgaata taaacttgtg gtagttggag ctggtgrcgt 60aggcaagagt
gccttgacga tacagctaat tcagaatcat tttgtggacg a 11165111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
65aacaagtggt tatagatggt gaaacctgtt tgttggacat actggataca gctggamaag
60aagagtacag tgccatgaga gaccaataca tgaggacagg cgaaggcttc c
11166111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 66caagtggtta tagatggtga aacctgtttg
ttggacatac tggatacagc tggacawgaa 60gagtacagtg ccatgagaga ccaatacatg
aggacaggcg aaggcttcct c 11167111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 67atatttcttc
atgaagacct cacagtaaaa ataggtgatt ttggtctagc tacagwgaaa 60tctcgatgga
gtgggtccca tcagtttgaa cagttgtctg gatccatttt g 11168111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
68gtatccacat cctcttcctc aggattgcct ttaccactca gagaaggagc tgtggragtg
60gcaccagaat ggattncaga gtncaggtaa gactgttgct gccagtgact a
11169111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 69caggacttgg gaggtatcca catcctcttc
ctcaggattg cctttaccac tcagayaagg 60agctgtggta gtggcaccag aatggattnc
agagtncagg taagactgtt g 11170111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 70cccacgtgtg
ccgcctgctg ggcatctgcc tcacctccac cgtgcagctc atcaygcagc 60tcatgccctt
cggctgcctc ctggactatg tccgggaaca caaagacaat a 11171111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
71ttactttgcc tccttctgca tggtattctt tctcttccgc acccagcagt ttggccygcc
60caaaatctgt gatcttgaca tgctgcggtg ttttcaccag tacgttcctg g
11172111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 72aggaagcaag tagtaattga tggagaaacc
tgtctcttgg atattctcga cacagkaggt 60cangaggagt acagtgcaat gagggaccag
tacatgagga ctggggaggg c 11173111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 73aagtagtaat
tgatggagaa acctgtctct tggatattct cgacacagca ggtcamgagg 60agtacagtgc
aatgagggac cagtacatga ggactgggga gggctttctt t 11174111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
74ccacaaaatg attctgaatt agctgtatcg tcaaggcact cttgcctacg ccacragctc
60caactaccac aagtttatat tcagtcattt tcagcaggcc ttataataaa a
11175111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 75tccacaaaat gattctgaat tagctgtatc
gtcaaggcac tcttgcctac gccarcagct 60ccaactacca caagtttata ttcagtcatt
ttcagcaggc cttataataa a 11176111DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 76attataaggc
ctgctgaaaa tgactgaata taaacttgtg gtagttggag ctggtgrcgt 60aggcaagagt
gccttgacga tacagctaat tcagaatcat tttgtggacg a 11177111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
77aacaagtggt tatagatggt gaaacctgtt tgttggacat actggataca gctggamaag
60aagagtacag tgccatgaga gaccaataca tgaggacagg cgaaggcttc c
11178111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 78caagtggtta tagatggtga aacctgtttg
ttggacatac tggatacagc tggacawgaa 60gagtacagtg ccatgagaga ccaatacatg
aggacaggcg aaggcttcct c 11179290DNAHomo sapiens 79gtactggtgg
agtatttgat agtgtattaa ccttatgtgt gacatgttct aatatagtca 60cattttcatt
atttttatta taaggcctgc tgaaaatgac tgaatataaa cttgtggtag
120ttggagctgg tggcgtaggc aagagtgcct tgacgataca gctaattcag
aatcattttg 180tggacgaata tgatccaaca atagaggtaa atcttgtttt
aatatgcata ttactggtgc 240aggaccattc tttgatacag ataaaggttt
ctctgaccat tttcatgagt 2908016DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 80cccgctgctc ctgtgc
168120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 81acccaacgca caacttagcg 208225DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
82cccgccttga tggatggaga tttac 258324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
83gcacccttgg tgaaacaatt gttg 248425DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
84cgccgaactg gaagtatttt gacag 258523DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
85tatccctcac agagagaagg gag 238619DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 86gcttgcaatg gctccaacc
198719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 87ggagccattg caagccaag 198821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
88tggcaagagc gatgagtact c 218925DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 89ctctcaaaag gtaactgccc
actta 259016DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 90ctcctgtgct gtcacc 169115DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
91cttagcggca cgcac 159218DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 92ggagatttac gcaatgtg
189317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 93acaattgttg agggggg 179420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
94aagtattttg acagctttac 209517DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 95cccggctgct cctgtgt
179620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 96gccgaacgca caacttagca 209725DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
97cccgccttga tggatggaga tttat 259825DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
98tgcacccttg gtgaaacaat tgttc 259925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
99cgccgaactg gaagtatttt gacat 2510019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
100atggccctga taagggaga 1910115DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 101agggcggtgc tcgag
1510219DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 102tgtcactcta cccagtaaa 1910321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
103agaatctgcc aattacctag a 2110421DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 104acaagtagct gataataaat g
2110517DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 105gctcctgtgt tgtcacc 1710616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
106cttagcagca cgcacc 1610719DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 107tggagattta tgcaatgtg
1910817DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 108acaattgttc agggggg 1710920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
109aagtattttg acatctttac 2011024DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 110gcctgatttt ggtctagcta
caga 2411119DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 111cgcgagaagg agctgtggt
1911221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 112ccgtgccttt accactcaga g 2111318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
113gcgcgtgcag ctcatcac 1811417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 114cggcagcagt ttggccc
1711521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 115gccggatatt ctcgacacag c 2111618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
116gcggacacag caggtcaa 1811718DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 117gcggacacag caggtcaa
1811818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 118cgccttgcct acgccact 1811916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
119cgctgcctac gccacg 1612017DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 120gcgttgccta cgccacc
1712119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 121cgcctcttgc ctacgccat 1912218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
122gcgtcttgcc tacgccag 1812318DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 123gcgtcttgcc tacgccac
1812420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 124cgcgtagttg gagctggtga 2012522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
125ccgatactgg atacagctgg aa 2212620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
126ccgtggatac agctggacaa 2012718DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 127ccgggtggtt ggagcaga
1812818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 128ccgggttgga gcaggtga 1812918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
129ccgggaggtt gtgaggca 1813023DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 130gccggatgac agaaacactt ttc
2313125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 131gcgtacaact acatgtgtaa cagtg
2513217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 132gccttcctgc atgggca 1713316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
133gccggcggca tgaacc 1613416DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 134gccgcggcat gaacca
1613522DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 135gccgccaaca gctttgaggt gc 2213623DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
136ccggattttg gtctagctac agt 2313718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
137gccagaagga gctgtggc 1813821DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 138ccgtgccttt accactcaga a
2113918DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 139gcgcgtgcag ctcatcat 1814017DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
140cggcagcagt ttggcca 1714122DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 141cgctggatat tctcgacaca gg
2214218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 142gcggacacag caggtcac 1814318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
143gcggacacag caggtcat 1814417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 144gcgttgccta cgccacc
1714517DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 145gcgttgccta cgccacc 1714617DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
146gcgttgccta cgccaca 1714718DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 147gcgtcttgcc tacgccac
1814818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 148gcgtcttgcc tacgccac 1814918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
149gcgtcttgcc tacgccaa 1815019DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 150gcctagttgg agctggtgg
1915122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 151ccgatactgg atacagctgg ac 2215221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
152cgcctggata cagctggaca t 2115317DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 153cgcgtggttg gagcagg
1715417DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 154cgcgttggag caggtgg 1715517DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
155ggcgaggttg tgaggcg 1715624DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 156gcctggatga cagaaacact tttt
2415726DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 157ggcctacaac tacatgtgta acagtt
2615816DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 158gcctcctgca tgggcg 1615916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
159gccggcggca tgaact 1616016DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 160gccgcggcat gaaccg
1616120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 161cgcgaacagc tttgaggtgt 2016219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
162cccgtggtct agctacaga 1916317DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 163ccccaaggag ctgtggt
1716420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 164cgccccttta ccactcagag 2016518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
165ggggatgcag ctcatcac 1816615DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 166ggcgcagttt ggccc
1516718DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 167cccgattctc gacacagc 1816817DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
168cgcccacagc aggtcaa 1716917DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 169tgcccacagc aggtcaa
1717015DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 170gccgcctacg ccact 1517115DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
171agcccctacg ccacg 1517215DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 172accccctacg ccacc
1517315DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 173ggctgcctac gccat 1517416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
174gggctgccta cgccag 1617515DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 175ggctgcctac gccac
1517618DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 176ccccgttgga gctggtga 1817719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
177cgggtggata cagctggaa 1917819DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 178gggcgataca gctggacaa
1917917DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 179gccctggttg gagcaga 1718017DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
180cccgttggag caggtga 1718116DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 181gccaggttgt gaggca
1618222DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 182tgccctgaca gaaacacttt tc 2218321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
183cgccctacat gtgtaacagt g 2118415DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 184ccgcctgcat gggca
1518515DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 185cccgcggcat gaacc 1518615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
186ccccggcatg aacca 1518717DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 187cgccagcttt gaggtgc
1718819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 188cccgtggtct agctacagt 1918916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
189ccccaggagc tgtggc 1619020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 190cgccccttta ccactcagaa
2019119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 191ggggagtgca gctcatcat 1919215DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
192ggcgcagttt ggcca 1519318DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 193cccgattctc gacacagg
1819417DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 194tgcccacagc aggtcac 1719517DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
195cgcccacagc aggtcat 1719614DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 196gcccctacgc cacc
1419715DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 197agcccctacg ccacc 1519815DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
198accgcctacg ccaca 1519916DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 199gggctgccta cgccac
1620015DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 200ggctgcctac gccac 1520116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
201gggctgccta cgccaa 1620217DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 202ccccttggag ctggtgg
1720318DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 203cgggggatac agctggac 1820419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
204gggcgataca gctggacat 1920516DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 205gcccggttgg agcagg
1620616DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 206cccttggagc aggtgg 1620716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
207gccaggttgt gaggcg 1620822DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 208tgccctgaca gaaacacttt tt
2220921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 209cgccctacat gtgtaacagt t 2121014DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
210ccgctgcatg ggcg 1421115DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 211cccgcggcat gaact
1521215DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 212ccccggcatg aaccg 1521317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
213cgccagcttt gaggtgt 1721426DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 214agcctcaatt cttaccatcc
acaaaa 2621522DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 215catggaacca gacagaaaag cg
2221622DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 216gtcactggca gcaacagtct ta 2221725DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
217tgggagccaa tattgtcttt gtgtt 2521823DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
218aggaacgtac tggtgaaaac acc 2321920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
219ccctccccag tcctcatgta 2022041DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 220tcttcaaatg atttagtatt
atttatggca aatacacaaa g 4122141DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 221tcttcaaatg atttagtatt
atttatggca aatacacaaa g 4122228DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 222tgtgtgacat gttctaatat
agtcacat 2822328DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 223tgtgtgacat gttctaatat agtcacat
2822428DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 224tgtgtgacat gttctaatat agtcacat
2822528DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 225tgtgtgacat gttctaatat agtcacat
2822628DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 226tgtgtgacat gttctaatat agtcacat
2822728DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 227tgtgtgacat gttctaatat agtcacat
2822822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 228ggtcctgcac cagtaatatg ca 2222929DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
229gcaaatgact tgctattatt gatggcaaa 2923029DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
230gcaaatgact tgctattatt gatggcaaa 2923124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
231agtggttctg gattagctgg attg 2423224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
232gtggttctgg attagctgga ttgt 2423318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
233gcaaccagcc ctgtcgtc 1823420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 234agaccccagt tgcaaaccag
2023524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 235ctggagtctt ccagtgtgat gatg 2423623DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
236ctggagtctt ccagtgtgat gat 2323717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
237tgtgcagggt ggcaagt 1723817DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 238tgtgcagggt ggcaagt
1723924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 239ctttcttgcg gagattctct tcct 2424019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
240cagacaactg ttcaaactg 1924116DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 241ctggcagcaa cagtct
1624217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 242tagtggcacc agaatgg 1724316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
243cccttcggct gcctcc 1624417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 244cagcatgtca agatcac
1724517DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 245ctggtccctc attgcac 1724616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
246ccctccccag tcctca 1624716DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 247ccctccccag tcctca
1624815DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 248cctgctgaaa atgac 1524915DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
249cctgctgaaa atgac 1525015DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 250cctgctgaaa atgac
1525115DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 251cctgctgaaa atgac 1525215DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
252cctgctgaaa atgac 1525315DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 253cctgctgaaa atgac
1525418DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 254tcgtccacaa aatgattc 1825518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
255ccttcgcctg tcctcatg 1825618DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 256ccttcgcctg tcctcatg
1825714DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 257cagtgcgctt ttcc 1425814DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
258cagtgcgctt ttcc 1425918DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 259ctgctcacca tcgctatc
1826017DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 260cctcaggcgg ctcatag 1726117DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
261atgggcctcc ggttcat 1726216DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 262accggaggcc catcct
1626316DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 263cacactggaa gactcc 1626416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
264cacactggaa gactcc 1626515DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 265ctgtgcgccg gtctc
1526616DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 266gctacagaga aatctc 1626717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
267gagctgtggt agtggca 1726816DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 268cactcagaga aggagc
1626916DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 269catcacgcag ctcatg 1627016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
270agtttggccc gcccaa 1627117DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 271ctcgacacag caggtca
1727218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 272gcaggtcaag aggagtac 1827318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
273gcaggtcaag aggagtac 1827414DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 274cgccactagc tcca
1427516DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 275ccacgagctc caacta 1627614DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
276acgccaccag ctcc 1427713DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 277cgccatcagc tcc
1327813DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 278cgccagcagc tcc 1327914DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
279cgccaccagc tcca 1428015DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 280gctggtgacg taggc
1528118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 281gctggaaaag aagagtac 1828217DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
282cagctggaca agaagag 1728317DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 283ttggagcaga tggtgtt
1728416DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 284tggagcaggt gatgtt 1628513DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
285tgaggcactg ccc 1328626DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 286gaaacacttt tcgacatagt gtggtg
2628720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 287tgtgtaacag tgcctgcatg 2028814DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
288gcatgggcag catg 1428915DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 289gcatgaaccg gaggc
1529015DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 290catgaaccag aggcc 1529118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
291ttgaggtgcg tgtttgtg 1829216DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 292gctacagtga aatctc
1629316DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 293agctgtggca gtggca 1629416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
294cactcagaaa aggagc 1629517DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 295tcatcatgca gctcatg
1729616DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 296agtttggcca gcccaa 1629717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
297ctcgacacag gaggtca 1729817DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 298caggtcacga ggagtac
1729918DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 299gcaggtcatg aggagtac 1830013DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
300cgccaccagc tcc 1330116DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 301ccaccagctc caacta
1630216DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 302acgccacaag ctccaa 1630313DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
303cgccaccagc tcc 1330413DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 304cgccaccagc tcc
1330515DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 305cgccaacagc tccaa 1530615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
306gctggtggcg taggc 1530718DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 307gctggacaag aagagtac
1830817DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 308cagctggaca tgaagag 1730916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
309tggagcaggt ggtgtt 1631016DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 310tggagcaggt ggtgtt
1631113DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 311tgaggcgctg ccc 1331227DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
312agaaacactt tttgacatag tgtggtg 2731321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
313atgtgtaaca gttcctgcat g 2131414DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 314gcatgggcgg catg
1431516DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 315gcatgaactg gaggcc 1631615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
316catgaaccgg aggcc 1531720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 317tttgaggtgt gtgtttgtgc
2031819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 318ccctggtagt tggagctgg 1931920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
319cgctgtggta gttggagctg 2032019DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 320gcccggtagt tggagctgg
1932120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 321cgccgtggta gttggagctg 2032219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
322ccccagttgg agctggtgg 1932317DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 323gccttgccta cgccaca
1732418DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 324gcctcttgcc tacgccaa 1832523DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
325aaagaatggt cctgcaccag taa 2332622DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
326ggtcctgcac cagtaatatg ca 2232723DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
327aaagaatggt cctgcaccag taa 2332823DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
328aaagaatggt cctgcaccag taa 2332923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
329aaagaatggt cctgcaccag taa 2333029DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
330gagtttgtat taaaaggtac tggtggagt 2933129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
331gagtttgtat taaaaggtac tggtggagt 2933219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
332ccctggtagt tggagctgc 1933320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 333cgctgtggta gttggagctc
2033419DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 334gcccggtagt tggagctga 1933520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
335cgccgtggta gttggagcta 2033619DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 336ccccagttgg agctggtga
1933717DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 337gccttgccta cgccacc 1733818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
338gcctcttgcc tacgccac 1833923DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 339atgcatatta aaacaagatt tac
2334020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 340aacaatagag gtaaatcttg 2034123DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
341atgcatatta aaacaagatt tac 2334223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
342atgcatatta aaacaagatt tac 2334323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
343atgcatatta aaacaagatt tac 2334420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
344accttatgtg tgacatgttc 2034520DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 345accttatgtg tgacatgttc
2034616DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 346tggagctgct ggcgta 1634716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
347tggagctcgt ggcgta 1634817DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 348ttggagctga tggcgta
1734920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 349tagttggagc tagtggcgta 2035017DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
350ttggagctgg tgacgta 1735114DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 351cgccaccagc tcca
1435215DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 352ctacgccacc agctc 1535316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
353tggagctggt ggcgta 1635416DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 354tggagctggt ggcgta
1635516DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 355tggagctggt ggcgta 1635616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
356tggagctggt ggcgta 1635716DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 357tggagctggt ggcgta
1635814DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 358cgccacaagc tcca 1435914DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
359tacgccaaca gctc 1436021DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 360tgattttggt ctagctacag a
2136116DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 361gagaaggagc tgtggt 1636218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
362tgcctttacc actcagag 1836315DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 363cgtgcagctc atcac
1536414DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 364cagcagtttg gccc 1436518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
365ggatattctc gacacagc 1836615DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 366gacacagcag gtcaa
1536715DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 367cttgcctacg ccact 1536816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
368ctcttgccta cgccat 1636917DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 369gtagttggag ctggtga
1737019DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 370atactggata cagctggaa 1937117DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
371tggatacagc tggacaa 1737220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 372gattttggtc tagctacagt
2037315DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 373agaaggagct gtggc 1537418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
374tgcctttacc actcagaa 1837515DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 375cgtgcagctc atcat
1537614DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 376cagcagtttg gcca 1437719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
377tggatattct cgacacagg 1937815DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 378gacacagcag gtcac
1537914DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 379ttgcctacgc cacc 1438015DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
380tcttgcctac gccac 1538116DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 381tagttggagc tggtgg
1638219DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 382atactggata cagctggac 1938318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
383ctggatacag ctggacat 1838424DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 384gcctgatttt ggtctagcta caga
2438519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 385cgcgagaagg agctgtggt 1938621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
386ccgtgccttt accactcaga g
2138718DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 387gcgcgtgcag ctcatcac 1838817DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
388cggcagcagt ttggccc 1738921DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 389gccggatatt ctcgacacag c
2139018DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 390gcggacacag caggtcaa 1839118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
391cgccttgcct acgccact 1839219DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 392cgcctcttgc ctacgccat
1939320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 393cgcgtagttg gagctggtga 2039422DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
394ccgatactgg atacagctgg aa 2239520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
395ccgtggatac agctggacaa 2039623DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 396ccggattttg gtctagctac
agt 2339718DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 397gccagaagga gctgtggc 1839821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
398ccgtgccttt accactcaga a 2139918DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 399gcgcgtgcag ctcatcat
1840017DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 400cggcagcagt ttggcca 1740122DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
401cgctggatat tctcgacaca gg 2240218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
402gcggacacag caggtcac 1840317DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 403gcgttgccta cgccacc
1740418DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 404gcgtcttgcc tacgccac 1840519DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
405gcctagttgg agctggtgg 1940622DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 406ccgatactgg atacagctgg ac
2240721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 407cgcctggata cagctggaca t 2140826DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
408agcctcaatt cttaccatcc acaaaa 2640922DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
409catggaacca gacagaaaag cg 2241022DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
410gtcactggca gcaacagtct ta 2241125DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
411tgggagccaa tattgtcttt gtgtt 2541223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
412aggaacgtac tggtgaaaac acc 2341320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
413ccctccccag tcctcatgta 2041441DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 414tcttcaaatg atttagtatt
atttatggca aatacacaaa g 4141528DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 415tgtgtgacat gttctaatat
agtcacat 2841628DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 416tgtgtgacat gttctaatat agtcacat
2841722DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 417ggtcctgcac cagtaatatg ca 2241829DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
418gcaaatgact tgctattatt gatggcaaa 2941929DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
419gcaaatgact tgctattatt gatggcaaa 2942019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
420cagacaactg ttcaaactg 1942116DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 421ctggcagcaa cagtct
1642217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 422tagtggcacc agaatgg 1742316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
423cccttcggct gcctcc 1642417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 424cagcatgtca agatcac
1742517DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 425ctggtccctc attgcac 1742616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
426ccctccccag tcctca 1642715DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 427cctgctgaaa atgac
1542815DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 428cctgctgaaa atgac 1542918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
429tcgtccacaa aatgattc 1843018DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 430ccttcgcctg tcctcatg
1843118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 431ccttcgcctg tcctcatg 1843214DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
432ctacagagaa atct 1443314DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 433ctgtggtagt ggca
1443414DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 434ctcagagaag gagc 1443513DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
435atcacgcagc tca 1343611DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 436ggcccgccca a
1143714DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 437gacacagcag gtca 1443816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
438caggtcaaga ggagta 1643913DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 439gccactagct cca
1344012DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 440gccatcagct cc 1244113DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
441tggtgacgta ggc 1344215DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 442gctggaaaag aagag
1544314DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 443ctggacaaga agag 1444414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
444ctacagtgaa atct 1444513DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 445tgtggcagtg gca
1344614DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 446ctcagaaaag gagc 1444714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
447catcatgcag ctca 1444812DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 448tggccagccc aa
1244914DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 449gacacaggag gtca 1445015DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
450aggtcacgag gagta 1545112DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 451ccaccagctc ca
1245211DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 452ccaccagctc c 1145313DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
453tggtggcgta ggc 1345414DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 454ctggacaaga agag
1445514DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 455ctggacatga agag 1445622DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
456gtgattttgg tctagctaca ga 2245719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
457tcagagaagg agctgtggt 1945821DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 458gattgccttt accactcaga g
2145918DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 459caccgtgcag ctcatcac 1846018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
460cacccagcag tttggccc 1846120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 461ttggatattc tcgacacagc
2046218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 462ctcgacacag caggtcaa 1846318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
463actcttgcct acgccact 1846418DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 464cactcttgcc tacgccat
1846519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 465tggtagttgg agctggtga 1946620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
466catactggat acagctggaa 2046721DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 467atactggata cagctggaca a
2146822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 468gtgattttgg tctagctaca gt 2246919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
469tcagagaagg agctgtggc 1947021DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 470gattgccttt accactcaga a
2147118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 471caccgtgcag ctcatcat 1847218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
472cacccagcag tttggcca 1847321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 473cttggatatt ctcgacacag g
2147419DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 474tctcgacaca gcaggtcac 1947518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
475actcttgcct acgccacc 1847618DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 476cactcttgcc tacgccac
1847719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 477tggtagttgg agctggtgg 1947820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
478catactggat acagctggac 2047920DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 479tactggatac agctggacat
2048026DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 480agcctcaatt cttaccatcc acaaaa
2648122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 481catggaacca gacagaaaag cg 2248222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
482gtcactggca gcaacagtct ta 2248325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
483tgggagccaa tattgtcttt gtgtt 2548423DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
484aggaacgtac tggtgaaaac acc 2348520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
485ccctccccag tcctcatgta 2048641DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 486tcttcaaatg atttagtatt
atttatggca aatacacaaa g 4148728DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 487tgtgtgacat gttctaatat
agtcacat 2848828DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 488tgtgtgacat gttctaatat agtcacat
2848922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 489ggtcctgcac cagtaatatg ca 2249029DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
490gcaaatgact tgctattatt gatggcaaa 2949129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
491gcaaatgact tgctattatt gatggcaaa 2949219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
492cagacaactg ttcaaactg 1949316DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 493ctggcagcaa cagtct
1649417DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 494tagtggcacc agaatgg 1749516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
495cccttcggct gcctcc 1649617DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 496cagcatgtca agatcac
1749717DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 497ctggtccctc attgcac 1749816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
probe 498ccctccccag tcctca 1649915DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 499cctgctgaaa atgac
1550015DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 500cctgctgaaa atgac 1550118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
501tcgtccacaa aatgattc 1850218DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 502ccttcgcctg tcctcatg
1850318DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 503ccttcgcctg tcctcatg 1850421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
504gctacagaga aatctcgatg g 2150517DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 505gagctgtggt agtggca
1750620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 506ttaccactca gagaaggagc 2050717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
507gctcatcacg cagctca 1750816DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 508ttggcccgcc caaaat
1650918DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 509gacacagcag gtcacgag 1851020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
510cagcaggtca agaggagtac 2051119DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 511ctacgccact agctccaac
1951216DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 512ctacgccatc agctcc 1651317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
513ctggtgacgt aggcaag 1751422DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 514ggatacagct ggaaaagaag ag
2251520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 515agctggacaa gaagagtaca 2051622DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
516tagctacagt gaaatctcga tg 2251715DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
517gagctgtggc agtgg 1551821DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 518ttaccactca gaaaaggagc t
2151920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 519cagctcatca tgcagctcat 2052019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
520gtttggccag cccaaaatc 1952118DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 521gacacaggag gtcaggag
1852219DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 522cagcaggtca cgaggagta 1952317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
523tacgccacca gctccaa 1752416DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 524tacgccacca gctcca
1652517DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 525ctggtggcgt aggcaag 1752621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
526atacagctgg acaagaagag t 2152720DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 527agctggacat gaagagtaca
2052822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 528ccccttttgg tctagctaca ga 2252918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
529gcccgaagga gctgtggt 1853020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 530cgccccttta ccactcagag
2053118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 531gcgggtgcag ctcatcac 1853217DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
532cgggagcagt ttggccc 1753321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 533gcccgatatt ctcgacacag c
2153418DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 534gcggacacag caggtcaa 1853516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
535cggtgcctac gccact 1653617DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 536cgccttgcct acgccat
1753719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 537gcccagttgg agctggtga 1953821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
538cgccactgga tacagctgga a 2153920DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 539ccgcggatac agctggacaa
2054022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 540ccccttttgg tctagctaca gt 2254118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
541gcccgaagga gctgtggc 1854220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 542cgccccttta ccactcagaa
2054318DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 543gcgggtgcag ctcatcat 1854417DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
544cgggagcagt ttggcca 1754521DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 545gcccgatatt ctcgacacag g
2154618DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 546gcggacacag caggtcac 1854716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
547cggtgcctac gccacc 1654817DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 548cgccttgcct acgccac
1754919DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 549gcccagttgg agctggtgg 1955021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
550cgccactgga tacagctgga c 2155120DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 551ccgcggatac agctggacat
2055226DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 552agcctcaatt cttaccatcc acaaaa
2655322DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 553catggaacca gacagaaaag cg 2255422DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
554gtcactggca gcaacagtct ta 2255525DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
555tgggagccaa tattgtcttt gtgtt 2555623DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
556aggaacgtac tggtgaaaac acc 2355720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
557ccctccccag tcctcatgta 2055841DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 558tcttcaaatg atttagtatt
atttatggca aatacacaaa g 4155928DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 559tgtgtgacat gttctaatat
agtcacat 2856028DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 560tgtgtgacat gttctaatat agtcacat
2856122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 561ggtcctgcac cagtaatatg ca 2256229DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
562gcaaatgact tgctattatt gatggcaaa 2956329DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
563gcaaatgact tgctattatt gatggcaaa 2956419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
564cagacaactg ttcaaactg 1956516DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 565ctggcagcaa cagtct
1656617DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 566tagtggcacc agaatgg 1756716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
567cccttcggct gcctcc 1656817DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 568cagcatgtca agatcac
1756917DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 569ctggtccctc attgcac 1757016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
570ccctccccag tcctca 1657115DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 571cctgctgaaa atgac
1557215DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 572cctgctgaaa atgac 1557318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
573tcgtccacaa aatgattc 1857418DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 574ccttcgcctg tcctcatg
1857518DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 575ccttcgcctg tcctcatg 1857614DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
576ctacagagaa atct 1457714DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 577ctgtggtagt ggca
1457814DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 578ctcagagaag gagc 1457913DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
579atcacgcagc tca 1358011DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 580ggcccgccca a
1158114DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 581gacacagcag gtca 1458216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
582caggtcaaga ggagta 1658313DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 583gccactagct cca
1358412DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 584gccatcagct cc 1258513DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
585tggtgacgta ggc 1358615DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 586gctggaaaag aagag
1558714DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 587ctggacaaga agag 1458814DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
588ctacagtgaa atct 1458913DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 589tgtggcagtg gca
1359014DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 590ctcagaaaag gagc 1459114DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
591catcatgcag ctca 1459212DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 592tggccagccc aa
1259314DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 593gacacaggag gtca 1459415DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
594aggtcacgag gagta 1559512DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 595ccaccagctc ca
1259611DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 596ccaccagctc c 1159713DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
597tggtggcgta ggc 1359814DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 598ctggacaaga agag
1459914DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 599ctggacatga agag 1460022DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
600ccccttttgg tctagctaca ga 2260118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
601gcccgaagga gctgtggu 1860220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 602cgccccttta ccactcagag
2060318DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 603gcgggtgcag ctcatcac 1860417DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
604cgggagcagt ttggccc 1760521DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 605gcccgatatt ctcgacacag c
2160626DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 606agcctcaatt cttaccatcc acaaaa
2660722DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 607catggaacca gacagaaaag cg 2260822DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
608gtcactggca gcaacagtct ta 2260925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
609tgggagccaa tattgtcttt gtgtt
2561023DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 610aggaacgtac tggtgaaaac acc 2361120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
611ccctccccag tcctcatgta 2061214DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 612ctacagagaa atct
1461314DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 613ctgtggtagt ggca 1461414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
614ctcagagaag gagc 1461513DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 615atcacgcagc tca
1361611DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 616ggcccgccca a 1161714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
617gacacagcag gtca 1461822DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 618ccccttttgg tctagctaca gu
2261918DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 619gcccgaagga gctgtggc 1862020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
620cgccccttta ccactcagaa 2062118DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 621gcgggtgcag ctcatcau
1862217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 622cgggagcagt ttggcca 1762321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
623gcccgatatt ctcgacacag g 2162419DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 624cagacaactg ttcaaactg
1962516DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 625ctggcagcaa cagtct 1662617DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
626tagtggcacc agaatgg 1762716DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 627cccttcggct gcctcc
1662817DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 628cagcatgtca agatcac 1762917DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
629ctggtccctc attgcac 1763014DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 630ctacagtgaa atct
1463113DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 631tgtggcagtg gca 1363214DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
632ctcagaaaag gagc 1463314DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 633catcatgcag ctca
1463412DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 634tggccagccc aa 1263514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
635gacacaggag gtca 14
* * * * *