U.S. patent application number 14/879156 was filed with the patent office on 2016-02-11 for methods, compositions, and kits for detecting allelic variants.
The applicant listed for this patent is Life Technologies Corporation. Invention is credited to Caifu CHEN, Ruoying Tan.
Application Number | 20160040256 14/879156 |
Document ID | / |
Family ID | 42310453 |
Filed Date | 2016-02-11 |
United States Patent
Application |
20160040256 |
Kind Code |
A1 |
CHEN; Caifu ; et
al. |
February 11, 2016 |
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) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Life Technologies Corporation |
Carlsbad |
CA |
US |
|
|
Family ID: |
42310453 |
Appl. No.: |
14/879156 |
Filed: |
October 9, 2015 |
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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14879156 |
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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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61138521 |
Dec 17, 2008 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 1/6858 20130101; C12Q 2600/156 20130101; C12Q 1/6858 20130101;
C12Q 2600/172 20130101; C12Q 2561/101 20130101; C12Q 2561/113
20130101; C07H 21/04 20130101; C12Q 2537/161 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for detecting a first allelic variant of a target
sequence in a nucleic acid sample, 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 that: a. is non-extendable at the 3' end;
b. is complementary to a region of the target sequence comprising a
second allelic variant of the target sequence, wherein said region
encompasses a position corresponding to the binding position of the
allele-specific nucleotide portion of the first allele-specific
primer; c. does not comprise a label; and d. comprises a minor
groove binder located at the 3'-end, the 5'-end and/or at an
internal position within said first allele-specific blocker and;
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 locus-specific detector
probe having at least one label; 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 locus-specific
detector probe, thereby detecting the first allelic variant of the
target gene in the nucleic acid sample at a sensitivity level of a
single copy of the first allelic variant in a background of about
1.times.10.sup.5 to about 1.times.10.sup.6 copies of the second
allelic variant.
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: vi. the nucleic acid sample; vii. 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;
viii. a second allele-specific blocker that: a. is non-extendable
at the 3' end; b. 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; c. does not comprise a label; and d. comprises a minor
groove binder located at the 3'-end, the 5'-end and/or at an
internal position within said second allele-specific blocker and;
ix. 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 x. a second locus-specific
detector probe having at least one label; 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 second
locus-specific 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 and/or second
allele-specific primer comprises a tail.
6. The method of claim 5, wherein said tail is GC-rich.
7. The method of claim 5, wherein said tail is between 2-30
nucleotides long.
8. The method of claim 5, wherein said tail is at the 5' end of
said first and/or second allele-specific primer.
9. The method of claim 1 or 3, wherein the Tm of said first and/or
second allele-specific primer is between 50.degree. C. to
55.degree. C.
10. The method of claim 1 or 3, wherein said concentration of said
first and/or second allele-specific primer is between 20-900
nM.
11. The method of claim 1 or 3, wherein said first and/or second
allele-specific primer is designed to comprise a highly
discriminating base at the 3' terminus.
12. The method of claim 1 or 3, wherein said allele-specific
nucleotide portion of said first and/or second allele-specific
primer is located at the 3' terminus of said first and/or second
allele-specific primer.
13. The method of claim 12, wherein A or G is used as the 3'
allele-specific nucleotide portion of said first and/or second
allele-specific primer if A/T is the allelic variant; or C or T is
used as the 3' allele-specific nucleotide portion of said first
and/or second allele-specific primer if C/G is the allelic
variant.
14. The method of claim 1 or 3, wherein said first and/or second
allele-specific primer and/or first and/or second allele-specific
blocker comprises at least one modified base.
15. The method of claim 14, 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).
16. The method of claim 14, wherein said modified base is a locked
nucleic acid (LNA) base.
17. The method of claim 14, wherein said modified base is any
modified base that increases the Tm between matched and mismatched
target sequences and/or nucleotides.
18. The method of claim 14, wherein the allele specific blocker
comprises a non-extendable blocker moiety at the 3' terminus.
19. The method of claim 18, wherein the non-extendable blocker
moiety is selected from an MGB, a modification of a ribose ring
3'-OH moiety of the allele specific blocker oligonucleotide, an
amine (NH.sub.2), a biotin, a PEG, a DPI.sub.3, and a PO.sub.4.
20. The method of claim 1, wherein said MGB moiety or moieties
is/are not cleaved from said first and/or second allele-specific
blocker during said amplification reaction.
21. The method of claim 1 or 3, wherein said first and/or second
locus-specific detector probe has two labels.
22. The method of claim 21, wherein said locus-specific detector
probe is a 5' nuclease probe.
23. The method of claim 1, wherein said nucleic acid sample is
genomic DNA (gDNA).
24. The method of claim 1 or 3, wherein said first and/or second
reaction mixture further comprises a polymerase, dNTPs, and/or
other reagents or buffers suitable for PCR amplification.
25. (canceled)
26. The method of claim 1 or 3, wherein said amplification reaction
is a real time polymerase chain reaction (PCR).
27. The method of claim 1, wherein said nucleic acid sample is
derived from a tumor sample, a blood sample comprising circulating
tumor cells, a breast or a lung cancer tumor sample, a tumor
comprises mutations in Ras, EGFR, Kit, pTEN, and/or p53, a tumor
wherein a Ras mutation is a KRAS or and NRAS mutation, a tumor
wherein a KRAS mutation is in codon 12 and/or codon 13 as depicted
in FIG. 6.
28-91. (canceled)
92. The method of claim 1 or 3 performed using a kit comprising:
two or more containers comprising the following components
independently distributed in one of the two or more containers: a)
the first allele-specific primer; and b) the first allele-specific
blocker; c) the locus-specific primer that is complementary to a
region of said target sequence that is 3' from said first allelic
variant and on the opposite strand; and d) a locus-specific
detector probe wherein the detector probe is a 5' nuclease
probe.
93.-118. (canceled)
119. The method of claim 1 or 3, wherein said method has a
selectivity of detection of at least 1:1000, 1:10,000, 1:100,000 or
1:1,000,000.
120. The method of claim 21, wherein the two labels of said
locus-specific detector probe comprise a fluorophore and a
quencher.
121. The method of claim 120, wherein said locus-specific detector
probe further comprises a minor groove binder.
122. The method of claim 1 or 3, wherein said first or second
allele-specific blocker is at a concentration that is less than the
concentration of said first or second allele-specific primer.
123. The method of claim 1 or 3, wherein said second allelic
variant is located 7-15 nucleotides away from said minor groove
binder when said allele-specific blocker is hybridized to said
target sequence.
124. The method of claim 1 or 3, wherein said allele-specific
blocker does not comprise a base analog.
125. The method of claim 1 or 3, wherein said allele-specific
primer and said allele-specific blocker both comprise one or more
base analogs.
126. The method of claim 1 or 3, wherein said allele-specific
blocker has a Tm that is between 60.degree. C. to 66.degree. C. and
said allele-specific primer has a Tm that is between 50.degree. C.
to 70.degree. C.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 12/641,321 filed Dec. 17, 2009, which claims
the benefit of priority under 35 U.S.C. 119 to U.S. Provisional
Application Ser. No. 61/258,582 filed Nov. 5, 2009; U.S.
Provisional Application Ser. No. 61/253,501 filed Oct. 20, 2009;
U.S. Provisional Application Ser. No. 61/251,623 filed Oct. 14,
2009; U.S. Provisional Application Ser. No. 61/186,775 filed Jun.
12, 2009; U.S. Provisional Application Ser. No. 61/164,230 filed
Mar. 27, 2009; and U.S. Provisional Application Ser. No. 61/138,521
filed Dec. 17, 2008, 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,
Buslov 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 A S-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
66.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 blocker 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 60.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), locked nucleic acid (LNA) or
2'-O,4'-C-ethylene nucleic acid (ENA) bases (FIG. 4b).
[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.,
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).
[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 Taq 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 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 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
66.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/T is the target 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/G is the target 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 NT 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 60.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 bust also selectivity. Such modified base(s) may
include, for example, 8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG
(ppG), locked nucleic acid (LNA) or 2'-O,4'-C-ethylene nucleic acid
(ENA) bases (FIG. 4b).
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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; (c), 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.
[0029] 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
66.degree. C. In some embodiments the first and/or second
allele-specific primer concentrations are between about 20-900
nM.
[0030] 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.
[0031] 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.
[0032] 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 NT is the target 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/G is the target 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.
[0033] 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 60.degree.
C. to 66.degree. C.
[0034] 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), locked
nucleic acid (LNA) or 2'-O,4'-C-ethylene nucleic acid (ENA) bases
(FIG. 4b).
[0035] 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 TaqMan.RTM. probe.
[0036] 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.
[0037] 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 Taq DNA polymerase.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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
[0042] 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.
[0043] 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
[0044] 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.
[0045] 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).
[0046] 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).
[0047] 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.
[0048] 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.)
[0049] FIG. 4B depicts some examples of modified bases of an MGB
blocker probe or allele-specific primer.
[0050] FIG. 5 depicts the TaqMan-like sensitivity and dynamic range
of one exemplary embodiment of cast-PCR.
[0051] 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.)
[0052] FIG. 7 depicts the specificity of KRAS mutation detection
using cast-PCR assays in one exemplary embodiment.
[0053] 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.
[0054] FIG. 9 depicts detection of the relative copy number of
mutant samples (KRAS-G12A) spiked in wild type samples using
cast-PCR methods.
[0055] FIG. 10 depicts a number of different tumor markers (SNPs)
detected in tumor samples using one exemplary embodiment of
cast-PCR.
[0056] FIG. 11A-11D shows a list of exemplary allele-specific
primers and probes used in cast-PCR assays.
DETAILED DESCRIPTION
I. Introduction
[0057] 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.
[0058] 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.
[0059] 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, 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 1,000,000.times.
fold difference, such as about 1000-10,000.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.
II. Definitions
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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
[0067] 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.)
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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) (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.)
[0072] 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 (Tm) 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).
[0073] 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.)
[0074] 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, 1996,
Nature Biotechnology 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
(Solinas 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; lsacsson 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; Broude 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).
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.).
[0082] 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).
[0083] 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.(Ct.sup.--.sup.condition1-(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.Ct or as a percentage using (1/2.sup.Ct 100).
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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
[0088] 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/or (d) a locus-specific primer. 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.
[0089] 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.
[0090] 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.
[0091] In further embodiments, the compositions can further include
a detector probe.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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 blocker 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.
[0096] 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.
[0097] 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 blocker 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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 blocker 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.
[0106] 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.
[0107] 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.
[0108] In other embodiments, the kits can further include a
detector probe.
[0109] 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 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.
[0110] 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.).
Allele-Specific Primers
[0111] 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. (e.g., 53.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 range in between. 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.
[0112] 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.
[0113] 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.
[0114] 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/T is the target allele), or C or T may be used as the
3' allele-specific nucleotide portion of the allele-specific primer
(e.g., if C/G is the target 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
Allele-Specific Blocker Probes
[0121] 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.
[0122] In some embodiments, the Tm of the blocker probes range from
60.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.
[0123] 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.")
[0124] 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.
[0125] In general, MGB moieties are molecules that binds 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.
[0126] 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.
[0127] 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).
[0128] 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.
[0129] 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.
[0130] 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).
[0131] In some embodiments, the allele-specific blocker probe can
comprise one or more modified bases in addition to the naturally
occurring bases adenine, cytosine, guanine, thymine and uracil. 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 (FIG. 4A).
[0132] 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. Such modified base(s) may include, for example,
8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG (ppG), locked nucleic acid
(LNA) or 2'-O,4'-C-ethylene nucleic acid (ENA) bases (FIG. 4B).
Other examples of modified bases include, but are not limited to,
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). These base analogues,
when present in an oligonucleotide, strengthen hybridization and
improve mismatch discrimination. All tautomeric forms of naturally
occurring bases, modified bases and base analogues may be included
in the oligonucleotide primer and probes of the invention.
[0133] Similarly, 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.
[0134] 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, phosphotriester, 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.
[0135] In addition, in some embodiments, the nucleotide units which
are incorporated into the oligonucleotides of the MGB 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.
[0136] The "sugar" or glycoside portion of some embodiments of the
MGB blocker probes of the present invention may comprise
deoxyribose, ribose, 2-fiuororibose, 2-0 alkyl or alkenylribose
where the alkyl group may have 1 to 6 carbons and the alkenyl group
2 to 6 carbons. In the naturally occurring nucleotides and in the
herein described modifications and analogs the deoxyribose or
ribose moiety forms a furanose ring. the glycosydic linkage is of
the configuration and the purine bases are attached to the sugar
moiety via the 9-position. the pyrimidines via the I-position and
the pyrazolopyrimidines via the I-position. The nucleotide units of
the oligonucleotide are interconnected by a "phosphate" backbone,
as is well known in the art. The oligonucleotide of the
oligonucleotide-MGB conjugates (MGB blocker probes) of the present
invention may include, in addition to the "natural" phosphodiester
linkages, phosphorothiotes and methylphosphonates.
[0137] 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.
Detector Probes
[0138] 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
range in between.
[0139] 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 Taq 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).
[0140] 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.
Locus-Specific Primers
[0141] 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.
[0142] 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.
Additional Components
[0143] 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. Bacteriol.
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, Taq DNA polymerase, Tne DNA polymerase, Tma DNA
polymerase, or mutants, derivatives or fragments thereof.
Various Sources and/or Preparation Methods of Nucleic Acids
[0144] 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.
[0145] 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.
[0146] The following examples are intended to illustrate but not
limit the invention.
EXAMPLES
I. General Assay Design
[0147] 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 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
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 tailed
allele-2-specific primer (ASP2), one MGB allele-1 blocker probe
(MGB1), one common locus-specific TaqMan probe (LST) and one common
locus-specific primer (LSP).
II. Reaction Conditions
[0148] 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 (as indicated), 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 each, then 58.degree. C. for 1 minute, then by 45
cycles at 95.degree. C. for 15 seconds each and then 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.
III. Nucleic Acid Samples
[0149] 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 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.
[0150] 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.
TABLE-US-00001 TABLE 1 SNP ID SEQUENCE CV11201742
GCTCTGCTTCATTCCTGTCTGAAGAAGGGCAGATAG
TTTGGCTGCTCCTGTG[C/T]TGTCACCTGCAATTC
TCCCTTATCAGGGCCATTGGCCTCTCCCTTCTCTCT
GTGAGGGATATTTTCTCTGACTTGTCAATCCACATC TTCC CV11349123
GGCTTGCAATGGCTCCAACCGGAAGGGCGGTGCTCG
AGCTGTGGTGCGTGC[C/T]GCTAAGTTGTGCGTTC CAGGGTGCACTCGC CV1207700
GCAACTATACCCTTGATGGATGGAGATTTA[C/T]G
CAATGTGTTTTACTGGGTAGAGTGACAGACCTT CV25594064
CCTGAACTTATTTGGCAAGAGCGATGAGTACTCTTA
AAATTACTATCTGGAAATTATATTATTTAGAATCTG
CCAATTACCTAGATCCCCCCT[C/G]AACAATTGTT TCACCAAGGAACTTCCTGAA
CV25639181 GAATTGGTTGTCTCCTTATGGGAACTGGAAGTATTT
TGACA[G/T]CTTTACCACATTTCTTCATGGGATAG
TAAGTGTTAAACAGCTCTGAGCCATTTATTATCAGC
TACTTGTAAATTAGCAGTAGAATTTTATTTTTATAC
TTGTAAGTGGGCAGTTACCTTTTGAGAGGAATACCT ATAG RNaseP
GCGGAGGGAAGCTCATCAGTGGGGCCACGAGCTGAG Control
TGCGTCCTGTCACTCCACTCCCATGTCCCTTGGGAA GGTCTGAGACTAGGG BRAF-1799TA
TACTACACCTCAGATATATTTCTTCATGAAGACCTC
ACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG
[T/A]GAAATCTCGATGGAGTGGGTCCCATCAGTTT
GAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGA
ATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCC CTGAGATGCTGCTGAGTT
CTNNB1-121AG TGCTAATACTGTTTCGTATTTATAGCTGATTTGATG
GAGTTGGACATGGCCATGGAACCAGACAGAAAAGCG
GCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGAC
CTCTGGAATCCATTCTGGTGCCACT[A/G]CACAGC
TCCTTCTCTGAGTGGTAAAGGCAATCCTGAGGAAGA GGATGTGGATACCTCCCAAGTC
CTNNB1-134CT TTTGATGGAGTTGGACATGGCCATGGAACCAGACAG
AAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTA
CCTGGACTCTGGAATCCATTCTGGTGCCACTACCAC
AGCTCCTT[C/T]TCTGAGTGGTAAAGGCAATCCTG
AGGAAGAGGATGTGGATACCTCCCAAGTCCTGTATG AGTGGGAA EGFR-2369CT
GTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATC
TGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCA
GCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCG
GGAACACAAAGACAATATTGGCTCCCAGTACCTGCT
CAACTGGTGTGTGCAGATCGCAAAGGTAATCAGGGA AGGGA EGFR-2573TG
GCATGAACTACTTGGAGGACCGTCGCTTGGTGCACC
GCGACCTGGCAGCCAGGAACGTACTGGTGAAAACAC
CGCAGCATGTCAAGATCACAGATTTTGGGC[T/G]G
GCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCAT GCAGAAGGAGGCAAAGTAAGGAGGTG
KRAS-176CG CAGGATTCCTACAGGAAGCAAGTAGTAATTGATGGA
GAAACCTGTCTCTTGGATATTCTCGACACAG[C/G]
AGGTCAAGAGGAGTACAGTGCAATGAGGGACCAGTA
CATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGC
CATAAATAATACTAAATCATTTGAAGATATTC KRAS-183AC
ACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTC
TCTTGGATATTCTCGACACAGCAGGTCA[A/C]GAG
GAGTACAGTGCAATGAGGGACCAGTACATGAGGACT
GGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAAT
ACTAAATCATTTGAAGATATTCACCATTATAGGTGG
GTTTAAATTGAATATAATAAGCTGACATTAA KRAS-34GA
TATTAACCTTATGTGTGACATGTTCTAATATAGTCA
CATTTTCATTATTTTTATTATAAGGCCTGCTGAAAA
TGACTGAATATAAACTTGTGGTAGTTGGAGCT
[G/A]GTGGCGTAGGCAAGAGTGCCTTGACGATACA
GCTAATTCAGAATCATTTTGTGGACGAATATGA KRAS-35GA
TATTAACCTTATGTGTGACATGTTCTAATATAGTCA
CATTTTCATTATTTTTATTATAAGGCCTGCTGAAAA
TGACTGAATATAAACTTGTGGTAGTTGGAGCTG
[G/A]TGGCGTAGGCAAGAGTGCCTTGACGATACAG
CTAATTCAGAATCATTTTGTGGACGAATATGATC KRAS-38GA
CATTATTTTTATTATAAGGCCTGCTGAAAATGACTG
AATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]C
GTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAG
AATCATTTTGTGGACGAATATGATCCAACAATAGAG
GTAAATCTTGTTTTAATATGCATATTACTGGTGCAG
GACCATTCTTTGATACAGATAAAGGTTTCTCTGACC ATTTTCATGAGTACTTAT NRAS-181CA
ATTCTTACAGAAAACAAGTGGTTATAGATGGTGAAA
CCTGTTTGTTGGACATACTGGATACAGCTGGA
[C/A]AAGAAGAGTACAGTGCCATGAGAGACCAATA
CATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGC
CATCAATAATAGCAAGTCATTTGCGGATATTAACCT
CTACAGGTACTAGGAGCATTATTTTCTCTGAAAGGA TG NRAS-183AT
TTACAGAAAACAAGTGGTTATAGATGGTGAAACCTG
TTTGTTGGACATACTGGATACAGCTGGACA[A/T]G
AAGAGTACAGTGCCATGAGAGACCAATACATGAGGA
CAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAATA
ATAGCAAGTCATTTGCGGATATTAACCTCTACAGGT ACTAGGAGCATTATTTTCTCTGAAAGGATG
NRAS-35GA TGGTTTCCAACAGGTTCTTGCTGGTGTGAAATGACT
GAGTACAAACTGGTGGTGGTTGGAGCAG[G/A]TGG
TGTTGGGAAAAGCGCACTGACAATCCAGCTAATCCA
GAACCACTTTGTAGATGAATATGATCCCACCATAGA GGTGAGGCCCAGTGGTAGCCCG
NRAS-38GA TTTCCAACAGGTTCTTGCTGGTGTGAAATGACTGAG
TACAAACTGGTGGTGGTTGGAGCAGGTG[G/A]TGT
TGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAA
CCACTTTGTAGATGAATATGATCCCACCATAGAGGT GAGGCCCAGTGGTAGCCC TP53-524GA
GGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAG
TCACAGCACATGACGGAGGTTGTGAGGC[G/A]CTG
CCCCCACCATGAGCGCTGCTCAGATAGCGATGGTGA
GCAGCTGGGGCTGGAGAGACGACAGGGCTGGTTGCC
CAGGGTCCCCAGGCCTCTGATTCCTCACTGATTGCT CTTAGGTCTGGCC TP53-637CT
CCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTG
CGTGTGGAGTATTTGGATGACAGAAACACTTTT
[C/T]GACATAGTGTGGTGGTGCCCTATGAGCCGCC
TGAGGTCTGGTTTGCAACTGGGGTCTCTGGGAGGAG GGGTTAAGGGTGGTTGTCAGTGGCCCTC
TP53-721TG CTTGGGCCTGTGTTATCTCCTAGGTTGGCTCTGACT
GTACCACCATCCACTACAACTACATGTGTAACAGT
[T/G]CCTGCATGGGCGGCATGAACCGGAGGCCCAT
CCTCACCATCATCACACTGGAAGACTCCAGGTCAGG
AGCCACTTGCCACCCTGCACACTGGCCTGCTGTGCC CCAGCCTC TP53-733GA
TAGGTTGGCTCTGACTGTACCACCATCCACTACAAC
TACATGTGTAACAGTTCCTGCATGGGC[G/A]GCAT
GAACCGGAGGCCCATCCTCACCATCATCACACTGGA
AGACTCCAGGTCAGGAGCCACTTGCCACCCTGCACA CTGGCCTGCTGTGCCCCAGCCTC
TP53-742CT CTGACTGTACCACCATCCACTACAACTACATGTGTA
ACAGTTCCTGCATGGGCGGCATGAAC[C/T]GGAGG
CCCATCCTCACCATCATCACACTGGAAGACTCCAGG
TCAGGAGCCACTTGCCACCCTGCACACTGGCCTGCT GTGCCCCAGCCTCTGCTTGCCTC
TP53-743GA TGACTGTACCACCATCCACTACAACTACATGTGTAA
CAGTTCCTGCATGGGCGGCATGAACC[G/A]GAGGC
CCATCCTCACCATCATCACACTGGAAGACTCCAGGT
CAGGAGCCACTTGCCACCCTGCACACTGGCCTGCTG TGCCCCAGCCTCTGCTTGCCTC
TP53-817CT CCTCTTGCTTCTCTTTTCCTATCCTGAGTAGTGGTA
ATCTACTGGGACGGAACAGCTTTGAGGTG[C/T]GT
GTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAG
GAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCAC
GAGCTGCCCCCAGGGAGCACTAAGCGAGGTAAGCAA
Data Analysis
[0151] An automatic baseline and manual threshold of 0.2 were used
to calculate the threshold cycle (CO which is defined as the
fractional cycle number at which the fluorescence passes the fixed
threshold. 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
[0152] The following example demonstrates that the application of
allele-specific primers comprising tails significantly improves the
discrimination of allelic variants.
[0153] 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 ACT 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.
[0154] Assays were performed using the general experimental design
and reaction conditions indicated above (with the exception that no
blocker probes were included), using 0.5 ng/uL genomic DNA
containing the hsv11711720 SNP comprising one of three alleles (A,
C, or T) as the nucleic acid template (see Table 2). The three
genotypes are indicated in Table 2a. Primers and probes were
designed according to the sequences shown in Table 3.
TABLE-US-00002 TABLE 2 Genomic DNA Sequence for hsv11711720 SNP
(target alleles are indicated in brackets).
AGAAAATAACTAAGGGAAGGAGGAAAGTGGGGAGGAAGGAAGAACAGTG
TGAAGACAATGGCCTGAAAACTGAAAAAGTCTGTTAAAGTTAATTATCA
GTTTTTGAGTCCAAGAACTGGCTTTGCTACTTTCTGTAAGTTTCTAATT
TACTGAATAAGCATGAAAAAGATTGCTTTGAGGAATGGTTATAAACACA
TTCTTAGAGCATAGTAAGCAGTAGGGAGTAACAAAATAACACTGATTAG
AATACTTTACTCTACTTAATTAATCAATCATATTTAGTTTGACTCACCT
TCCCAG[A/C/T]ACCTTCTAGTTCTTTCTTATCTTTCAGTGCTTGTCC
AGACAACATTTTCATTTCAACAACTCCTGCTATTGCAATGATGGGTACA
ATTGCTAAGAGTAACAGTGTTAGTTGCCAACCATAGATGAAGGATATAA
TTATTCCTGTCCCAAGATTTGCTATATTCTGGGTAATTACAGCAAGCCT
GGAACCTATAGCCTGCAAAACAAAACAAATTAGAGAAATTTTAAAAATA
TTATCTTCACAACTCATGCTTCTATTTTCTGAAAACTCACCTTCATGAG
ACTATATTCATTATTTTAT
TABLE-US-00003 TABLE 2a Genotypes of Genomic DNA Sequence for
hsv11711720 SNP Genomic DNA ID Genotype NA17203 AA NA17129 CC
NA17201 TT
[0155] Table 3: List and Sequences of Primers and Probes:
conventional allele-specific primers ("ASP-tail"); tailed
allele-specific primers ("ASP"); 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).
TABLE-US-00004 TABLE 3 Primer/ Tm Probe ID Sequence (5' to 3')
(.degree. C.) 17129-ASP ATATTTAGTTTGACTCACCTTCCCAGC 63.2
17129-tailASP accACTCACCTTTCCCAGC 63.0 17203-ASP
ATATTTAGTTTGACTCACCTTCCCAGA 62.0 17203-tailASP accACTCACCTTTCCCAGA
63.7 17201-ASP ATATTTAGTTTGACTCACCTTCCCAGT 62.2 17201-tailASP
accACTCACCTTTCCCAGT 64.0 LST (6-FAM)-TGGACAAGCACTGAAAGA- 67.4 (MGB)
LSP GCAGGAGTTGTTGAAATGAAAATGTTG 62.5
[0156] 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, 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.
[0157] 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)-(.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").
TABLE-US-00005 TABLE 4 Specificity .DELTA.Ct .DELTA.Ct Improvement
NT mismatch (ASP - tail) (ASP) (fold difference) CA 0.9 11.5 1552.1
CT 1.2 11.5 1278.3 AC 10.0 11.9 3.7 AG 9.8 11.9 4.3 TG 2.3 11.5
588.1 TC -0.1 11.5 3104.2 Average 4.0 11.6 1088.5
Example 2
Low Primer Concentrations Improve Discrimination of Allelic
Variants
[0158] 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-D.
[0159] 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
[0160] 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. 11A-D.
[0161] 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
[0162] 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 PCR
reactions.
[0163] 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
blocker probes. Assay primers and probes were designed according to
the sequences shown in FIG. 11A-D.
[0164] 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.)
[0165] 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 .DELTA.Ct .DELTA.Ct Specificity (no MGB (+MGB
Improvement SNP ID 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
[0166] 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. 11A-D.
[0167] 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 SNP allele-1 SNP
allele-2 ASP design .DELTA.Ct Specificity .DELTA.Ct Specificity 3'
NT of 3' NT of Allele (Ct_mismatch - (fold Allele (Ct_mismatch -
(fold SNP ID ASP1 ASP2 NT Ct_match) difference) NT Ct_match)
difference) KRAS- G A C 13.4 10809 T 8.2 294 38GA NRAS- C A G 27.5
189812531 T 9.8 891 181CA NRAS- A T T 17.9 244589 A 23.4 11068835
183AT TP53- C T G 12.3 5043 A 8.3 315 742CT
Example 6
Determination of the Sensitivity and Dynamic Range for Cast-PCR
[0168] 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.
[0169] 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.
11A-D.
[0170] 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
[0171] 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.
[0172] 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
NRAS-181CA SNP target sequence (see Table 1). Assay primers and
probes were designed according to the sequences shown in FIG.
11A-D.
[0173] 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 alleles A1 and A2. 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
alleles A1 and A2, respectively.
Example 8
Cast-PCR is Able to Detect a Single Copy Mutant DNA in One Million
Copies of Wild Type DNA
[0174] 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.
[0175] 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. 11A-D, and cast-PCR reactions were
performed using wild type or mutant ASP and corresponding MGB
blocker probes.
[0176] 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
[0177] 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.
[0178] The mutant DNA contained the KRAS-G12A mutation. 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.
[0179] 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.
11A-D.
[0180] 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
[0181] 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.
[0182] 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. 11A-D.
[0183] 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 80% for a
sample containing the EGFR-2573TG SNP.
[0184] It is 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."
[0185] 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.
[0186] 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
2231144DNAArtificial 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 2737289DNAHomo
sapiens 37gtactggtgg agtatttgat agtgtattaa ccttatgtgt gacatgttct
aatatagtca 60cattttcatt atttttatta taaggcctgc tgaaaatgac tgaatataaa
cttgtggtag 120ttggagctgg tggcgtaggc aagagtgcct gacgatacag
ctaattcaga atcattttgt 180ggacgaatat gatccaacaa tagaggtaaa
tcttgtttta atatgcatat tactggtgca 240ggaccattct ttgatacaga
taaaggtttc tctgaccatt ttcatgagt 2893816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38cccgctgctc ctgtgc 163920DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 39acccaacgca caacttagcg
204025DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 40cccgccttga tggatggaga tttac 254124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
41gcacccttgg tgaaacaatt gttg 244225DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
42cgccgaactg gaagtatttt gacag 254323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
43tatccctcac agagagaagg gag 234419DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 44gcttgcaatg gctccaacc
194519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 45ggagccattg caagccaag 194621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
46tggcaagagc gatgagtact c 214725DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 47ctctcaaaag gtaactgccc
actta 254816DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 48ctcctgtgct gtcacc
164915DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49cttagcggca cgcac 155018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 50ggagatttac gcaatgtg 185117DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 51acaattgttg agggggg 175220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 52aagtattttg acagctttac 205317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
53cccggctgct cctgtgt 175420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 54gccgaacgca caacttagca
205525DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 55cccgccttga tggatggaga tttat 255625DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
56tgcacccttg gtgaaacaat tgttc 255725DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
57cgccgaactg gaagtatttt gacat 255819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
58atggccctga taagggaga 195915DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 59agggcggtgc tcgag
156019DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 60tgtcactcta cccagtaaa 196121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
61agaatctgcc aattacctag a 216221DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 62acaagtagct gataataaat g
216317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 63gctcctgtgt tgtcacc 176416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 64cttagcagca cgcacc 166519DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 65tggagattta tgcaatgtg 196617DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 66acaattgttc agggggg 176720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 67aagtattttg acatctttac 206824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
68gcctgatttt ggtctagcta caga 246919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
69cgcgagaagg agctgtggt 197021DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 70ccgtgccttt accactcaga g
217118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 71gcgcgtgcag ctcatcac 187217DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
72cggcagcagt ttggccc 177321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 73gccggatatt ctcgacacag c
217418DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 74gcggacacag caggtcaa 187518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
75gcggacacag caggtcaa 187618DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 76cgccttgcct acgccact
187716DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 77cgctgcctac gccacg
167817DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 78gcgttgccta cgccacc 177919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
79cgcctcttgc ctacgccat 198018DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 80gcgtcttgcc tacgccag
188118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 81gcgtcttgcc tacgccac 188220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
82cgcgtagttg gagctggtga 208322DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 83ccgatactgg atacagctgg aa
228420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 84ccgtggatac agctggacaa 208518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
85ccgggtggtt ggagcaga 188618DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 86ccgggttgga gcaggtga
188718DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 87ccgggaggtt gtgaggca 188823DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
88gccggatgac agaaacactt ttc 238925DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 89gcgtacaact acatgtgtaa
cagtg 259017DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 90gccttcctgc atgggca 179116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
91gccggcggca tgaacc 169216DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 92gccgcggcat gaacca
169319DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 93gccaacagct ttgaggtgc 199423DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
94ccggattttg gtctagctac agt 239518DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 95gccagaagga gctgtggc
189621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 96ccgtgccttt accactcaga a 219718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
97gcgcgtgcag ctcatcat 189817DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 98cggcagcagt ttggcca
179922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 99cgctggatat tctcgacaca gg 2210018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
100gcggacacag caggtcac 1810118DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 101gcggacacag caggtcat
1810217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 102gcgttgccta cgccacc 1710317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
103gcgttgccta cgccacc 1710417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 104gcgttgccta cgccaca
1710518DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 105gcgtcttgcc tacgccac 1810618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
106gcgtcttgcc tacgccac 1810718DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 107gcgtcttgcc tacgccaa
1810819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 108gcctagttgg agctggtgg 1910922DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
109ccgatactgg atacagctgg ac 2211021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
110cgcctggata cagctggaca t 2111117DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 111cgcgtggttg gagcagg
1711217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 112cgcgttggag caggtgg 1711317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
113ggcgaggttg tgaggcg 1711424DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 114gcctggatga cagaaacact tttt
2411526DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 115ggcctacaac tacatgtgta acagtt
2611616DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 116gcctcctgca tgggcg 1611716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
117gccggcggca tgaact 1611816DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 118gccgcggcat gaaccg
1611920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 119cgcgaacagc tttgaggtgt 2012026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
120agcctcaatt cttaccatcc acaaaa 2612122DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
121catggaacca gacagaaaag cg 2212222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
122gtcactggca gcaacagtct ta 2212325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
123tgggagccaa tattgtcttt gtgtt 2512423DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
124aggaacgtac tggtgaaaac acc 2312520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
125ccctccccag tcctcatgta 2012641DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 126tcttcaaatg atttagtatt
atttatggca aatacacaaa g 4112741DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 127tcttcaaatg atttagtatt
atttatggca aatacacaaa g 4112828DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 128tgtgtgacat gttctaatat
agtcacat 2812928DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 129tgtgtgacat gttctaatat agtcacat
2813028DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 130tgtgtgacat gttctaatat agtcacat
2813128DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 131tgtgtgacat gttctaatat agtcacat
2813228DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 132tgtgtgacat gttctaatat agtcacat
2813328DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 133tgtgtgacat gttctaatat agtcacat
2813422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 134ggtcctgcac cagtaatatg ca 2213529DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
135gcaaatgact tgctattatt gatggcaaa 2913629DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
136gcaaatgact tgctattatt gatggcaaa 2913724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
137agtggttctg gattagctgg attg 2413824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
138gtggttctgg attagctgga ttgt 2413918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
139gcaaccagcc ctgtcgtc 1814020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 140agaccccagt tgcaaaccag
2014124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 141ctggagtctt ccagtgtgat gatg 2414223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
142ctggagtctt ccagtgtgat gat 2314317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
143tgtgcagggt ggcaagt 1714417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 144tgtgcagggt ggcaagt
1714524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 145ctttcttgcg gagattctct tcct 2414619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
146cagacaactg ttcaaactg 1914716DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 147ctggcagcaa cagtct
1614817DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 148tagtggcacc agaatgg 1714916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
149cccttcggct gcctcc 1615017DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 150cagcatgtca agatcac
1715117DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 151ctggtccctc attgcac 1715216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
152ccctccccag tcctca 1615316DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 153ccctccccag tcctca
1615415DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 154cctgctgaaa atgac 1515515DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
155cctgctgaaa atgac 1515615DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 156cctgctgaaa atgac
1515715DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 157cctgctgaaa atgac 1515815DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
158cctgctgaaa atgac 1515915DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 159cctgctgaaa atgac
1516018DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 160tcgtccacaa aatgattc 1816118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
161ccttcgcctg tcctcatg 1816218DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 162ccttcgcctg tcctcatg
1816314DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 163cagtgcgctt ttcc 1416414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
164cagtgcgctt ttcc 1416518DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 165ctgctcacca tcgctatc
1816617DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 166cctcaggcgg ctcatag 1716717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
167atgggcctcc ggttcat 1716816DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 168accggaggcc catcct
1616916DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 169cacactggaa gactcc 1617016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
170cacactggaa gactcc 1617115DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 171ctgtgcgccg gtctc
1517216DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 172gctacagaga aatctc 1617317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 173gagctgtggt agtggca 1717416DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 174cactcagaga aggagc 1617516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 175catcacgcag ctcatg 1617616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 176agtttggccc gcccaa 1617717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 177ctcgacacag caggtca 1717818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 178gcaggtcaag aggagtac 1817918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 179gcaggtcaag aggagtac 1818014DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 180cgccactagc tcca 1418116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 181ccacgagctc caacta 1618214DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 182acgccaccag ctcc 1418313DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 183cgccatcagc tcc 1318413DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 184cgccagcagc tcc 1318514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 185cgccaccagc tcca 1418615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 186gctggtgacg taggc 1518718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 187gctggaaaag aagagtac 1818817DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 188cagctggaca agaagag 1718917DNAArtificial
SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 189ttggagcaga tggtgtt
1719016DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 190tggagcaggt gatgtt 1619113DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 191tgaggcactg ccc 1319226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 192gaaacacttt tcgacatagt gtggtg
2619320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 193tgtgtaacag tgcctgcatg
2019414DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 194gcatgggcag catg 1419515DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 195gcatgaaccg gaggc 1519615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 196catgaaccag aggcc 1519718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 197ttgaggtgcg tgtttgtg 1819816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 198gctacagtga aatctc 1619916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 199agctgtggca gtggca 1620016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 200cactcagaaa aggagc 1620117DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 201tcatcatgca gctcatg 1720216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 202agtttggcca gcccaa 1620317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 203ctcgacacag gaggtca 1720417DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 204caggtcacga ggagtac 1720518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 205gcaggtcatg aggagtac 1820613DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 206cgccaccagc tcc 1320716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 207ccaccagctc caacta 1620816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 208acgccacaag ctccaa 1620913DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 209cgccaccagc tcc 1321013DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 210cgccaccagc tcc 1321115DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 211cgccaacagc tccaa 1521215DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 212gctggtggcg taggc 1521318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 213gctggacaag aagagtac 1821417DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 214cagctggaca tgaagag 1721516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 215tggagcaggt ggtgtt 1621616DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 216tggagcaggt ggtgtt 1621713DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 217tgaggcgctg ccc 1321827DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 218agaaacactt tttgacatag tgtggtg
2721921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 219atgtgtaaca gttcctgcat g
2122014DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 220gcatgggcgg catg 1422116DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 221gcatgaactg gaggcc 1622215DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 222catgaaccgg aggcc 1522320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 223tttgaggtgt gtgtttgtgc 20
* * * * *