NZ620654B2 - Compositions and methods for detecting allelic variants - Google Patents
Compositions and methods for detecting allelic variants Download PDFInfo
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- NZ620654B2 NZ620654B2 NZ620654A NZ62065412A NZ620654B2 NZ 620654 B2 NZ620654 B2 NZ 620654B2 NZ 620654 A NZ620654 A NZ 620654A NZ 62065412 A NZ62065412 A NZ 62065412A NZ 620654 B2 NZ620654 B2 NZ 620654B2
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- New Zealand
- Prior art keywords
- allele
- specific
- nucleic acid
- specific primer
- allelic variant
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/107—Modifications characterised by incorporating a peptide nucleic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/186—Modifications characterised by incorporating a non-extendable or blocking moiety
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
- C12Q2535/125—Allele specific primer extension
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
- C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
- C12Q2537/163—Reactions characterised by the reaction format or use of a specific feature the purpose or use of blocking probe
Abstract
method for detecting or quantitating a first allelic variant of a target sequence in a nucleic acid sample suspected of having at least a second allelic variant of the target sequence is disclosed. The a reaction mixture is formed by combining (i) the nucleic acid sample; (ii) an allele-specific primer, wherein an allele-specific nucleotide portion of the allele-specific primer is complementary to the first allelic variant of the target sequence, and the allele-specific primer comprises at least one nucleic acid modification; (iii) an allele-specific blocker probe that is complementary to a region of the target sequence comprising the second allelic variant and the allele-specific blocker probe comprises a non-extendable 3’-hexanediol, blocker moiety and at least one nucleic acid modification; (iv) a detector probe; and (v) locus-specific primer that is complementary to a region of the target sequence where the region of the target sequence complementary to the locus-specific primer is 3’ from the first allelic variant; and on the opposite strand. Then an amplification reaction on the reaction mixture using the locus-specific primer and the allele-specific primer to form an amplicon is carried out. The amplicon is detected by detecting a change in a detectable property of the detector probe, thereby detecting the first allelic variant of the target gene in the nucleic acid sample. A composition comprises (a) 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 and the allele-specific primer comprises at least one nucleic acid modification; and (b) an allele-specific blocker probe that is complementary to a region of the target sequence comprising a second allelic variant, and the allele-specific blocker probe comprises a non-extendable 3’-hexanediol, blocker moiety and at least one nucleic acid modification. The composition can further comprise (c) a detector probe; and/or (d) a locus-specific primer that is complementary to a region of the target sequence that is 3' from the first allelic variant and on the opposite strand. A reaction mixture comprising the above described composition and (e) a nucleic acid molecule is also disclosed. primer, wherein an allele-specific nucleotide portion of the allele-specific primer is complementary to the first allelic variant of the target sequence, and the allele-specific primer comprises at least one nucleic acid modification; (iii) an allele-specific blocker probe that is complementary to a region of the target sequence comprising the second allelic variant and the allele-specific blocker probe comprises a non-extendable 3’-hexanediol, blocker moiety and at least one nucleic acid modification; (iv) a detector probe; and (v) locus-specific primer that is complementary to a region of the target sequence where the region of the target sequence complementary to the locus-specific primer is 3’ from the first allelic variant; and on the opposite strand. Then an amplification reaction on the reaction mixture using the locus-specific primer and the allele-specific primer to form an amplicon is carried out. The amplicon is detected by detecting a change in a detectable property of the detector probe, thereby detecting the first allelic variant of the target gene in the nucleic acid sample. A composition comprises (a) 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 and the allele-specific primer comprises at least one nucleic acid modification; and (b) an allele-specific blocker probe that is complementary to a region of the target sequence comprising a second allelic variant, and the allele-specific blocker probe comprises a non-extendable 3’-hexanediol, blocker moiety and at least one nucleic acid modification. The composition can further comprise (c) a detector probe; and/or (d) a locus-specific primer that is complementary to a region of the target sequence that is 3' from the first allelic variant and on the opposite strand. A reaction mixture comprising the above described composition and (e) a nucleic acid molecule is also disclosed.
Description
COMPOSITIONS AND METHODS FOR DETECTING ALLELIC
VARIANTS
CROSS-REFERENCES TO RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Application No.
61/525,137, filed August 18, 2011, and U.S. Provisional Application No. 61/588,151, filed
January 18, 2012, the disclosures of which are hereby incorporated by reference in their
entirety for all purposes.
BACKGROUND OF THE INVENTION
Single nucleotide polymorphisms (SNPs) 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 DMA ( wok, Ann. Rev. Genom. Hum. Genet.. 2:235-
258 (2001)). SNPs have been implicated in genetic disorders, susceptibility to different
diseases, predisposition to adverse reactions to drags, 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.
Numerous approaches for SNP genotyping have been developed based on methods
involving hybridization, ligation, or DNA polymerases (Chen et al, Pharmacogenomics J.,
3:77-96 (2003)). For example, aliele-specific polymerase chain reaction (AS-PCR) is a
widely used strategy for detecting DNA sequence variation (Wu et al, Proc. Nail Acad. Sci.
USA, 86:2757-2760 (1989)). AS-PCR, as its name implies, is a PCR-bascd 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. S-PCR exploits the
fidelity of DNA polymerases, which extend primers with a mismatched 3 base at much
lower efficiency, from 0 to 100,000 fold less efficient, than that with a matched 3' base
(Chen et al, Pharmacogenomics J., 3:77-96 (2003)). The difficulty in extending mismatched
primers results in diminished PGR amplification that can be readily detected.
The specificity and selectivity of AS-PCR, however, is largely dependent on the
nature of exponential amplification of PGR 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 et al.,
Anal. Biochem., 284:l1-18 (2000)), 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 et al., Hum. Mut., 6:163-169 (1995); Imyanitov et al.,
Biotechniques, 33:484-490 (2002); McKinzie et al., Mut. Res., 517:209-220 (2002); Latorra et
al., Hum. Mut., 22:79-85 (2003)).
Another technology involving probe hybridization methods used for discriminating
allelic variations is TaqMan® genotyping. However, like AS-PCR, selectivity using this method
is limited and not suitable for detecting rare (1 in ≥ l,000) alleles or mutations in a mixed
sample.
[0005a] Any discussion of the prior art throughout the specification should in no way be
considered as an admission that such prior art is widely known or forms part of common
general knowledge in the field.
As such, there is a need in the art for improved compositions and methods to detect
single point substitutions (e.g., SNPs), insertions, or deletions against a background of wild-
type allele in thousand-fold or greater excess with increased sensitivity and specificity.
[0006a] It is an object of the present invention to overcome or ameliorate at least one of the
disadvantages of the prior art, or to provide a useful alternative.
SUMMARY OF THE INVENTION
[0006b] According to a first aspect, the present invention provides a method for detecting or
quantitating a first allelic variant of a target sequence in a nucleic acid sample suspected of
having at least a second allelic variant of the target sequence, said method comprising:
(a) forming a reaction mixture by combining:
(i) the nucleic acid sample;
(ii) an allele-specific primer, wherein an allele-specific nucleotide portion of the allele-
specific primer is complementary to the first allelic variant of the target sequence, and
wherein the allele-specific primer comprises at least one nucleic acid modification;
(iii) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising the second allelic variant, wherein the allele-specific blocker
- 2a -
probe comprises a non-extendable, 3’-hexanediol, blocker moiety and at least one
nucleic acid modification;
(iv) a detector probe; and
(v) a locus-specific primer that is complementary to a region of the target sequence,
wherein the region of the target sequence complementary to the locus-specific primer
3’ from the first allelic variant; and
on the opposite strand; and
(b) carrying out an amplification reaction on the reaction mixture using the locus-specific
primer and the allele-specific primer to form an amplicon; and
(c) detecting the amplicon by detecting a change in a detectable property of the detector
probe, thereby detecting the first allelic variant of the target gene in the nucleic acid sample.
[0006c] According to a second aspect, the present invention provides a composition
comprising:
(a) 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, and
wherein the allele-specific primer comprises at least one nucleic acid modification; and
(b) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising a second allelic variant, wherein the allele-specific blocker probe
comprises a non-extendable, 3’-hexanediol, blocker moiety and at least one nucleic acid
modification.
[0006d] According to a third aspect, the present invention provides a reaction mixture
comprising the composition of the invention and further comprising:
(e) a nucleic acid molecule.
[0006e] According to a fourth aspect, the present invention provides a kit comprising two or
more containers comprising the following components independently distributed in one of the
two or more containers:
(a) 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, and
wherein the allele-specific primer comprises at least one nucleic acid modification; and
(b) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising a second allelic variant, wherein the allele-specific blocker probe
comprises a non-extendable, 3’-hexanediol, blocker moiety and at least one nucleic acid
modification.
- 2b -
[0006f] Unless the context clearly requires otherwise, throughout the description and the
claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive
sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of
“including, but not limited to”.
The present invention provides compositions, methods, and kits for discriminating
sequence variation between different alleles. More specifically, in some embodiments, the
present invention provides compositions, methods, and kits for determining the presence
and/or level (e.g., quantitating) of rare (e.g., mutant) allelic variants, such as single nucleotide
polymorphisms (SNPs) or nucleotide insertions or deletions, in samples comprising abundant
(e.g., wild-type) allelic variants with high sensitivity and/or specificity. As such, in certain
embodiments, the present invention provides a highly selective method for the detection of
somatic mutations, e.g., in samples containing abundant levels of a wild-type allele compared
to very low levels of a mutant allele.
In one aspect, the present invention provides compositions for use in identifying
and/or quantitating allelic variants in nucleic acid samples. In certain embodiments, the
compositions of the invention can comprise one, two, three or more of the following: (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, 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 eytosine residues ("GC-rieh"). In some embodiments, the melting
temperature T m ) of the entire allele-specific primer ranges from about 50°C to about
67°C. n some embodiments, the a!leie-specific primer concentration is between about 20-
900 M
n some embodiments, the allele-specific nucleotide portion of the allele-specific
primer is located at the 3' terminus. As a non-limiting example, is used as the 3 allele-
specific nucleotide portion of the allele-specific primer when detecting and/or quantifying a
polymorphic site in which "A" is the mutant allele. As another non-limiting example, "C" is
used as the 3' allele-specific nucleotide portion of the allele-specific primer when detecting
and/or quantifying a polymorphic site in which G" is the mutant allele.
In some embodiments, the allele-specific blocker probe comprises a non-extendable
blocker moiety at the 3' terminus. The blocker moiety can comprise any modification of the
ribose ring 3'- of the oligonucleotide which prevents addition of further bases to the 3'-
end of the oligonucleotide sequence by a polymerase. In some exempiary embodiments, the
non-extendable blocker moiety includes, without limitation, an optionally substituted
- C 2
aikyl dioi (e.g., a 3'-hexanediol modification), an optionally substituted C2-C24 alkenyl dioi,
an optionally substituted alkynyi dioi, a minor groove binder (MGB), an amine (N ),
biotin, PEG, P . , and combinations thereof. In preferred embodiments, the non-extendable
blocker moiety comprises an optionally substituted C - 2 aikyl dioi (e.g., a 3'-hexanedioI
modification). In certain instances, the allele-specific nucleotide portion of the allele-specific
blocker probe is located from about 5 to about 1 or from about 5 to about 10, such as about
, 6, 7, 8, 9, 10, 1, 12, 3, 14, or 5 nucleotides away from the blocker moiety of the allele-
specific blocker probe. In certain other instances, the allele-specific blocker probe is not
cleaved during P R amplification. In further instances, the Tm of the allele-specific blocker
probe ranges from about 58°C to about 66°C.
In certain embodiments, the non-extendable blocker moiety does not comprise or
include a minor groove binder (MGB). In certain other embodiments, the non-extendable
blocker moiety does not comprise or include a PO4 group. In further embodiments, the non-
extendable blocker moiety consists essentially of or consists of an optionally substituted -
C24 aikyl dioi (e.g.. a 3'-hexanediol modification), an optionally substituted C2-C 2 alkenyl
dioi, or an optionally substituted alkynyi dioi.
n some embodiments, the a!leie-specific blocker probe and/or a!leie-specific primer
comprises at least 1, 2 3, 4, 5, or 6 (e.g., non-consecutive) base, sugar, and/or backbone
odifications certain instances, the modification(s) may increase the difference in the T
between matched and mismatched target sequences and/or decrease mismatch priming
efficiency, thereby improving assay specificity a d/or selectivity. Non-limiting examples of
such modifications include locked nucleic acid (LNA), peptide nucleic acid (PNA), threose
nucleic acid (TNA), zip nucleic acid (Z A) triazole nucleic acid, ' eth l-deoxycytidine,
2'-fluoro, 8-azadeaza-dA (ppA), 8-azadeaza-dG (ppG), 2'-deoxypseudoisocytidine (iso
dC), 5-fluoro-2'-deoxyuridine (fdU), and 2'-0,4'-C-ethylene bridged nucleic acid (ENA)
modifications, and combinations of these modifications. In preferred embodiments, the
modification present on the allele-specific blocker probe and or allele-specific primer
comprises one or more LNA modifications. n certain embodiments, the modification is
located (a) at the 3'-end, (b) at the '-end, (c) at an internal position, or at any combination of
(a) (b) or (c) within the allele-specific blocker probe and/or the allele-specific primer. In
some preferred embodiments, the modification (e.g., LNA) is located at the allele-specific
nucleotide portion of the allele-specific primer, such that the nucleoside of the modified
residue comprises the nucleobase sed to discriminate between allelic variants in other
preferred embodiments, the modification (e.g., LNA) is located at the allele-specific
nucleotide portion of the allele-specific blocker probe, such that the nucleoside of the
modified residue comprises the nucleobase used to discriminate between allelic variants.
in some embodiments, the detector probe comprises a sequence-based or locus-
specific detector probe in other embodiments, the detector probe comprises a 5' nuclease
probe. In some exemplary embodiments, the detector probe comprises an MGB moiety , a
reporter moiety (e.g., FAM™, TET™, JOE™ VIC™, or SYBR Green), a quencher moiety
(e.g.. Black Hole Quencher™ or TAMRA™), and/or a passive reference (e.g., ROX™). In
some embodiments, the detector probe is designed according to the methods and principles
described in U.S. Patent No. 6,727 356, the disclosure of which is incorporated herein by
reference in its entirety. In particular embodiments, the detector probe comprises a TaqMan^
probe (Applied Biosystems, Foster City, CA).
In some embodiments, the compositions of the invention can further comprise a
polymerase; deoxyribonueleotide 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 D A, such as genomic D A (gDNA) or
complementary DNA (cDNA). In other embodiments, the template sequence or nucleic acid
sample ca be R A, such as messenger R (mRNA).
In another aspect, the present invention 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
(a lel -i ; (b) hybridizing an allele-specific blocker probe to a second nucleic ac d molecule
comprising a second allele (allel --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) PGR amplifying
the first nucleic acid molecule comprising allele -
In yet 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) hybridizing a first allele-specific
primer to a first nucleic acid molecule comprising a first allele (allele- ) in a first reaction
mixture and hybridizing a second allele-specific primer to a . first nucleic acid molecule
comprising a second allele (ai e e-2) in a second reaction mixture, wherein a eie-2
corresponds to the same locus as allele- ; (b) hybridizing a first allele-specific blocker probe
to a second nucleic acid molecule comprising alleie-2 in the first reaction mixture and
hybridizing a second allele-specific blocker probe to a second nucleic acid molecule
comprising allele- in the second reaction mixture; (c) hybridizing a first detector probe to
the first nucleic acid molecule in the first reaction mixture and hybridizing a second detector
probe to the first nucleic acid molecule in the second reaction mixture: (d) hybridizing a first
locus-specific primer to the extension product of the first allele-specific primer in th first
reaction mixture and hybridizing a second locus-specific primer to the extension product of
the second allele-specific primer in the second reaction mixture; (e) P R amplifying the first
nucleic acid molecule to form a first set or sample of amplicons and PG 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- in the sample
comprising ailele-2 and/or alleie-2 in the sample comprising allele- .
n some embodiments, 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 T of the entire first and/or second allele-specific primer ranges from about 50°C to
about 67°C. In some instances, the concentration of the first and/or second allele-specific
primer is between about 20-900 nM.
In some embodiments, 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 port on of the second allele-specific primer are the same sequence.
In so e embodiments, 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 aliele-specific primer comprise the same sequence. In other
embodiments, the 5' tai 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.
In some embodiments, the allele-specific nucleotide portion of the first allele-
specific primer is specific to a first allele (allele- ) of a SNP and the allele-specific nucleotide
portion of t e second allele-specific primer is specific to a second allele (allele-2) of the same
SNP. In some embodiments, 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.
In certain other embodiments, the first and/or second allele-specific blocker probe
independently comprises a non-extendable blocker moiety at the 3' terminus. The blocker
moiety can comprise any modification of the ribose ring 3'-OH of the oligonucleotide which
prevents addition of further bases to the '-end of the oligonucleotide sequence by a
polymerase. In exemplary embodiments, the non-extendable blocker moiety includes,
without limitation, an optionally substituted Ci-C alkyl dio (e.g., a 3'-hexanediol
modif i cation) an optionally substituted C2-C24 alkenyl diol, an optionally substituted C2-C24
alkynyl diol, a minor groove binder (MGB), an amine (NH , biotin, PEG, PO , and
combinations thereof. In preferred embodiments, the non-extendable blocker moiety
comprises an optionally substituted C -C24 alkyl diol (e.g.. a 3'-hexanediol modification). In
certain instances, the allele-specific nucleotide portion of the first and/or second aliele-
specific blocker probe is located from about 5 to about 15 or from about 5 to about 10, such
as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the blocker moiety of
the first and/or second allele-specific blocker probe. In certain other instances, the first
and/or second aliele-specific blocker probe is not cleaved during PGR amplification. In
further instances, the Tm of the first and/or second aliele-specific blocker probe ranges from
about 58°C to about 66°C.
In certain embodiments, the non-extendable blocker moiety in the first and/or
second aliele-specific blocker probe does not comprise or include a minor groove binder
(MGB) and/or a O group. In certain other embodiments, the non-extendable blocker
moiety in the first and/or second aliele-specific blocker probe consists essentially of or
consists of an optionally substituted C¾-C¾ a ky dio (e.g., a 3'-hexanediol modification), an
optionally substituted C?-C? a k n diol, or an optionally substituted C 2-C 2 alkynyl diol.
In some embodiments, the first and/or second aliele-specific blocker probe and/or
the first and' r second aliele-specific primer comprises at least one nucleic acid modification.
In some embodiments, the modification's ) may increase the difference in the Tm between
matched and mismatched target sequences and/or decrease mismatch priming efficiency,
thereby improving assay specificity and/or selectivity. Examples of such modification(s)
include, without limitation, the modified bases, nucleic acid analogs, and ribose-modified
nucleic acids described herein such as locked nucleic acids (LNA;, peptide nucleic acids
P A), threose nucleic acids (TNA) zip nucleic acids (ZNA), and triazole nucleic acids
(TzNA). In particular embodiments, one or more (e.g., 2 to 6, or 2, 3, 4, 5, or 6) nucleic acid
modifications such as L As are present on the aliele-specific blocker probe and/or allele-
specific primer. In preferred embodiments, the plurality of modifications on the probe or
primer is non-consecutive or non-contiguous, e.g., two modifications such as LNAs are not
next to each other in the probe or primer sequence. In certain embodiments, the nucleic acid
modification's) is located (a) at the 3'-end, (b) at the '-end, (c) at an internal position, or at
any combination of (a), (b) or (c) within the first and/or second aliele-specific blocker probe
and/or the first and/or second aliele-specific primer. In some preferred embodiments, one
modification (e.g., LNA) is located at the aliele-specific nucleotide portion of the first and' r
second aliele-specific primer, such that this modification comprises the nucleobase used to
discriminate between allelic variants. In other preferred embodiments, one modification
(e.g., LNA) is located at the aliele-specific nucleotide portion of the first and/or second allele-
specific blocker probe, such thai this modification comprises the nucleobase that is used to
discriminate between allelic variants.
In some embodiments, the specificity of allelic discrimination is improved by the
inclusion of a nucleic acid modification in the first and/or second aliele-specific primer
and/or first and/or second aliele-specific blocker probe as compared to the use of a non-
modified allelic-speeific primer or blocker probe n some embodiments, the improvement in
specificity s at leas about 2 fold (e.g.. at least about 2 fold, 3 fold, 4 fold, 5 old, 10 fold, 15
fold, 20 fold, etc:).
In other embodiments, the specificity of allelic discrimination is at least about 2 fold
(e.g., at least about 2 fold, 3 fo d, 4 fold, 5 fold, 10 fold. fold, 20 fold, etc.) better than the
specificity of allelic discrimination using A e-Specii f c PGR w h a Blocking reagent (ASB-
PCR) methods described in Marian et al, PloS ONE, 4:e4584 (2009).
in some embodiments, the methods further comprise a 2-stage cycling protocol In
some embodiments, the number of cycles in the first stage of the 2-stage cycling protocol
comprises fewer cycles than the number of cycles used in the second stage. In other
embodiments, the number of cycles in the first stage is about 90% fewer cycles than the
number of cycles in the second stage. In yet other embodiments, the number of cycles in the
first stage is between 3-7 cycles and the number of cycles in the second stage is between 42-
48 cycles.
in some embodiments, the annealing/extension temperature used during the first
cycling stage of the 2-stage cycling protocol is between about 1 -3°C lower than the
annealing/extension temperature used during the second stage. n certain embodiments, the
annealing/extension temperature used during the first cycling stage of the 2-stage cycling
protocol is between 56-59°C and the annealing/exte n temperature used during the second
stage is between 60-62°C.
In some embodiments, the methods further comprise a pre-amplificaiion step. In
certain embodiments, the pre-ampiification step comprises a multiplex amplification reaction
that uses at least two complete sets of a e e-spe ific primers and locus-specific primers,
wherein each set is suitable or operative for amplifying a specific polynucleotide of interest.
In other embodiments, the products of the multiplex amplification reaction are divided into
secondary suigle-plex amplification reactions, wherein each single-plex reaction contains at
least one primer set previously used in the multiplex reaction. In yet other embodiments, the
multiplex amplification reaction further comprises a plurality of allele-specific blocker
probes. In some embodiments, the multiplex amplification reaction is carried out for a
number of cycles suitable to keep the reaction within the linear phase of amplification.
In some embodiments, 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 he first and/or second detector probe s a 5' nuclease probe n some
exemplary embodiments, the first and/or second detector probes comprises an MGB moiety,
a reporter moiety (e.g.. FAM™, TET™, JOE™, VIC™, or SYBR* Green), a quencher
moiety (e.g., Black Hole Quencher™ or TAMRA™), and/or a passive reference (e.g.,
ROX™). in some embodiments, the first and/or second detector probe is designed according
to the methods and principles described in U.S. Patent No. 6,727,356 the disclosure of which
is incorporated herein by reference in its entirety. In particular embodiments, the first and/or
second detector probe comprises a TaqMan* probe (Applied Biosystems, Foster City, CA).
n some embodiments, 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.
In some embodiments, the first and/or second reaction mixtures 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 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 .
I some embodiments, the first al e e-specif 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. n 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.
In some exemplary embodiments, the first allele is a rare (e.g., inor) or mutant
allele. In other exemplary embodiments, the second allele is an abundant (e.g., major) or
wild-type allele.
n another aspect, the present invention provides kits for detecting or quantitating a
first allelic variant in a sample comprising a second allelic variant comprising one or more of
the following: (a) a first alle!e-specific primer; (b) a second allele-specific primer; (c), a first
locus-specific primer; (d) a second locus-specific primer; (e) a first alleie-specific blocker
probe; (f a second alleie-specific blocker probe; (g) a first locus-specific detector probe; and
(h) a second locus-specific detector probe.
In some embodiments, the first and/or second alleie-specific primer comprises a
target-specific portion and an alleie-specific nucleotide portion. In some embodiments, the
f i rst and/or second alleie-specific primer may further comprise a tai . Other embodiments
with respect to the first and/or second alleie-specific primers in the kits of the invention are
described abo ve.
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. n 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
In particular embodiments, the compositions, methods, and kits of the invention
provide improved detection and discrimination of allelic variants using one, two, three or
more of the following components: (a) an alleie-specific primer comprising a nucleic acid
modification such as a locked nucleic acid (LNA) at the position of the discriminating base
(e.g., an alleie-specific primer containing a 3'-end LNA at the polymorphic site); (b) an
alleie-specific blocker probe comprising a non-extendable blocker moiety such as a C -C24
alkyl diol (e.g., hexanediol) modification at the 3' terminus and a nucleic acid modification
such as a locked nucleic acid (LNA) at the position of the discriminating base (e.g., a blocker
oligonucleotide containing a hexanediol chemical group at the 3'-end and a single LNA at the
polymorphic site at a position that is about 5-15 (e.g., about 10) nucleotides away from the
blocking moiety, e.g., in the middle of the blocker probe); (c) a detector probe such as a
TaqMan probe (e.g., a TaqMan MGB FAM probe); and (d) a locus-specific primer such as
a reverse primer. In certain instances, the blocker moiety comprises a C -C24 alkyl diol (e.g.,
hexanediol) that is conjugated to the 3'-end of the alleie-specific blocker oligonucleotide
sequence via a phosphoramidite linkage. In certain other instances, the assay methods of the
invention are performed on an ABI 7900HT Real Time PCR Instrument, although any type of
real time PCR instrument known to one of ordinary skill in the art can be used. In particular
embodiments, the reaction characteristics comprise the following: Stage 1: 95.0°C for 10:00
min; Stage 2 : Repeats: 40 95.0°C for 0:20 min 60.0°C for 0:45 min.
Other objects, features, and advantages of the present invention will be apparent to
one of skill in the art from the following detailed description and figures.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts one embodiment of the somatic mutation detection assays of the
present invention.
Figure 2 illustrates that the use of an allele-specific primer comprising a locked
nucleic acid (LNA) modification at the 3'-end ("+A" in "G12S ASP-LNA") and an allele-
specific blocker probe comprising a LNA modification in the middle of the oligonucleotide
sequence ("+G" in "G12S blocker-LNA") and a 3'-hexanediol modification ("C6" in "G12S
blocker-LNA") improves the discrimination of allelic variants at the KRAS G12S SNP.
Figure 3 illustrates that the use of an allele-specific primer comprising a LNA
modification at the 3'-end ("+C" in "G12R ASP-LNA") and an allele-specific blocker probe
comprising a LNA modification in the middle of the oligonucleotide sequence ("+G" in
"G12R blocker-LNA") and a 3'-hexanediol modification ("C6" in "G12R blocker-LNA")
improves the discrimination of allelic variants at the KRAS G12R SNP.
Figure 4 illustrates that the use of an allele-specific primer comprising a LNA
modification at the 3'-end ("+G" in "H1047R ASP-LNA") and an allele-specific blocker
probe comprising a LNA modification in the middle of the oligonucleotide sequence ("+A"
in "H1047R blocker-LNA") and a 3'-hexanediol modification ("C6" in "H1047R blocker-
LNA") improves the discrimination of allelic variants at the PIK3CA H1047R SNP.
Figure 5 illustrates improved allelic variant discrimination at the EGFR T790M
polymorphic site using the LNA-modified allele-specific primers and probes of the present
invention.
Figure 6 illustrates improved allelic variant discrimination at the EGFR L858R
polymorphic site using the LNA-modified allele-specific primers and probes of the present
invention.
Figure 7 illustrates the effect of an abundant amount of wild-type DNA from whole
blood on the interference of detecting the PIK3CA H1047R variant allele in H1047R-positive
KPL4 cells.
Figure 8 illustrates the effect of an abundant amount of wild-type DNA from whole
blood on the interference of detecting the KRAS G12R variant allele in G12R-positive PSNl
cells.
Figure 9 shows that screening of 150 CRC tissue samples indicated that there was
no interference observed from the negative samples and that detection of a weak signal can
be validated by titration.
Figure 10 shows that the DxS/Qiagen Scorpion assay can only detect 1000 cells in
the mixture of whole blood spiked with a serial dilution of SW1 116 (G12A-positive) cells.
Figure 11 illustrates that the Inostics BEAMing assay made the incorrect call and
identified 14 out of 15 mutant samples as wild-type samples.
Figure 12 illustrates that the somatic mutation genotyping assay of the invention had
a detectable signal as low as 50 to 100 positive cells in the whole blood mixture.
Figure 13 illustrates an exemplary embodiment of the invention in which the rare
allele, and not the wild-type allele is selectively amplified using the methods described
herein. The wild-type allele does not produce an amplification product because the blocker
probe with LNA (C in Figure 13) and hexanediol modifications hybridizes to the wild-type
allele, thus impeding real-time PCR amplification. The allele-specific primer with a LNA
modification (T in Figure 13) preferably hybridizes to the mutant allele rather than the
blocker probe, and thus facilitates the generation of a real-time PCR amplification product.
Figure 14 also shows that an allele-specific primer can amplify both the wild-type
(negative control) and mutant (positive control) alleles. The amplification plots illustrate that
the assay with the allele-specific LNA primer has high sensitivity. The primer with LNA
selective amplifies the mutant allelic variant.
Figure 15 shows that strategically placed LNA modifications on an allele-specific
primer can improve amplification and lower the Ct value. The performance of the assay can
be improved by the used of more than one LNA.
Figure 16 shows that three or more LNA modifications placed consecutively on an
allele-specific primer do not produce an amplification product.
Figure 17 shows an exemplary LNA molecule and other modified LNAs that can be
used in the present invention. The allele-specific primer of the present invention can
comprise 2 to 6 LNAs.
Figure 18 shows an exemplary PNA-DNA duplex (left) and an exemplary allele-
specific primer with a PNA modification (T in Figure 18; right).
Figure 19 shows an exemplary TNA-containing oligonucleotide (left) and an
exemplary allele-specific primer with a TNA modification (T in Figure 19; right).
Figure 20 shows an exemplary ZNA oligonucleotide (left) and an exemplary ZNA
modified allele-specific primer (right; T in Figure 20).
Figure 21 shows an exemplary TzDNA molecule (left) and an exemplary allele-
specific primer with a TzDNA modification (right; T in Figure 21).
Figure 22 illustrates an exemplary blocker probe for the G12A KRAS assay of the
present invention. The blocker probe (oligonucleotides) is specifically designed to hybridize
to the wild-type allele and inhibits the amplification of contaminating wild-type genomic
DNA efficiently and selectively, without affecting the amplification of the allelic variant.
Figure 23 illustrates that the KRAS G12A assay comprising a LNA primer and a
blocker probe with hexanediol specifically amplified the allelic variant (A) and inhibited
amplification of the wild-type allele (G). The selectivity of the assay is improved with the
use of a blocker in combination with the presence of LNA on the allele-specific primer.
Figure 24 shows a comparison between a blocker probe with a hexanediol
modification (3' carbon tail) and one with a phosphate group. The blocker hexanediol probe
performed better with a lower Ct of 28.4, compared to a Ct of 31.2 for the phosphorylated
blocker.
Figure 25 shows that a blocker probe with LNA modification has a lower Ct value
compared to one without LNA. Figure 25 also illustrates that the method of the present
invention that employs a blocker probe with LNA efficiently and selectively amplified the
mutant variant, and shows excellent allelic discrimination.
Figure 26 shows that an exemplary method of the present invention that uses LNA-
containing allele-specific primer and LNA-containing blocker probe had high specificity for
the mutant allele and generated a specific amplification product. Figure 26 also shows that
the wild-type allele was not amplified.
Figure 27 shows the difference in melting temperature (Tm) and Ct value between
two blocker probes with the same sequence and 3' hexanediol modification, but different
locations of LNA on the blocker sequence. This illustrates how the position of the LNA in
the blocker sequence influences the performance of the assay. LNA placed at the base of the
allelic variance improves the performance of the assay with the lower Ct, compared to the
blocker with LNA placed away from the allelic variant nucleotide.
Figure 28A shows the influence of Tm of the blocker probe on the performance of
the assay. Figure 28B also shows the difference in Tm and Ct value between a blocker
without LNA and one with two LNAs. The increase in Tm due to the presence of LNA,
consequently improves the sensitivity and selectivity of the assay of the present invention.
With the higher Tm, the blocker probe remained annealed to its target during extension,
thereby efficiently blocking the wild-type allele from interfering with amplification and
allowing the variant to be amplified preferentially and selectively. The Tm of the same
blocker sequence increased from 59.9°C to 68°C with the addition of two LNAs.
Figure 29 shows that the placement of 6 consecutive LNA modifications including
LNA located at the a e e-spe i nucleotide position on the blocker probe completely
arrested amplification during PCR cycling.
Figure 30 shows that the ACt values can be used to determine the feasibility of the
assay and its selectivity. The higher ACt value obtained with the LNA-containing primers
and probes indicate the feasibility and selectivity of the assay of the present invention.
Figure 31 shows how the AACt values are calculated from the Ct values from
various somatic mutation genotyping assays. Assay A of the figure that was designed with an
LNA-containing primer and blocker performed better than the other assays that contained
primers and probes with consecutive LNAs.
Figure 32 illustrates the use of the PIK3CA E545K assay of the present invention to
quantify the percentage of the mutant variant present in an unknown sample. Figure 32A
shows that the standard curve for the PIK3CA E545K allelic variant and the MCF 7 cell line.
It was created using methods described herein. Figure 32B shows amplification curves for
two unknown samples from patients with colorectal cancer (Samples A and B) and the
positive control (MCF 7 cell line) generated using the genotyping assay. Figure 32C shows
the amount and the percentage (percent mutation) of the mutant variant E545K present in the
samples as determined by the calculator.
Figure 33 illustrates the use of the KRAS G12D assay of the present invention to
quantify the percentage of the mutant variant present in an unknown sample. Figure 33A
shows the amplification plot and the standard curve for the KRAS G12D genotyping assay
and the LS 174T cell line. Figure 33B shows that amplification plots for two unknown
samples from patients with pancreatic cancer (Samples A and B) and a positive control (LS
174T cell line) that were generated using methods of the present invention. Figure 33C
shows the amount of DNA in Sample A expressing the mutant variant was determined using
the calculator to be 3.25 ng or 9.6%, relative to the positive control.
Figure 34 illustrates the use of the EGFR E746-A750 deletion EGF assay of the
present invention to quantify the percentage of the mutant variant present in an unknown
sample. Figure 34A shows the amplification plot and standard curve for the E746-A750
deletion of the EGFR gene for the H I650 cell line. Figure 34B shows that the amplification
plot for an unknown sample (Sample A) from a patient with lung cancer and a positive
control (HI 650 cell line) that were generated using methods of the present invention. Figure
34C shows the amount of DNA in Sample A expressing the EGFR deletion variant was
determined using the calculator to be 3.25 ng or 9.6%, relative to the positive control.
Figure 35 illustrates the use of the V600E BRAF assay of the present invention to
quantify the percentage of the allelic variant present in an unknown sample. Figure 35A
shows the amplification plot and standard curve for the BRAF V600E allelic variant for the
HT 29 cell line. Figure 35B shows that the amplification plot for an unknown sample
(Sample A) from a patient with lung cancer and a positive control (HI 650 cell line) that were
generated using methods of the present invention. Figure 35C shows the amount of DNA in
Sample A expressing the V600E variant of BRAF was calculated to be 0.18 ng or 1.2%,
relative to the positive control.
Figure 36 shows H&E stained frozen sections of a non-small cell lung cancer
(NSCLC) tumor sample. Figure 36A shows a section that has a high percentage of tumor
cells (the white arrow indicates tumor cells). Figure 36B shows a section composed of a
mixture of tumor cells (white arrow), stroma with blood vessels (black arrow), inflammatory
cells (e.g., lymphocytes; red arrow); and a lung alveolus filled with macrophages (green
arrow).
Figure 37 illustrates the relationship between cytokeratin (CK) levels and the
expression of allelic variants in either gastric tumor samples or pancreatic tumor samples.
Figure 37A illustrates that in gastric tumor samples #1, 2 and 4 there was a high level of CK
and a low percent mutation for the EGFR T790M, KRAS G12V, KRAS Q61H, or PIK3CA
E545K allelic variant. These results show that in sample #1, 2 and 4 few of the tumor cells
are likely to carry the EGFR T790M, KRAS G12V, KRAS Q61H, or PIK3CA E545K SNPs.
Yet, in sample #5 there was a high level of CK and a high percent mutation (e.g., 90%) for
the KRAS G12D mutation. This result shows that most of the tumor cells in sample #5 are
likely to carry the G13D mutation. Figure 37B shows that in pancreatic tumor sample # 1
there was a high level of CK and a high percent mutation (100%) for the KRAS G12D
variant, thus indicating that most of the tumor cells are likely to carry the mutant allele. In
contrast, in pancreatic tumor sample #3 there was a high level of CK but a low percent
mutation (e.g., 5%) for the KRAS G12D SNP. Few tumor cells of sample #3 are likely to
carry the G12D mutation.
Figure 38 illustrates the sensitivity of the somatic mutation genotyping assay of the
present invention (e.g., KRAS G12A assay) compared to Life Technologies' castPCR™
Mutation Assay. Figure 38A illustrates the amplification curves for the genotyping assays of
the present invention. Figure 38B illustrates the amplification curves for Life Technologies'
castPCR™ Mutation Assays performed on the same test samples. Figure 38C shows that
assay of the present invention detected the G12A mutation when the test sample contained as
few as 250 positive tumor cells. By comparison, a larger number of tumor cells were needed
to detect the mutation using Life Technologies' castPCR™ Mutation Assay.
Figure 39 illustrates the sensitivity of the somatic mutation genotyping assay of the
present invention (e.g., KRAS G12A assay) compared to Life Technologies' castPCR™
Mutation Assay. Figure 39A shows the amplification curves of the test samples using the
genotyping assay of the present invention . Figure 39B shows the amplification curves for
Life Technologies' castPCR™ Assay. Figure 39C shows that assay of the present invention
detected the G12S KRAS allelic variant in as few as 100 positive tumor cells, while Life
Technologies' castPCR™ Mutation Assay could not.
Figure 40 shows the results obtained by using the methods of the present invention
to detect (e.g., presence or absence) and/or quantitate (e.g., percent mutation) the following
SNPs in breast cancer samples: PIK3CA E542K, E545D, E545K, H1047R; EGFR T790M,
L858R; KRAS G12A, G12C, G12D, G12R, G12S, G12V, and G13D; and BRAF V600E.
This figure shows that the PIK3CA H1047R SNP was expressed at different percentages in
the breast cancer samples.
Figure 4 1 shows that PIK3CA SNPs (E542K, E545D, E545K and H1047R) were
also detected and quantitated (e.g., percent mutation) in an additional set of breast cancer
samples. 45 breast cancer samples were screened for the PIK3CA E542K, E545D, E545K
and H1047R allelic variants.
Figure 42 shows that lung tumor samples can be screened for SNPs using methods
of the present invention. In this embodiment, the presence and percent mutation of various
SNPs were determined in 25 lung tumor samples. The SNPs included PIK3CA E542K,
E545D, E545K and H1047R; EGFR T790M, L858R and E746 deletion; KRAS G12C, G12R,
G12S, G12D, G12A, G12V, G13C, G13D, and Q61H; and BRAF V600E.
Figure 43 illustrates the results obtained from using the methods of the present
invention on an additional 32 human lung tumor samples. The SNPs included PIK3CA
E542K, E545D, E545K and H1047R; EGFR T790M, L858R and E746 deletion; KRAS
G12C, G12R, G12S, G12D, G12A, G12V, and G13D; and BRAF V600E.
Figure 44 shows that gastric tumor samples can be screened using methods of the
present invention to detect the presence and percent mutation of various SNPs. The SNPs
included PIK3CA E542K, E545D, E545K and H1047R; EGFR T790M, L858R and E746
deletion; KRAS G12C, G12R, G12S, G12D, G12A, G12V, G13D, and Q61H; and BRAF
V600E. In this embodiment, each assay was run with 40 ng of sample (e.g., DNA).
Figure 45 shows the results obtained from using the methods of the present
invention with xenograft samples to detect the presence and percent mutation of various
SNPs. The SNPs included PIK3CA E542K, E545D, E545K and H1047R; EGFR T790M,
L858R and E746 deletion; KRAS G12C, G12R, G12S, G12D, G12A, G12V, G13C, G13D,
and Q61H; and BRAF V600E. The EGFR E746 deletion was present in samples #585-588
and predicted to be in 100% of the cells in the sample. The PIK3CA H1047R allele was
detected in samples #581-584 at a percentage of mutation of 3.4%, 1.2%, 1% and 1.8%,
respectively.
Figure 46 illustrates that KRAS, BRAF and PIK3CA allelic variants can be detected
and quantitated (e.g., percent mutation) in colorectal cancer samples using the methods of the
present invention. The SNPs included PIK3CA E542K, E545D, E545K and H1047R; KRAS
G12C, G12R, G12S, G12D, G12A, G12V, and G13D; and BRAF V600E.
Figure 47 illustrates that KRAS, BRAF and PIK3CA allelic variants can be detected
and quantitated in additional colorectal cancer samples using the methods of the invention.
Figure 48 illustrates that liver tumor and colon tumor tissues from patients with
colorectal cancer can be screened for KRAS, BRAF and PIK3CA allelic variants using the
methods of the present invention. The results show that some of the samples had a plurality
of SNPs.
Figure 49 illustrates that samples from patients with pancreatic cancer can be
screened for SNPs and the percent mutation can be determined according to methods of the
present invention. In this embodiment, fine needle aspirate samples were from obtained from
patients and screened using the SNP genotyping assays described herein. In the pancreatic
cancer samples tested, various KRAS mutations were detected, but PIK3CA (e.g., E542K,
E545D, E545K, H1047R), EGFR (e.g., T790M, L858R) and BRAF (e.g., V600E) mutations
were not detected.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
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.
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 ACt value between a mismatched and matched allele-specific primer
is indicative of greater discriminating power between allelic variants. Such factors found to
impro ve 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) ow allele-specific
primer concentration; (c) allele-specific primers designed to have lower Trn's; 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 nucleic acid modifications such as, e.g., modified bases, nucleic acid
analogs, and/or ribose-modified nucleic acids, in order to increase the delta T between
matched and mismatched target sequences.
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 invention relates generally to novel amplification methods which utilize one
or more of the factors described above to improve the discrimination of allelic variants during
PC R, e.g., by increasing ACt values.
In certain aspects, the present invention is based on locked nucleic acid (LNA)
chemistry using allele-specific real time PCR with a blocker oligonucleotide containing a
hexanediol 3' modification to prevent the amplification of the wild-type allele. The limit of
detection of the present methods is advantageously 2-10 DNA copies. The present invention
also provides selective and robust detection of a large panel of somatic mutations. As a non-
limiting example, the present invention enables the detection of a very low copy number
mutant allele (e.g., 0.01%-0.1%) in a whole blood background. In some instances, surrogate
samples for use in the mutation assays described herein include, but are not limited to, blood,
serum, plasma, tissues (e.g., FNA, CTCs, core biopsy, FFPE tissue), and mixtures thereof.
In particular aspects, the mutation assays of the invention are based on an allele-
specific PCR. In certain embodiments, the detection is a real time method using TaqMan
probe technology. In preferred embodiments, an allele-specific primer (ASP) containing a
single LNA base mutation at its 3'-end can be used to specifically detect the mutant allele. In
these embodiments, a blocking oligonucleotide (blocker) complementary to the wild-type
sequence can be used to suppress any non-specific amplification of the wild-type allele. This
blocker may contain a single LNA variant situated at the wild-type nucleotide position. In
some instances, the blocker comprises a hexanediol chemical group at the 3'-end to prevent
any extension. In other instances, the present invention further includes a reverse primer to
complete the reaction. Without being bound to any particular theory, the presence of a LNA
modified base increases the discrimination between wild-type and mutant alleles, enabling
greater target allele specificity and blocking efficacy. In particular embodiments, LNA is a
modified base used to increase the specificity of PCR probes and the thermal stability of
duplexes. LNA modified bases are capable of single nucleotide discrimination, thereby
minimizing the possible mismatch between the AS primer and wild-type allele. In certain
embodiments, the allele-specific primer and/or blocking oligonucleotide comprises an LNA
modified base at the position of the allelic variant and 1, 2, 3, 4, 5, 6, 7, or more additional
non-consecutive or non-contiguous LNA modifications and/or an LNA modified base at the
'-end.
Accordingly, the compositions and methods of the present invention advantageously
enable the detection of very low levels of mutant (somatic) DNA in samples including blood,
plasma, serum, and/or tissues (FNA, CTCs, core biopsy, FFPE tissue, etc.). In particular, the
assays of the present invention are significant improvements on previously described allelic-
specific PCR mutation detection by virtue of the use of LNA chemistry in combination with a
novel blocking oligonucleotide design. As such, the present invention provides mutation
analysis with a 10 to 100 fold higher sensitivity than detection technologies known in the art
such as the BEAMing (Beads, Emulsions, Amplification, and Magnetics) assay (Inostics), the
Scorpions/ARMS reaction (Qiagen), and the castPCR Assay (Life Technologies). In
addition, the present invention provides a method with the ability to detect oncogenic
resistant mutants such as EGFR T790M, EGFR E746-A750 deletion, KRAS G12A, KRAS
G12D, KRAS G12S, E545K PIK3CA, and V600E BRAF.
II. Definitions
As used herein, the following terms have the meanings ascribed to them unless
specified otherwise.
As used herein, the term "allele" includes alternative D A sequences at the same
physical locus on a seg en of DNA, such as, for example, on homologous chromosomes.
An allele can refer o 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 ("allelic variant '). In so e instances, an allele
can correspond to a single nucleotide difference at a particular physical locus. In other
instances, an allele can correspond to a nucleotide (single or multiple) insertion or deletion.
The term "allele-specifie primer' includes an oligonucleotide sequence that
hybridizes to a sequence comprising an allele of interest, and which when used in PGR can be
extended to effectuate first strand cD A synthesis. Allele-specifie 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-specifie primers may comprise an
allele-specifie nucleotide portion, a target-specific portion, and/or a tail
As used herein, the terms "allele-specifie nucleotide portion" or "'allele-specifie
target nucleotide" include a nucleotide or nucleotides in an allele-specifie primer that can
selectively hybridize and be extended from o e 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.
The term "target-specific portion" includes the region of an allele -specific primer
that hybridizes to a target polynucleotide sequence. In some embodiments, the target-specific
portion of the allele-specifie 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-specifie primer may comprise the allele-specifie nucleotide portion. In
other instances, the target-specific portion of he aiiele-specific primer is adjacent to the 3'
al le- pec f i c nucleotide portion.
As vised herein, th terms "tail" or " '-tail" include the non-3 ' end of a primer. Tins
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-
, 4-6, 5-8, 6-12, 7- , 10-20, 15-25 or 20-30 nucleotides, or any range in between, in length.
The terms "aiiele-specific blocker probe" or "blocker probe" or "blocker' include
an oligonucleotide sequence that binds to a strand of D A comprising a particular allelic
variant which is located on the same, opposite or complementary strand as that bound by an
alleiic-specific primer, and reduces or prevents amplification of that particular allelic variant.
As discussed herein, aiiele-specific blocker probes generally comprise modifications, e.g., at
the 3' -OH of the ribose ring, which prevent primer extension by a polymerase. The aiiele-
specific blocker probe can be designed to anneal to the same or opposing strand of what the
aiiele-specific primer anneals to and can be modified with a blocking group (eg., 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 th 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 aiiele-specific primer. In illustrative examples, the aiiele-specific blocker probes do not
include a label, such as a fluorescent, radioactive, or chemiluminescent label.
As used herein, the terms "non-extendable blocker moiety" or "blocker moiety"
include 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 PGR reaction. Examples of blocker moieties include, but are
not limited to, modifications of the ribose ring 3' -OH of the oligonucleotide, which prevent
addition of further bases to the 3'-end of the oligonucleotide sequence by a polymerase. n
particular embodiments, the non-extendable blocker moiety includes, without limitation, an
optionally substituted C - a ky diol (eg., a 3'-hexanediol modification), an optionally
substituted CVC¾ alkeny! diol, an optionally substituted C2-C24 aikynyl diol, a minor groove
binder (MGB), an amine H ), biotin, PEG, P0 , and combinations thereof. Examples of
GB' s include CC1065 analogs, lexitropsins, distamycin, netropsin, berenii, duocarmycin,
pentamidine, 4, 6-diaminophenylindo1e and pyrrolo[2 ,1-c][^benzodiazepines, and DPI3.
As used herein, the term "modified base" includes 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 et al. Hum. Mu , 2:79-85 (2003); Nakiandwe
et al, Plant Method, 3:2 (2007).
The terms "locked nucleic acid" or "LNA" include a class of nucleic acid analogues
in which the ribose ring is "locked" by a methylene bridge connecting the 2'-0 atom and the
4'-C atom. LNA nucleosides contain the common nucleobases (T, C, G, A, U, and mC) and
are able to form base pairs according to standard Watson-Crick base pairing rules. However,
by "locking" the molecule with the methylene bridge, the LNA is constrained in the ideal
conformation for Watson-Crick binding.
The terms "peptide nucleic acid", "peptidic nucleic acid" or "PNA" include a on-
naturally occurring and artificially synthesized nucleic acid analog or mimic comprising
various naturally-occurring or non-naturally-occurring nucleobases attached to a backbone of
repeating N-(2-aminoethyl)-glycine units linked by amide bonds. The purine and pyrimidine
bases are attached to the uncharged backbone through methylene carbonyl linkages. Like
with DNA, Watson-Crick base pairing rules apply to peptide nucleic acids.
The terms "zip nucleic acids" or "ZNAs" include oligonucleotides conjugated with
one or a plurality of cationic spermine moieties that decrease electrostatic repulsions with
target nucleic acid strands and increase the affinity of the oligonucleotides for their targets.
The terms "triazole nucleic acids", "TzNAs", "triazole deoxynucleic acid",
"TzDNA", "triazole-linked analogue of deoxyribonucleic acid" or "TLDNA" include an
oligonucleotide comprising a non-naturally occurring triazole linkage.
The terms "(3'-2') -L-threose nucleic acid", "threose nucleic acid" or "TNA"
include a non-naturally occurring nucleic acid discovered during investigations of nucleic
acids that obey Watson-Crick base-pairing rules and are bound to alternative sugar-phosphate
backbones (see, e.g., Ichida et al, Nucleic Acids Res., 33: 5219-5225 (2005)). TNAs have a
repeat unit one atom shorter than natural nucleic acids, yet they can base pair with DNA,
RNA, and itself. While not wanting to be bound by a particular theory, it is believed that
TNA hybridizes strongly with DNA and even more strongly with RNA because TNA is a
good mimic of the A-form of DNA and of RNA. The increased stability of TNA-DNA
duplexes compared to analogous DNA-DNA complexes results in improved mismatch
discrimination of allelic variants.
As vised herein, the term "detector probe' includes any of a variety of signaling
molecules indicative of amplification. For example, SYBR Green and other DMA-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 and include, but are not limited to, TaqMan probes described
herein (see also, U.S. Patent No. 5,538,848), various stem- loop molecular beacons (see, e.g.,
U.S. Patent Nos. 6,103,476 and 5,925,517; Tyagi eta!.. Nature Biotech., 1996, 14:303-308),
stemless or linear beacons (see. e.g., PCT Publication No. WO 99/21881), PNA Molecular
Beacons™ (see, e.g., U.S. Patent Nos. 6,355,421 and 6,593,091), linear PNA beacons (see,
e.g., Kubista et al, 2001, SP E 4264:53-58), non-FRET probes (see, e.g., U.S. Patent No.
'® '
6,150,097). Sunrise /AmplifSuor probes (see, e.g., U.S. Patent No. 6,548,250), stem-loop
and duplex Scorpion™ probes (see, e.g., So nas et al, 200 , Nucl. Acids Res., 29:E96; U.S.
Patent No. 6,589,743), bulge loop probes (see, e.g., U.S. Patent No. 6,590,091), pseudo knot
probes (see, e.g., U.S. Patent No. 6,589,250), cyclicons (see, e.g., U.S. Patent No. 6,383,752),
MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (see, e.g., U.S. Patent No.
6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanopariieie probes,
and ferrocene-modified probes as described, for example in U.S. Patent No. 6,485,90! :
Mbianga / , 2001, Methods, 25:463-471; Whitcombe et al, 99, Nature Biotechnol,
17:804-807; isacsson et a ., 2000, Molecular Cell Probes, 4:32 1-328; Svanvik et al. , 2000,
AnalBiochem., 281 :26-35; Wolffs et , 2001, Biotechniques, 766:769-771 ; Tsourkas et ,
2002, Nucleic Acids Research, 30:4208-4215; Riccelii et al, 2002, Nucleic Acids Research,
:4088-4093; Zhang et al, 2002 Shanghai, 34:329-332; Maxwell etal, 2002, J. Am. Chem.
Soc, 124:9606-9612; Broude et al, 2002, Trends Biotechnol, 20:249-56: Huang et al, 2002,
Chem Res. Toxicol, 15:1 18-126; and Yu et al, 2001, . Am. Chem. Soc, 14:1 1155-1 1161.
Detector probes can comprise reporter dyes such as, for example, 6-carboxyfluorescein (6-
FAM) or tetrachlorofluorescin (TBI). Detector probes can also comprise quencher moieties
such as tetramethylrhodamine (TAMRA), Black Hole Quenchers (Biosearch), Iowa Black
( DT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate
Quenchers (Epoch Biosciences). In some embodiments, detector probes can comprise two
probes, wherein for example a fluor is on one probe, and a quencher is on the other probe,
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?, instead of the
carboxylate group, phosphoramidite forms of fluorescein, phosphoramtdite forms of CY5
(Amersharn Bioscienees-GE Healthcare).
The term "iocus-specific primer" includes an oligonucleotide sequence that
hybridizes to products derived from the extension of a first primer (such as an allele-specific
primer) in a PGR. reaction, and which can effectuate second strand cDNA synthesis of the
product. Accordingly, in some embodiments, the allele-specific primer serves as a forward
PGR primer and the locus -specific primer serves as a reverse PGR primer, or vice versa. In
some preferred embodiments, iocus-specific primers are present at a higher concentration as
compared to the allele-specific primers.
As used herein, the term "rare allelic variant" includes 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 about
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. e.g., less than about 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, 1.0,000, 25,000, 50,000, 75,000, .100,000,
250,000, 500,000, 750,000, or 1,000,000 copies per 1, 10, 100, or 1,000 micro liters of a
sample or a reaction volume.
The term "abundant allelic variant" includes 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
about 10X, 100X, I ,OOOC , 10,000X, 100,000X, I ,OOO ,OOOC , I O ,OOO ,OOOC , IOO ,OOO ,OOOC , or
I ,OOO ,OOO ,OOOC compared to another allelic variant for a given SNP or gene. Alternatively,
the abundant allelic variant can be, for example, greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10,
, 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 , 10, 100, 1,000 micro
liters of a sample or a reaction volume.
In certain embodiments, 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,
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.
As v sed 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., ceils) comprising wild-type alleles, some of which have been mutated to form a
minor or rare allele. In some instances, the first and second allelic variants 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. In certain instances, the first and second allelic variants can be contained on
gD A, as well as mRNA and cD A, and generally any target nucleic acids that exhibit
sequence variability due to, e.g., SNP or nucleotide(s) insertion and/or deletion mutations.
The terms "thermostable or "thermostable polymerase include an enzyme that is
heat stable or heat resistant and catalyzes polymerization of deoxyribonucieotides 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 desiabihzation of single -stranded nucleic acids
or denaturation of double-stranded nucleic acids during PGR 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°-100°C under
conditions such as is typically required for PGR amplification.
As used herein, the terms "PGR amplifying ' or "PGR amplification ' include
cycling polymerase-mediated exponential amplification of nucleic acids employing primers
that hybridize to complementary strands, as described, for example, in Innis el «/., PGR
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. Patent Nos. 5,928,907; 6,015,674; 6,174,670: and
6,814,934 and include, but are not limited to, the AB Prism 7700 Sequence Detection
System (Applied Biosystems, Foster City, CA), the ABI GeneAmp 5700 Sequence
Detection System (Applied Biosystems), the ABI GeneAmp 7300 Sequence Detection
System (Applied Biosystems), the ABI GeneArnp^ 7500 Sequence Detection Syste
(Applied Biosystems), the StepOne™ Real-Time PGR System (Applied Biosystems), and he
ABI GeneAmp* 7900 Sequence Detection System (Applied Biosystems).
The terms "pre-amplification" or "pre-ampHfy" include a process wherein a
plurality of primer pairs are included in multiplexed PGR amplification reaction, and the
multiplexed amplification reaction undergoes a limited number of cycles so that the PCR-
based pre-amplification reaction ends prior to the PGR plateau and/or reagent depletion. The
term "PCR-based pre-amplification" can be considered to indicate that a secondary
amplification reaction is subsequently performed, typically of lower p exy level than the
PCR-based pre-amplification reaction. This secondary amplification reaction, typically a
plurality of separate secondary amplification reactions, can employ primer pairs encoded by
the primers used in the multiplexed PCR-based pre-amplification reaction. However, each
secondary amplification reaction typically comprises a single or a few primer pairs. Further
examples of PCR-based pre-amplification approaches can be found, for example, in U.S.
Patent No. 6,605,451 and in U.S. Application No. 10/723,520, the disclosures of which are
herein incorporated by reference in their entireties for all purposes.
As used herein, the terms "Tin"' or "melting temperature' of an oligonucleotide
include 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
T of a primer or probe can be determined empirically by means of a melting curve. n
some embodiments th T n can also be calculated using formulas well kno in th art (See,
e.g., Maniatis et al, Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor, MY:1982).
As used herein, the term "sensitivity ' includes 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" includes the ability of an assay to distinguish
between amplification from a matched template versus a mismatched template. Frequently,
specificity is expressed as AC; Ch, - C - An improvement in specificity or
f l ,, , l
"specificity improvement" or "fold difference" expressed herein as 2 -
C __ fx! i ¾
The term "selectivity" includes 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 thai can detect 1 mutant template in the presence of 100 wild-type templates is said to
have a selectivity of 1:100 or % . As used herein, assay selectivity can also be calculated as
1/2 or as a percentage using (1/2 x 100)
The term "Ct" or "Ct value' includes the threshold cycle and signifies the cycle of a
PCR amplification assay in which sig al from a reporter that is indicative of amplicon
generation (e.g., fluorescence) f i rs becomes detectable above a background level In some
embodiments, the threshold cycle or "Ct" is the cycle number at which PCR amplification
becomes exponential
As used herein, the term "delta Ct" or "ACt" includes 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. In some embodiments, 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
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
a lele discrimination power.
According to various embodiments, a Ct value may be determined usi g 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 to, s positive inflection of a second derivative,
a negative inflection of a second derivative, a zero crossing of the second derivative, or a
positi ve inflection of a first derivative. In some 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 no limited to, various embodiments of a fit poi t
method, and various embodiments of a sigmoidai method. See, e.g., U.S. Patent Nos.
6,303,305; 6,503,720; 6,783,934, 7,228,237 and U.S. Publication No. 2004/0096819; the
disclosures of which are herein incorporated by reference in their entireties for all purposes.
The term "sample" as used herein includes any biological specimen obtained from a
patient. Samples include, without limitation, whole blood, plasma, serum, red blood cells,
white blood cells (e.g., peripheral blood mononuclear cells), ductal lavage fluid, nipple
aspirate, lymph (e.g., disseminated tumor cells of the lymph node), bone marrow aspirate,
ascites, pleural efflux, saliva, urine, stool (i.e., feces), sputum, bronchial lavage fluid, tears,
fine needle aspirate (FNA) (e.g., harvested by random periareolar fine needle aspiration), any
other bodily fluid, a tissue sample (e.g., tumor tissue) such as a biopsy of a tumor (e.g.,
needle biopsy) or a lymph node (e.g., sentinel lymph node biopsy), a tissue sample (e.g.,
tumor tissue) such as a surgical resection of a tumor, and cellular extracts thereof. In some
embodiments, the sample is whole blood or a fractional component thereof such as plasma,
serum, or a cell pellet. In other embodiments, the sample is obtained by isolating circulating
cells of a solid tumor from whole blood or a cellular fraction thereof using any technique
known in the art. In yet other embodiments, the sample is a formalin fixed paraffin
embedded (FFPE) tumor tissue sample, e.g., from a solid tumor.
The term "subject" or "patient" or "individual" typically includes humans, but can
also include other animals such as, e.g., other primates, rodents, canines, felines, equines,
ovines, porcines, and the like.
III. Description of the Embodiments
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 a ele-spe ifi blocker probe; (c) a detector
probe; (d) a locus-specific primer; and (e) any combinations thereof. In some embodiments,
the compositions may further comprise a polymerase, dNTPs, reagents and/or buffers suitable
for PGR. amplification and/or a template sequence or nucleic acid sample. In some instances,
the polymerase can be thermostable.
in another aspect, the present invention provides compositions for use in identifying
and/or quantitating an allelic variant in a . nucleic acid sample, wherein the compositions can
comprise: (i) 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 and
comprises a nucleic acid modification; and/or (ii) an ailele-specific blocker probe, wherein an
al le-specific nucleotide portion of the ailele-specific blocker probe is complementary to a
second allelic variant of the target sequence and comprises a nucleic acid modification, and
wherein the alleie-specific blocker probe comprises a non -extendable blocker moiety at the 3'
terminus.
In some illustrative embodiments, the compositions can further comprise a locus-
specific primer that is complementary to a region of the target sequence that is ' from the
first allelic variant and on the opposite strand. In yet other embodiments, the compositions
further comprise a detector probe.
I another aspect, the present invention provides methods for amplifying an ailele-
specific sequence. Some of these methods can comprise: (a) hybridizing an ailele-specific
primer to first nucleic acid molecule comprising a target allele; (b) hybridizing an ailele-
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 alleie-specific primer; and (e) PGR amplifying
the target allele. In particular embodiments, the alleie-specific blocker probe comprises a
non-extendable blocker moiety at the 3' terminus. In other particular embodiments, both the
alleie-specific primer and the ailele-specific blocker probe independently comprise a nucleic
acid modification such as, for example, a modified base (e.g., LNA, PNA, TNA, ZNA and
TzDNA), a nucleic acid analog, or a ribose-modified nucleic acid, at the position of the target
allele and the alternative allele, respectively.
A. LNA, PNA, TNA, ZNA or TzNA Modifications of Oligonucleotides for
Primers and/or Probes
In one aspect, the present invention provides oligonucleotide compositions, wherein
the oligonucleotides comprise at least one nucleic acid modification and/or a non-extendable
blocker moiety at the 3' terminus.
Non-limiting examples of nucleic acid modifications include locked nucleic acid
(LNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), zip nucleic acid (ZNA),
triazole nucleic acid (TzNA), and combinations thereof.
In preferred embodiments, the present invention comprises an oligonucleotide
comprising at least one locked nucleic acid (LNA). LNA includes a class of nucleic acid
analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'-0
atom and the 4'-C atom. LNA nucleosides contain the common nucleobases (T, C, G, A, U,
and mC) and are able to form base pairs according to standard Watson-Crick base pairing
rules. When incorporated into a DNA oligonucleotide, LNA makes the pairing with a
complementary nucleotide strand more rapid and increases the stability of the resulting
duplex. The affinity-enhancing effect of incorporation of LNA monomers into an
oligonucleotide is demonstrated by an increase in the duplex melting temperature of 2-8°C
per LNA monomer. In some instances, LNA refers to modifications of LNA, such as, but not
limited to oxy-LNA, thio-LNA, and amino-LNA. See, e.g., Johnson el al, Nucl. Acid Res.,
2004, 32, e55; Latorra el al. Hum. Mu , 2003, 22, 79; Chou t al, Biotech., 2005, 39, 644.
In some embodiments, the present invention comprises an oligonucleotide
comprising at least one peptide nucleic acid (PNA). PNA is a non-naturally occurring and
artificially synthesized nucleic acid analog or mimic comprising various naturally-occurring
or non-naturally-occurring nucleobases attached to a backbone of repeating N-(2-
aminoethyl)-glycine units linked by amide bonds. It is appreciated by those skilled in the art
that a PNA-DNA duplex binds with greater strength, higher stability, more quickly and with
more specificity compared to an analogous DNA-DNA duplex, due to the lack of electrostatic
repulsion between the PNA strand and DNA strand. The greater stability is reflected by a
higher Tm for the PNA-DNA duplex versus the analogous DNA-DNA duplex. PNA
complexes are more thermally stable and less susceptible to degradation by nucleases,
proteases and peptidases. It has been shown that the Tm of PNA-DNA duplexes is in part
independent of salt concentration. In addition, it is more likely that single base mismatches
can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA
-mer lowers the melting point (Tm) by 8°-20°C vs. 4°-16°C for the DNA/DNA 15-mer
duplex. This has the effect of improving the discrimination between matched and
mismatched sequences. See, e.g., Nielsen, P.E. and Egholm, M., Current Issues Molec. Biol.
1; 89-104 (1999); Orum et al. "Peptide Nucleic Acid". Laboratory Methods for the Detection
of Mutations and Polymorphisms in DNA ed. Graham R. Taylor. CRC Press, 1997; Nielsen,
P.E. and Egholm, M., Current Issues Molec. Biol. 1; 89-104 (1999); Gaylord et al, Proc.
Natl. Acad. Set, 102: 34-39 (2005).
PNA can specifically block primer annealing and chain elongation on a perfectly
matched template without interfering with reactions on templates with mismatched bases.
PNA can be used to improve mutation detection by suppressing wild-type allele amplification
in SNP analysis such as, but not limited to asymmetric PCR clamping, melting curve analysis
(see, e.g., Oh et al. J. Mol. Diagn., 12: 418-424 (2010); Orum et al, Nucleic Acids Res, 21:
5332-5336 (1993); Luo et al., Nucleic Acids Res, 34:el2 (2006); Karkare et al, Appl.
Microbiol. Biotechnol., 71:575-586 (2006)).
In some embodiments, the present invention can comprise an oligonucleotide
comprising at least one zip nucleic acid (ZNA). ZNA is an oligonucleotide conjugated with
one or a plurality of cationic spermine moieties. This structure decreases electrostatic
repulsion with its target nucleic acid strand and increases the affinity of the oligonucleotide
for its targets. The number of cationic units attached at any position of the oligonucleotide
can modulate the global charge of the molecule, which can raise the corresponding Tm of a
ZNA duplex (e.g., ZNA-ZNA duplex, ZNA-DNA duplex, and ZNA-RNA duplex) in a linear
and predictable manner. They are efficient at low magnesium concentration and at high
annealing temperatures, which can be advantageous for accurate detection of allelic variants.
ZNAs can be single-labeled or dual-labeled with fluorescent moieties and fluorescent
quenchers. ZNAs are commercially available from e.g., Sigma-Aldrich. See, e.g., Voirin et
al, Nat. Protoc, 2:1360-1367 (2007), Noir et al, J. Am. Chem. Soc, 130 ;13500-13505
(2008), Moreau et al, Nucleic Acids Res., 37: el30 (2009); Paris et al, Nucleic Acids Res.,
38: e95 (2010).
In other embodiments, the present invention comprises an oligonucleotide
comprising at least one triazole nucleic acid (TzNA). TzNA oligonucleotides can be
synthesized using click chemistry (e.g., copper-catalyzed azide-alkyne cycloaddition
reaction). Oligonucleotides containing AZT -based triazole linkages can be used as PCR
templates with a variety of polymerases for amplification (El-Sagheer et al., J. Am. Chem.
Soc, 131: 3958-3964 (2009)). Genes containing trizole linker can also be functional in
Escherichia coli (El-Sagheer et al., Proc. Natl. Acad. Scl, 108: 11338-1 1343 (201 1)). See,
e.g., Isobe et al, Org. Lett., 10: 3729-3732 (2008); Fujino et al, Tetrahedron Lett., 50: 4101-
4103 (2009); von Matt et al, Bioorg. Med. Chem. Letts., 7 : 1553-1556 (1997).
In yet other embodiments, the present invention comprises an oligonucleotide
comprising at least one threose nucleic acid (TNA). TNAs have a repeat unit one atom
shorter than natural nucleic acids, yet they can base pair with DNA, RNA, and itself. While
not wanting to be bound by a particular theory, it is believed that TNA hybridizes strongly
with DNA and even more strongly with RNA because TNA is a good mimic of the A-form of
DNA and of RNA. The increased stability of TNA-DNA duplexes compared to analogous
DNA-DNA complexes results in improved mismatch discrimination of allelic variants. See,
e.g., Ichida et al, Nucleic Acids Res., 33: 5219-5225 (2005).
Modified bases are considered to be those that differ from the naturally-occurring
bases by addition or deletion of one or more functional groups, differences in the heterocyclic
ring structure (i.e., substitution of carbon for a heteroatom, or vice versa), and/or attachment
of one or more linker arm structures to the base. In some embodiments, all tautomeric forms
of naturally-occurring bases, modified bases and base analogues ay also be included in the
oligonucleotide primers and probes of the invention.
in further embodiments modified sugars or sugar analogs can be present in one or
more of the nucleotide subunits of an oligonucleotide in accordance with the invention. Sugar
modifications include, but are not limited to, attachment o bstit ents to the 2', 3' and/or 4
carbon atom of the sugar, different epimeric forms of the sugar, differences in the cx- or b -
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.
In certain embodiments, one or more modified intenmcleotide or backbone linkages
can be present in the oligonucleotides of the present invention. Such modified linkages
include, but are not limited to, peptide, phosphate, phosphodiester, phosphotriester,
alkylphosphate, alkanephosphonate, thiophosphate, phosphorothioate, phosphorodithioate,
m tbylphosphonate, pbosphoramidate, substituted phosphoramidate, and the like. Additional
modifications of bases, sugars and/or internucleotide linkages that are compatible wit their
use in oligonucleotides serving as probes and/or primers, will be apparent to those of skill in
the art.
The non-extendable blocker moiety can comprise any modification of t e ribose
ring 3' -OH of the blocker probe which prevents addition of further bases to the 3' -end of the
oligonucleotide sequence by a polymerase. n some embodiments, the blocker moiety can
include, without limitation, a optionally substituted I- a ky dio (e.g., a 3'-hexanediol
modification) an optionally substituted C2-C24 alkenyl diol, an optionally substituted C2-C24
alkynyl diol, a minor groove binder (MGB , an amine ( H2), biotin, PEG, PO4, and mixtures
thereof. In particular embodiments, the optionally substituted Ci-C alkyl diol comprises a
methanediol, etbanediol, ,3-propanedio , l ,4~butanediol, ,5-pentanedioi, 1,6-hexanedioi,
1,7-heptanediol, or 1,8-octanediol modification to the 3'-end of the allele-specific blocker
probe. In some embodiments, the non-extendable blocker moiety comprises an optionally
substituted C1-C20, C -C , C -C , C ~C , C -Ci , C4-C10, C -C , , C . C , , C , C , C ,
2 2 20 2
Cs, Co, , j , or C alkyl diol. In other embodiments, the -extendable blocker moiety
comprises an optionally substituted C , C -C- , C.r C . - -C3, C C , C , C5, Ce,
C , Cg, , , C , or C12 alkenyl diol or alkynyl dio .
In certain embodiments, the non-extendable blocker moiety does not comprise or
include a minor groove binder (MGB) and/or a P0 group. In certain other embodiments, the
non-extendable blocker moiety consists essentially of or consists of an optionally substituted
; . : alkyl diol (e.g., a met anedio ethanedioi, 1,3 -propanediol, 1,4-butanediol, 1,5-
pentanedioL ,6- exanedio i ,7 hepta edioi, or L8-octanediol modification, or an optionally
substituted C1-C20, C -€i , C -C , d -, C -C -C10, C -€ , , C C , C , C , C , C
Cg, , , , or C ; ai yi diol modification), an optionally substituted C C ?, alkenyl diol
or an optionally substituted C -C alkynyl diol (e.g., an optionally substituted C2-C20, C2-C12,
C 4-C 2 -Cio, C2, C C7, Cs, C j , C j , or C alkenyl or alkynyl diol
C C 4 , C4, C5, C9,
modif i cation) or mixtures thereof.
The term "optionally substituted" includes the replacement of at least one hydrogen
atom with a substituent. In the case of an "oxo" substituent (=0), two hydrogen atoms are
replaced. Non-limiting examples of substituents include oxo, halogen, heterocycle, -CN,
x x y x y x y x x x y x
-OR , -NR R , -NR C(=0)R , -NR S0 R , -C(=0)R , -C(=0)OR , -C(=0)NR R , -SO„R ,
x y x y
and -SO NR R , wherein n is 0, 1, or 2, wherein R and R are the same or different and are
independently hydrogen, alkyl, or heterocycle, and wherein each of the alkyl and heterocycle
substituents may be further substituted with one or more of the substituents described herein.
The term "optionally substituted," when used before a list of substituents, means that each of
the substituents in the list may be optionally substituted as described herein.
In certain embodiments, the allele-specific nucleotide portion of the allele-specific
blocker probe is located from about 5 to about 15 or from about 5 to about 10, such as about
, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the blocker moiety of the allele-
specific blocker probe. In certain other instances, the allele-specific blocker probe is not
cleaved during PCR amplification. In further instances, the Tm of the allele-specific blocker
probe ranges from about 58°C to about 66°C.
In some embodiments, the allele-specific blocker probe and/or allele-specific primer
comprises at least about 1, 2, 3 4, 5, or 6 (e.g., 2to 6) nucleic acid modifications. In certain
instances, the one or more modifications may increase the difference in he I m between
matched and mismatched target sequences and/or decrease mismatch priming efficiency,
thereby improving assay specificity and/or selectivity. In certain other instances, the one or
more modifications improve allelic discrimination of samples of circulating tumor cells.
Non-limiting examples of such modifications include locked nucleic acid (LNA), peptide
nucleic acid (PNA), threose nucleic acid (TNA), zip nucleic acid (ZNA), triazole nucleic acid
zNA), 5'me yl-deoxyc>t idine. 2'-fluoro-modified nucleic acid, 8-azadeaza-dA (ppA),
8-aza.-deaza-dG (ppG), lH--pyrazolo[4,4-d] pyrimidin-4(5H)--6(7H)--dione (ppX), 2'-
deoxypseudoisocytidine (iso dC), 5-f oro- ' -deoxyuridine (fdU), and 2' -0,4' -C-ethylene
bridged nucleic acid (ENA) modifications, and combinations of these modifications. In
certain embodiments, the LNA modifications present on the alleie-spceifie blocker probe
and/or ailele-specific primer are non-consecutive or non-contiguous, such that two LNA
bases are not next to each other in the sequence.
In preferred embodiments, the nucleic acid modification th a is present o the ailele-
specific blocker probe and/or ailele-specific primer comprises one or more LNA nucleotides.
In certain embodiments, the modification is located (a) at the '-end, (b) at the '-end, (c) at
an internal position, or at any combination of (a), (b) or (c) within the ailele-specific blocker
probe and/or the ailele-specific primer. In some preferred embodiments, one modification
(e.g., LNA) is located at the ailele-specific nucleotide portion of the ailele-specific primer,
such that this modification comprises the nucleobase used to discriminate between allelic
variants. In other preferred embodiments, one modification (e.g.. LNA) is located at the
alleie-specific nucleotide portion of the ailele-specific blocker probe, such that this
modification comprises the nucleobase used to discriminate between allelic variants. In yet
other preferred embodiments, the nucleic acid modifications (e.g., LNA) are not placed in
consecutive or contiguous positions of the alleie-specific primer and/or blocker probe.
in some embodiments, the nu eic acid modifications present on the alleie-specific
blocker probe and/or ailele-specific primer independently comprise one or more (at least L 2,
3, 4, 5, 6, 7, 8, 9, 10. , 12, 13, 4, . or all) PNA, ZNA, TNA, and/or TzDNA nucleotides.
Other examples of nucleic acid modifications that can be used in the invention are
described, e.g., in U.S. Patent No. 7,5 ,978, the disclosure of which is incorporated herein
by reference in its entirety for a l purposes.
Many modified nucleic acid moieties, including, for example, LNA, PNA, ZNA,
TNA, TzDNA, ppA, ppG, and 5-Fluoro-dU (fdU), are commercially available and ca be
used in oligonucleotide synthesis methods well known in the art. in some embodiments,
synthesis of modified primers and probes can he carried out using standard chemica l means
also well known in the a t For example, the modified moiety or base can be introduced by
use of (a) a modified nucleoside as a DNA synthesis support, (b) a modified nucleoside as a
phosphoramidite, (c) reagent during DMA synthesis (e.g., benzylamine treatment of a
convertible amidite when incorporated i to a D A sequence), or (d) by post-synthetic
odification
In addition, in some embodiments, the nucleotide units which are incorporated into
the oligonucleotides of the allele-specific primers and/or allele-specific blocker probes of the
present invention may have a cross-linking function (an alkylating agent) covalently bound to
one or more of the bases, e.g., through a linking arm.
in yet another aspect, the present invention provides methods for detecting and/or
quantitating an allelic variant in a mixed sample. Some of these methods can comprise: (a)
hybridizing a first allele-specific primer to a firs nucleic ac d molecule comprising a first
allele (allele- ) in a first reaction mixture and hybridizing a second allele-specific primer to a
first nucleic acid molecule comprising a second allele (a e e-2) in a second reaction mixture,
wherein allele-2 corresponds to the same loci as allele- : (b) hybridizing a first allele-specific
blocker probe to a second nucleic acid molecule comprising a ie-2 in the first reaction
mixture and hybridizing a second allele-specific blocker probe to a second nucleic acid
molecule comprising allele- in the second reaction mixture; (c) hybridizing a first detector
probe to the first nucleic acid molecule in the first reaction mixture and hybridizing a second
detector probe to the first nucleic acid molecule in the second reaction mixture; (d)
hybridizing a first locus -specific primer to the extension product of the first allele-specific
primer in the first reaction mixture and hybridizing a second locus-specific primer to the
extension product of the second allele-specific primer in the second reaction mixture; (e)
PGR amplifying the first nucleic acid molecule to form a first set or sample of amplicons and
PGR 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- in the sample comprising allele-2 and/or allele-2 in the sample comprising
allele- .
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. Non-limiting examples of such methods include
forming a reaction mixture by combining one or more of the following components: i) a
nucleic acid sample; (ii) an allele-specific primer, wherein an allele-specific nucleotide
portion of the allele-specific primer is complementary to the first allelic variant of the target
sequence and comprises a nucleic acid modification as described herein; ( i) an allele-
specific blocker probe that is complementary to a region of the target sequence comprising
the second allelic varia t wherein the region encompasses a position corresponding to the
binding position of the allele-specific nucleotide portion of the allele-specific primer, and
wherein the alleie-specific blocker probe comprises a non-extendable blocking moiety and a
nucleic acid modification as described herein; (iv) a locus-specific primer that is
complementary to a region of he target sequence that is 3' from the first allelic variant and
that is on the opposite strand; and/or (v) a detector probe. In certain instances, the first allelic
variant comprises a mutant allele and the second allelic variant comprises the wild-type
allele.
Next an amplification reaction, typically a PGR amplification reaction, is carried
out on the reaction mixture using the locus-specific primer and the allele-specific primer to
form an amplieon. Then, the amplieon is detected by a change in a detectable property of the
detector probe upon binding to the amplieon, thereby detecting the first allelic variant of the
targe gene in fee nucleic acid sample. The detector probe in illustrative embodiments is a 5'
nuclease probe and the detectable property in illustrative embodiments is fluorescence.
In some embodiments, the 3' nucleotide position of the 5' target region of the allele-
specific primer is an allele-specific nucleotide position. In other embodiments, the allele-
specific nucleotide portion of the allele-specific blocker probe is located in the center of the
allele-specific blocker probe.
In certain embodiments, the quantity of the first allelic variant is determined by
evaluating a change in a detectable property of the detector probe.
In some embodiments, the methods of the invention for detecting an allelic variant
in a target sequence in a nucleic acid sample comprises the following cycling protocol:
(a) forming a reaction mixture comprising one or more of the following components:
(i) nucleic acid sample;
(ii) an allele-specific primer, wherein an allele-specific nucleotide portion of the
allele-specific primer is complementary to a first allelic variant of the target sequence and
comprises a . nucleic acid modification at the location of the first allelic variant as described
herein;
ii) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising a second allelic variant, wherein the region encompasses a position
corresponding to the binding position of the allele-specific nucleotide portion of the allele-
specific primer, and wherein the allele-specific blocker probe comprises a blocking moiety
and a nucleic acid modification at the location of the second allelic variant as described
herein;
(iv) a locus-specific primer that is complementary to a region of the target sequence
tha is 3' from the first allelic variant and on the opposite strand; and
(v) a detector probe:
(b) PGR amplifying the target sequence using a cycling protocol comprising a number of
cycles run at an annealing/exteasion temperature; and
(c) detecting a change in a detectable property of the detector probe in the amplified
products of the target sequence produced by step (b).
There are several major advantages of the methods of the present invention. First
the genotyping assays described herein improve the detection sensitivity by lowering the Ct
value for matched targets or alleles. Next, the genotyping assays described herein improve
specificity by increasing the Ct between Ct values of matched and mismatched sequences.
In addition, the genotyping assays described herein improve uniformity of efficiency across
various assays.
In other embodiments, the methods of the present invention may include a 2-stage
cycling protocol. In some embodiments, the methods for detecting an allelic variant In a
target sequence in a nucleic acid sample comprises:
(a) forming reaction mixture comprising one or more of the following components:
(i) a nucleic acid sample;
(ii) an allele-specific primer, wherein an allele-specific nucleotide portion of the
allele-specific primer is complementary to a first allelic variant of the target sequence and
comprises a nucleic acid modification at the location of the first allelic variant as described
herein;
(iii) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising a second allelic variant, wherein the region encompasses a position
corresponding to the binding position of the allele-specific nucleotide portion of the allele-
specific primer, and wherein the allele-specific blocker probe comprises a blocking moiety
and a nucleic acid modification at the location of the second allelic variant as described
herein;
(iv) a locus-specific primer that is complementary to a region of the target sequence
that is 3' from the first allelic variant and on the opposite strand: and
(v) a detector probe;
(b) PCR amplifying the target sequence using a 2-stage cycling protocol comprising:
(i) a first amplification step comprising a first number of cycles run at a first
annealing/extension temperature; and
(ii) a second amplification step comprising a second number of cycles run at a second
annealing/extension temperature; and
(c) delecting a change in detectable property of the detector probe in he amplified
products of the target sequence produced by step (b).
In some instances, the first number of cycles in step (b) is fewer than the second
number of cycles and the first annealing/extension temperature is lower than the second
annealing/extension temperature. In some embodiments, the number of cycles used in the
first stage of the cycling protocol is about 2 -20 . 4%-l8%, 6%-16%, 8%-l4%, 0%-12%,
or any percent n between , of the total number of cycles used in the second stage. n other
embodiments, the first stage employs between about 1 to 10 cycles, 2 to 8 cycles, 3 to 7
cycles, 4 to 6 cycles, or any number of cycles in between, e.g., 2, 3, 4, 5, 6, or 7 cycles.
In some embodiments, the number of cycles used in the second stage of the cycling
protocol is about 5 times, 6 times, 8 times, 0 times, times, 8 times, 25 times, 30 times,
times, 40 times, 45 times, 50 times the number of cycles used in the first stage. In some
embodiments, the second stage employs between about 30 to 50 cycles, 35 to 48 cycles, 40 to
46 cycles, or any number of cycles in between, e.g.. 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45,
or 46 cycles.
In some embodiments, the lower annealing/extension temperature used during the
first cycling stage is about 1°C, about 2°C, about 3°C, about 4°C, or about 5°C lower than the
annealing/extension temperature used during the second cycling stage. In some instances, the
annealing/extension temperature of the first stage is between about 50°C to 60°C, 52°C to
58°C, or 54°C to 56°C, e.g., 53°C, 54°C, 55°C, or 55°C. In certain other embodiments, the
annealing/extension temperature of the second stage is between about 56°C to 66°C, 58°C to
64°C, or 60°C to 62°C, e.g., 58 C, 60 C, 62 C, or 64°C.
In another aspect, the present invention provides a reaction mixture that comprises
the following components: (i) a nucleic acid molecule; (ii) an allele-specific primer, wherein
an allele-specific nucleotide portion of l re allele-specific primer is complementary to a first
allelic variant of a target sequence and comprises a nucleic acid modification; (iii) an allele-
specific blocker probe, wherein an allele-specific nucleotide portion of the allele-specific
blocker probe is complementary to a second allelic variant of the target sequence and
comprises a nucleic acid modification, and wherein the allele-specific blocker probe
comprises a blocker moiety at the 3'-end of the oligonucleotide sequence; (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/or (v) a . detector probe.
n some embodiments, the methods of the present invention are used to detect a first
allelic variant that is present at a frequency of less than about 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 i / ,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 of the present invention are used to detect a first allelic variant that is present in
less than about 2, 3, 4, 5 6, 7, 8, 9, 10, 15, 20 25, 30, 35, 40, 45, 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 10, 300, 500, or 1,000 micro liters, and any fractional
ranges in between, of a sample or a reaction volume.
n certain embodiments, the first allelic variant is a mutant allele and the second
allelic variant is a wild-type allele. In some embodiments, the present methods can involve
detecting one mutant molecule in a background of at least about 1,000 to 1,000,000, such as
about L O O 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 efficiency that is at least comparable to TaqMan^-based assays.
I another aspect, the present invention provides kits for quantitating a first allelic
variant in a sample comprising an alternative second allelic variant that include: (a) an ailele -
speciiic primer; (b) an ailele-speeific blocker probe: (c) a locus-specific primer: (d) a detector
probe; and/or (e) a polymerase.
n yet another aspect, the present invention provides kits comprising two or more
containers comprising the following components independently distributed in one of the two
or more containers: (i) 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
and comprises a nucleic acid modification as described herein; and (ii) an allele-specific
blocker probe that is complementary to a region of the target sequence comprising a second
allelic variant, wherein the region encompasses a position corresponding to th binding
position of the ailele-speeific nucleotide portion of the allele-specific primer, and wherein the
allele-specific blocker probe comprises a non-extendable blocker moiety and a nucleic acid
modification as described herein. n particular embodiments, the allele-specific blocker
probe comprises an allele-specific nucleotide portion tha is complementary to a second
allelic variant of the target sequence, wherein the allele-specific nucleotide portion comprises
a nucleic acid modification, and wherein t allele-specific blocker probe comprises a
blocker moiety at the '-end of the oligonucleotide sequence.
In some embodiments, the kits can further comprise a locu -specific primer tha is
complementary to a region of the target sequence that is 3' from the first allelic variant and
that is on the opposite strand. In other embodiments the kits can further comprise a detector
probe. In yet other embodiments, the kits can further comprise additional components used
for pre-ampiification.
In some embodiments, the compositions, methods, and/or kits of the invention are
useful for detecting tumor cells in samples such as blood or fine needle aspirates (FNA) for
early cancer diagnosis. n other embodiments, the compositions, methods and/or kits of the
invention are useful for cancer or disease-associated genetic variation or somatic mutation
detection and validation. In yet other embodiments, the compositions, methods, and/or kits
can be used for genotyping di-ailelic, tri-a e ic or tetra-alielic SNPs. In other embodiments,
the compositions, methods, and/or kits of the invention can be used for identifying single or
multiple nucleotide insertion or deletion mutations. In some embodiments, the compositions,
methods, and/or kits of the invention can be used or DNA typing from mixed DMA samples
for QC and human identification assays, ceil line Q for ceil 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.
B. Allele-Specific Primers
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°C to 70°C, such as about 52°C to 68°C, about 54°C to 66°C, about 56°C
to 64°C, about 58°C to 62°C, or any temperature in between (e.g., 53°C, 54°C, 55°C, 56°C).
In other embodiments, the m of the allele-specific primers is about 3°C to 6°C higher than
the anneal/extend temperature of the PG cycling conditions employed during amplification.
In certain instances, allele-specific primers designed with low Tm's increase discrimination
of allelic variants.
In some embodiments of the invention, ow allele- specific primer concentration
improves selectivity in certain instances, a reduction in concentration of allele-specific
primers below 900 M increases the delta Ct between matched and mismatched sequences.
some embodiments, the concentration of allele-specifie primers ranges from about 20 nM
to 900 nM, such as about 50 nM to 700 nM, about 00 nM to 500 nM. about 200 nM to 400
nM, about 200 nM to 300 nM, about 400 nM to 500 nM, or any range in between.
In some embodiments, the allele-specifie primers of the invention can comprise an
allele-specifie nucleotide portion that is specific to the target allele of interest. The allele-
specifie nucleotide portion of an allele-specifie primer is complementary to one allele of a
gene, but not another allele of the gene. In other words, the al e e speeif e nucleotide portion
binds to one or more variable nucleotide positions of a gene that are 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-specifie nucleotide portion is one nucleotide in length. In some embodiments, the
allele-specifie nucleotide portion of an allele-specifie primer is located at the 3 terminus of
the allele-specifie primer. In other embodiments, the allele-specifie nucleotide portion is
located about 1-2, 3-4, 5-6, 7-8, 9- 1, - 5, or 16-20 nucleotides in from the 3' most-end of
the allele-specifie primer.
Allele-specifie primers designed to target discriminating bases can also improve
discrimination of allelic variants. In so e embodiments, the nucleotide of the allele-specifie
nucleotide portion targets a highly d cri nating 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-specifie nucleotide
portion of the allele-specifie primer (e.g., if A or T is the major allele), or C or T may be used
as the 3' allele-specifie nucleotide portion of the allele-specifie primer (e.g., if C or G is the
major allele). In other embodiments, A may be used as the nucleotide-specifiic portion at the
3' end of the allele specific primer (e.g., the allele-specifie nucleotide portion) when detecting
and/or quantifying A/T SNPs. In yet 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.
n some embodiments, the allele-specifie primer can comprise a target-specific
portion that is specific to the polynucleotide sequence (or locus) of interest. In other
embodiments, the target-specific portion is about 75-85%, 85-95%, 95-99%, or 00%
complementary to t target polynucleotide sequence of interest. In some embodiments the
target-specific portion of the allele-specifie primer can comprise the allele-specifie nucleotide
portion. n other embodiments, the target-specific portion is located 5 to the allele-specifie
nucleotide portion. The target-specific portion can be about 4-30, about 5-25, about 6-20,
about 7- , or about 8-10 nucleotides in length. In some embodiments, the Trn of the target
specific portion is about 5°C below the anneal/extend temperature used for PGR cycling. In
some embodiments, the m of the target specific portion of the ailele-specific primer ranges
from about 53°C to 60°C, about 52°C to 59°C, about 53°C to 58°C, about 54°C to 57°C,
about 55°C to 56°C, or about 50°C to about 60°C.
n embodiments where two ailele-specific primers are used, the target- specific
portion of the first ailele-specific primer and the target-specific portion of the second ailele-
specific primer comprise the same sequence or are the same sequence.
In some embodiments, the ailele-specific primer can comprise one or ore modified
nucieobases or nucleosidic bases different from the naturally occurring bases (i.e., adenine,
cytosinc, guanine, thymine and uracil). In some embodiments, the modified bases are still
abb to effectively hybridize to nucleic acid units that contain adenine, guanine, cytosine,
uracil or thymine moieties. In some embodiments, the modified base(s) may increase the
difference in the T between matched and mismatched target sequences and/or decrease
mismatch priming efficiency, thereby improving assay specificity, selectivity and
reproducibility. In so e embodiments, the modified base(s) may increase the binding
affinity of the ailele-specific primer towards its complementary DNA target.
In particular embodiments, the ailele-specific primer comprises at least one, two, or
more modified bases. Examples of modified bases include, without limitation, locked nucleic
acid (LNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), zip nucleic acid (ZNA)
and triazole DNA (TzDNA), 8-azadeaza-dA (ppA), 8-azadeaza-dG (ppG), 2'-
deoxypseudoisocytidine (iso dC), 5--fluoro-2"- deoxyuridine (fdU), 2'··0,4' -C-ethylene
bridged nucleic acid (ENA) bases, and combinations thereof.
In some embodiments, the ailele-specific primer comprises 2 to 6 LNAs, PNAs,
TNAs, Z s or ribose modified nucleic acids. These modified bases exhibit thermal
stability towards complementary DNA and RNA, which allows for excellent mismatch
discrimination methods of the present invention. The high binding affinity of these
modified bases can be used in hybridization assays that require high specificity, selectivity
and/or reproducibility.
In some embodiments, the modified base present on the ailele-specific primer
comprises one or more LNA modifications. In these embodiments, LNA modifications are
not placed in consecutive positions on the ailele-specific primer. In certain embodiments, the
modified base is located (a) at the 3'-end, (b at the '-end, (c) a an internal position, or at
any combination of (a), (b) or (c) within the alleie- pe i primer. In some embodiments, the
modified base (e.g., LNA nucleoside) is located at the aUele-specific nucleotide portion of the
alleie-specific primer, such that the LNA nucleoside comprises the nucleobase used to
discriminate between allelic variants.
In preferred embodiments, a LNA modification is located at the 3' end of the aliele-
specific primer. With the LNA at. the allelic variant at the 3 end, the melting temperature
(Tin) of the alleie-speeifie primer increases, thereby enhancing the selectivity of the assay of
the present invention towards the allelic variant n addition, selective amplification of the
allelic variant can occur at a higher amplification temperature during PGR cycling. In some
instances, the presence of the LNA at the 3'-end may also help slow down the 3' fi 5'
proofreading exonuclease activity of DNA polymerase. In other instances, the presence of
the LNA at the penultimate positions may provide protection against 3'fi 5' exonuclease
activity of DNA polymerase (see, e.g., Giusto, D and King, G, Nucleic Acids Res., 32:3, e 32,
1-8 (2004)).
In some embodiments, a plurality of LNAs are present in the primer and are spaced
anywhere between 5 to 10 bases from the 3'-end. In other embodiments, the primer can
contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 LNAs spaced anywhere between 2, 3, 4, 5, 6, 7, 8, 9,
or 10 bases from the 3'-end. In yet other embodiments, a LNA modification is positioned at
the penultimate position relative to the 3' end of the primer, at the 3'-end, at the 5'-end, and
combinations thereof. n these embodiments, LNA modifications are typically not placed in
consecutive positions of the primer sequence.
LNA modifications in the oligonucleotide increases the binding affinity towards the
complementary DNA target. The higher affinity is affiliated to the reduced conformational
flexibility of the locked 3'-endo conformation of the ribose. Like DNA bases, LNA bases are
linked in by the same phosphate backbone allowing the LNA-DNA primer to bind efficiently
to its complementary D , thus resulting in a higher I m of the duplex. The discriminatory
effect dependents on the ability of primer binding to the genomic DNA and staying bound
during PG cycling. This is achieved due to the high binding affinity of the LNA-DNA
primer. The higher Tm of the LNA-DNA duplex correlates to its better binding affinity.
AUele-specific primers with LNA allow for higher reaction temperatures during PGR cycling,
which enhances the specificity of the genotyping assay, compared to non-LNA primers with a
lower T .
In some embodiments, the allele-specific primer, blocker probe and/or detector
probe comprises PNA modifications. PNA modifications on an allele-specific primer offers
high specificity and sensitivity to its DNA target. The higher binding affinity and higher Tm
of PNA offers high specificity and reproducibility to the methods described herein.
It is appreciated by those skilled in the art that a PNA-DNA duplex binds with
greater strength, higher stability, more quickly and more specificity compared to a DNA-
DNA duplex, due to the lack of electrostatic repulsion between a PNA strand and DNA
strand. The greater stability is reflected by a higher Tm for a PNA-duplex versus the
analogous DNA-DNA duplex. PNA complexes are more thermally stable and less
susceptible to degradation by nucleases, proteases and peptidases. It has been shown that the
Tm of PNA-DNA duplexes is in part independent of salt concentration. In some
embodiments, an allele-specific primer and/or blocker probe with a PNA can bind its nucleic
acid target in the absence of salt despite the presence of secondary structure. See, e.g.,
Nielsen, P.E. and Egholm, M., Current Issues Molec. Biol. 1; 89-104 (1999); Gaylord et ah,
Proc. Natl. Acad. Set, 102: 34-39 (2005)), the disclosures of each of which are incorporated
herein by reference in their entireties for a l purposes.
In some embodiments, the allele-specific primer comprises one or a plurality of zip
nucleic acid (ZNA) molecules. A ZNA primer has increased affinity for its complementary
target DNA due to decreased electrostatic repulsion between the nucleic acid strands. The
presence ofone or a plurality of ZNA modifications on the allele-specific primer can increase
the Tm of the ZNA-DNA duplex and improve allelic discrimination in methods of the present
invention.
In some embodiments, the alleie-specific primer comprises one or a plurality of (3'-
2') a -L-threose nucleic acids (TNAs). TNA can hybridize strongly with DNA and is a good
mimic of the A-form of DNA and of RNA. Due to the increased stability of a TNA-D A
duplex compared to analogous DNA-DNA complexes, using a TNA aliele-speeific primer in
methods of the present invention can improve mismatch discrimination of allelic variants.
In other embodiments, the allele-specific primer comprises one or a plurality of
triazole linked DNA (TzDNA) molecules. Since TzDNA has increase thermal stability
towards matched itsDNA target compared to analogous DNA primer, TzDNA allele-specific
primers can be used in methods of the present invention for improved allelic variant
discrimination.
n some embodiments, the a!leie-specific primer comprises a ta some instances,
al le- pec f i c primers comprising tails enable the overall length of the primer to be reduced,
thereby lowering the Tm without significant impact on assay sensitivity. In some instances,
the- tail is on the 5 terminus of the aliele-specific primer. In other instances, the tai is
located 5' of the target-specific portion and/or ailele-specific nucleotide portion of the aliele-
specific primer. In some embodiments, the tai 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 tai can be about 2-40, such as about 4-30, about 5-25,
about 6-20, about 7- , or about 8-10 nucleotides in length. In some embodiments, th tai 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.
The tail of the aliele-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. As a non-limiting example, the tail of the aliele-specific primer can hybridize to the
complementary sequence in an extension product resulting from extension of a locus-specific
primer.
In embodiments where two aliele-specific primers are used, the tail of the first
a!leie-speeific primer and the tail of the second ailele-specific primer comprise the same
sequence or are the same sequence.
C. Allele-Specific Blocker Probes
In some embodiments, the ailele-specific blocker probe of the present invention is
specifically designed to hybridize to the a first allele and inhibit the amplification of the first
allele efficiently and selectively, without affecting the amplification of the second variant. In
some aspects of the present invention, the blocker probe is specifically designed to hybridize
to the wild-type allele and inhibit its amplification, without affecting the amplification of the
mutant allele.
Ailele-specific blocker probes may be designed as short oligomers that are single-
stranded and have a length of about 100 nucleotides or less, about 50 nucleotides or less,
about 30 nucleotides or less, about 20 nucleotides or less, or about 5-20 nucleotides
n some embodiments, the Tm of the blocker probes range from 58°C to 70°C, 6 1 C
to 69°C, 62°C to 68°C, 63°C to 67°C, 64°C to 66°C, or about 60°C to about 63°C, or any
range in between. n yet other embodiments, he T of the alleie-specific blocker probes is
about 3°C to 6 C higher than the anneal/extend temperature in the PGR cycling conditions
employed during amplification.
In some embodiments, the blocker probes are not cleaved during PGR amplification.
In some embodiments, the blocker probes comprise a non-extendable blocker moiety at their
3'-ends. In other e bodiments the blocker probes further comprise other moieties including,
but not limited to, additional non-extendable blocker moieties, quencher moieties, fluorescent
moieties, etc. at their 3 -end, '-end, and/or any interna position in between. In certain other
embodiments, the allele position is located about 5-15, such as about 5- , about 6-10, about
7-9, about 7- . or about 9-1 , such as about 6, about 7, about 8, about 9, about 10, or about
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 include, without limitation, an optionally substituted 1- 2 alky io
e.g. a '-hexanediol modification), an optionally substituted C2-C24 alkenyl diol or C2-C24
alkynyi diol a . minor groove binder (MGB), an amine ( H2) biotin, PEG, PO , and mixtures
thereof. As disclosed herein, the use of non-extendable blocker moieties such as, e.g., 3'-
hexanediol modifications in alleie-specific blocker probes can increase the specificity of
alleie-specific PGR. Suitable methods for conjugating non-extendable blocker moieties to
alleie-specific blocker probes are known to one of ordinary skill in the art. For example, a
blocker moiety comprising a C1-C24 alkyl diol (e.g., hexanediol) can be conjugated to the 3'-
end of an alleie-specific blocker probe via a phosphoramidite linkage.
In some aspects, the blocker probe with hexanediol modification performs better
than one with phosphorylation. The flexibility and the hydrophobicity of the carbon chain
allows the blocker to hybridize to its wild-type target without being sterically hindered.
Although the phosphate group at the 3' end binds to the target wild-type sequence, efficiency
may be diminished by the bulkiness and the ionic nature of the phosphate group.
In certain embodiments, the non-extendable blocker moiety does not comprise or
include a minor groove binder (MGB). In certain oilier embodiments the non-extendable
blocker moiety does not comprise or include a P group. In further embodiments, the non-
extendable blocker moiety consists essentially of or consists of an optionally substituted C -
C 4 alkyl diol (e.g., a 3'-hexanediol modification), an optionally substituted C -C alkenyl
diol, or an optionally substituted C2-C24 aikynyl diol.
In some embodiments, the blocker probe has a dideoxycytidine (ddC) moiety, which
is a 3' chain terminator that prevents 3' extension by DNA polymerases.
In some embodiments, the allele-specific blocker probe can comprise one or more
modified nucleobases or c eosidic bases different from the naturally occurring bases (i.e.,
adenine, cyiosine, guanine, thymine and uracil) n some embodiments, the modified bases
are still able to effectively hybridize to nucleic acid units that contain adenine, guanine,
cyiosine, uracil or thymine moieties. In some embodiments, the modified base(s) may
increase the difference in the T between matched and mismatched target sequences and/or
decrease mismatch priming efficiency, thereby improving assay specificity and selectivity.
It is appreciated by those in the art that the mismatch discrimination ability of a
probe relies upon the difference in melting temperatures (ATm) between matched and
mismatched probe-target duplexes. ATm typically increases when the probe size descreases
because the mismatch has a more destabilizing effect on the duplex. In general, allele-
specific probes that are better at discriminating matched and mismatched sequences have
greater ATm values.
In particular embodiments, the allele-specific blocker probe of the present invention
comprises at ast one, two. or more modified bases. Non-limiting examples of modified
bases include locked nucleic acid (LNA), 8-azadeaza-dA (ppA), 8-azadeaza-dG (ppG),
2'-deoxypseudoisocytidine (iso dC), 5-fluoro-2 '-deoxyuridine (fdU), 2'-0,4'-C-ethylene
bridged nucleic acid (ENA) bases, and combinations thereof. In preferred embodiments, the
modified base present on the allele-specific blocker probe comprises one or more LNA
modifications. In certain embodiments, the modified base is located (a) at the "-end, (b) at
the 5 -end, (c) at an internal position, or at any combination of (a), b) or (c) within the allele-
specific blocker probe. In some preferred embodiments, the modified base (e.g., LNA
nucleoside) is located at the allele-specific nucleotide portion of the allele-specific blocker
probe, such that the L nucleoside comprises the nucleobase used to discriminate between
allelic variants.
In some embodiments, the inhibitory effect of the blocker probe increases as the Tm
of the blocker probe increases. Depending on the position of the LNA in the blocker, the Tm
can increase by 1-8°C (see, e.g., Koshkin et al, Tetrahedron, 54: 3607-3630 (1998), Obika et
al, Tetrahedron Lett, 39: 5401-5404 (1998), Wang et al, Bioorg. Med. Chem. Lett., 9 : 1147-
1150 (1999)). A blocker with higher Tm can remain annealed to the wild-type allele during
extension, thereby inhibiting efficient PCR amplification. Kinetic studies of duplex
formation have shown that LNA-containing DNA duplexes have a slower dissociation rate
compared to duplexes of native DNA. It has also been shown that the rigid structure of LNA
affects its interactions with Taq DNA polymerase (Larotta et ah, Molecular and Cellular
Probes, 17: 253-259 (2003)).
In some aspects of the present invention, the blocker probe can possess one or a
plurality of LNA modifications. In some embodiments, LNA is placed at the mid-position of
the blocker sequence, the penultimate position, the 5' end, different intervals of the blocker
sequence, and/or combinations thereof. In some embodiments, LNA is placed at the variant
nucleotide complementary to the wild-type variant. In other embodiments, a blocker probe
does not contain consecutive LNAs.
In embodiments where two allele-specific blocker probes are used, 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 or complementary
sequence as the first allele-specific primer.
In some embodiments, the allele-specific primer and the blocker probe both
comprise LNA. The presence of the modified base on both the primer and the blocker probe
enhances the specificity of the assay of the present invention. It has been shown in kinetic
studies of duplex formation that the slower dissociation rate of LNA-containing complexes is
due to differences in its hybridization performance as compared to that of native DNA. The
rigid structure of LNA molecule could alter the way it interacts with Taq DNA polymerase
(Larotta et al, Mol. Cell. Probes, 17: 253-259 (2003)).
D. Detector Probes
In some aspects, the methods of the present invention require the use of a short
detector probe length with a high melting temperature. Shorter length probes with high Tm
are necessary for good allelic discrimination, especially when dealing with difficult mutations
such as G A or G T. Examples of detector probes include, but are not limited to, minor
grove binding (MGB) probes, Zen probes (IDT, Coralville, IA), zip nucleic acid (ZNA)
probes, and protein nucleic acid (PNA) probes.
In some embodiments, the detector probe is designed as short oligomers ranging
from about 5-30 nucleotides, such as about 6, about , about 22. about 24, about 30, or
any number in between. In some embodiments, the Tm of th detector probe ranges from
about 60°C to 70°C, about 61 C to 69°C, about 62°C to 68°C, about 63°C to 67°C, about
64°C to 66°C, o any temperature In between.
In some embodiments, the detector probe is a locus-specific detector probe (LST).
In other embodiments, the detector probe is a 5' nuclease probe. In some embodiments, the
detector probe can comprises an MGB moiety, a reporter moiety (e.g., FAM™, TET™,
JOE™, VIC™, or SYBR Green), a quencher moiety (e.g., Blac Hole Quencher™ or
TAMRA™), and/or a passive reference (e.g.. RQX™). In some exemplary embodiments, the
detector probe is designed according to the methods and principles described in U.S. Patent
No. 6,727,356, the disclosure of which Is incorporated herein by reference in its entirety for
a l purposes.
In particular embodiments, the detector probe is a TaqMan* probe (Applied
Biosystems, Foster City, CA). Taqman probes are designed with MGB iigands (see, e.g.,
Afonina et al. Nucleic Acid Res., 25:2657-2660 (1997)) to help the probe to form super
stabilized duplexes with its complementary DNA target The increased stability of the
duplexes is associated with a higher Tm. For example, the m (65°C) of a 12-mer Taqman
probe with MGB is almost identical to the Tm (66°C) of 27-mer DNA probe without MGB.
It has been shown that with shorter probes, MGB contributes more to the overall stability of
the probe. It has been shown that 3 -MGB DNA probes increase sequence specificity during
P R cycling K tyavin et al, Nucleic Acid es., 28:655-661(2000)). MGB probes of the
present invention are designed to detect a genomic sequence either specific to the allelic
variant, near the allelic variant, or away fro the allelic variant. For instance, the MGB
probe can detect the 3 end of the amplification product generated by PGR cycling using an
allele-specific primer and a blocker probe.
n other embodiments, the detector probe is a Zen probe (IDT, Coralville, IA). A
ZEN double-quenched probe comprise an oligonucleotide probe, a 5' FAM fluorophore, a 3'
IBFq quencher and an internal ZEN quencher. The ZEN quencher lowers background and
generates higher signal compared to traditional dye-quencher probes (e.g., 5' FAM-3'
TAMRA, 5' FAM-3' IBFQ, 5' FAM-3' Eclipse, 5' FAM- 3' BHQ-1). The internal ZEN
quencher decreases the length between the dye and quencher to only 9 base pairs which
significantly reduces background fluorescence and provides more thorough quenching. The
sensitivity of the ZEN probe increases as the endpoint signal increases and the Ct (threshold
cycle; the fractional cycle number where fluorescence increases above the threshold) values
reduce.
In yet other embodiments, the detector probe comprises one or a plurality of zip
nucleic acid (ZNA) and/or peptide nucleic acid (PNA) modifications. Due to the increase
stability of the cationic charges of a ZNA probe, methods of the present invention can
comprise short dual-labeled ZNA probes. ZNA probes have enhanced target recognition,
greater sensitivity, high specificity, and increased Tm over standard detector probes. In
another embodiment, the detector probe is a PNA probe. Short PNA probes of the present
invention can offer high specificity because a PNA modification affords a high level of
discrimination at the single base level.
In some embodiments, the detector probe uses oligonucleotides with modified bases
or nucleic acid analogs comprising 2 to 6 LNAs, PNAs, TNAs, ZNAs or ribose modified
nucleic acids. These modified bases exhibit thermal stability towards complementary DNA
and RNA, which allows for excellent mismatch discrimination in methods of the present
invention.
In other embodiments, t e locus-specific detector probe is designed according to the
principles and methods described in U.S. Patent o. 6,727,356, the disclosure of which is
incorporated herein by reference in its entirety for all purposes. For example, tluorogenic
probes can b prepared with a quencher at the 3' terminus of a single DNA strand and a
fiuorophore 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 fiuorophore from the quencher
and releasing the fluorescent signal In some embodiments, the detector probes are hybridized
to the template strands during primer extension step ofPCR amplification (e.g., at 60°-65°C).
In other embodiments, an MGB is covalently attached to the quencher moiety of the locus-
specific detector probes (e.g., through a linker).
in embodiments where two detector probes are used, the first and second detector
probes are the same and/or comprise the same sequence or are the same sequence.
E. Locus-Specific Primers
In some embodiments, the iocus-specific primer is designed as a short oligomer
ranging from about 15-30 nucleotides, such as about 16, about 18, about 22, about 24, about
, or any number in between. In some embodiments, the Tm of the locus-specific primer
ranges from about 60 C to 70°C, about 6 °C to 69 C, about 62°C to 68°C, about 63 C to
67°C, or about 64°C to 66°C, or any range in between.
In embodiments where two locus-specific primers are used, he first locus-specific
primer and/or the second locus-specific primer comprise the same sequence or are the same
sequence.
F. Additional Components
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.) usi g
methods tha are well-known to o e of ordinary skill in the art. See, e.g., U.S. Patent 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 et
aL, . Bacterid., 98(i):289-297 1969); Oshima etcil.Jnt. . Syst. B ct ri , 24(1): 102-11 2
( 974). Suitable for use as sources of thermostable polymerases are the thermophilic bacteria
Thermus aquaticus, The us thermophilus, Thermococcus iitoralis, Pyrococcus furiosus,
Pyrococcus oosii and other species of the Pyrococcus genus. Bacillus stearothermophiius,
Su folobu acidocaldarius, Tliermoplasma acidophilum, Thermus flavus, Thermus ruber,
Thermus broekianus, 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, T e DNA polymerase, T na DNA polymerase, or mutants, derivatives, or
fragments thereof.
G. Quantitation of Allelic Variants
In certain aspects, the methods of quantitating an allelic variant of a target sequence
in a nucleic acid sample comprise determining an amount and/or a percentage of the allelic
variant present in the sample using a standard curve established from a cell line positive for
the allelic variant. In particular embodiments, the amount of DNA (e.g., amount of nucleic
acid carrying the allelic variant) present in the sample is calculated (e.g., in nanogram (ng) or
any other unit of weight) from the standard curve using the Ct value. In some instances, the
standard curve is based on the cell line carrying a 100% mutation for the allelic variant. In
other instances, the amount of DNA derived from the standard curve (e.g., amount of nucleic
acid carrying the allelic variant in the sample) is converted to a percent mutation of the allelic
variant based on the cell line.
The assays of the present invention have high selectivity and can differentiate and
quantitate a rare variant allele from the wild-type allele. The data from the assays are also
linear and can be used to derive quantitative information of the allelic variant, e.g., to detect,
determine, or calculate the amount or percentage of the allelic variant present in a sample.
In some aspects, a standard curve is generated for an allelic variant from a cell line
positive for the variant. In some embodiments, a standard curve is generated for KRAS
allelic variants from the following positive cell lines: G12A from the SW1 116 cell line;
G12C from the NCI-H23 cell line; G12D from the LS 174T cell line; G12R from the PSN1
cell line; G12S from the A 549 cell line; G12V from the SW 403 cell line; G13C from the H
1734 cell line G13D from the T 84 cell line; and/or Q61H from the H 460 cell line. In other
embodiments, a standard curve is generated for PIK3CA allelic variants from the following
positive cell lines: E542K from the SW948 cell line; E545D from the Sup T l cell line;
E545K from the MCF 7 cell line; and/or H1047R from the KPL4 cell line. In yet other
embodiments, a standard curve is generated for EGFR allelic variants from the following
positive cell lines: T790M and L858R from the H1975 cell line and/or E746-A750 deletion
(E746 del) from the H I650 cell line. In yet other embodiments, a standard curve is generated
for the BRAF V600E variant from the HT 29 cell line. In some instances, a standard curve is
created from performing the assays of the invention on a series of dilution of DNA (e.g., 100,
, 1, 0.1, and/or 0.01 ng) from the positive cell line.
In other aspects, the methods of quantitating an allelic variant of a target sequence
in a nucleic acid sample comprise determining an amount and/or a percentage of the allelic
variant present in the sample using a calculator based on a standard curve established from a
cell line positive for the allelic variant. In certain embodiments, a percent mutation calculator
specific to an allelic variant is established from the standard curve of the allelic variant. The
calculator can be used to calculate the amount of mutation in a nucleic acid sample from the
Ct value obtained from the methods of the invention. In some instances, the percent mutation
of the allelic variant can be calculated based on the assumption that the positive cell line has a
percent mutation of 100% for the allelic variant.
In particular embodiments, the amount or percentage of an allelic variant present in
a sample can be quantitated by determining a Ct value obtained when the genotyping assay
described herein is performed on nucleic acid obtained from the sample. A standard curve
for the allelic variant can be generated by performing the genotyping assay described herein
on a serial dilution of nucleic acid sample from a cell line positive for the allelic variant. The
standard curve can then be used to determine a Ct value for a specific amount of nucleic acid
present in the positive control (e.g., cell line) sample. The standard curve can also be used to
determine the line slope and/or line intercept values when Ct values obtained for the positive
control samples are plotted as a function of the quantity of DNA per positive control reaction.
Figures 32-35 provide non-limiting examples of standard curve plots (e.g., for the
positive control samples), amplification curves (e.g., for unknown (test) and positive control
samples), and calculators for quantitating the amount and the percent (e.g., percent mutation)
of the alleleic variant present in a sample based upon information obtained from the standard
and/or amplification curves. In certain embodiments, the percent mutation is calculated from
the amount of mutation with respect to the starting amount of nucleic acid (e.g., DNA) in the
sample. The starting amount of nucleic acid (e.g., DNA) in the sample can be expressed as a
logio value (e.g., in nanograms). In other embodiments, the calculator quantitates the amount
and/or the percent (e.g., percent mutation) of the alleleic variant present in a sample based
upon information such as Ct values, line slope values, and/or line intercept values that are
obtained from the standard and/or amplification curves generated using the genotyping assay
described herein. In further embodiments, the percent mutation calculated for an alleleic
variant present in an unknown (test) sample is relative to the positive control (e.g., compared
to the positive cell line with a percent mutation of 100% for the allelic variant).
IV. Exemplary Embodiments
In one aspect, the present invention provides a method for detecting or quantitating
a first allelic variant of a target sequence in a nucleic acid sample suspected of having at least
a second allelic variant of the target sequence, said method comprising:
(a) forming a reaction mixture by combining:
(i) the nucleic acid sample;
(ii) an allele-specific primer, wherein an allele-specific nucleotide portion of the
allele-specific primer is complementary to the first allelic variant of the target
sequence, and wherein the allele-specific primer comprises at least one nucleic
acid modification (e.g., one or a plurality of nucleic acid modifications);
(iii) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising the second allelic variant, wherein the allele-specific blocker
probe comprises a non-extendable blocker moiety and at least one nucleic acid
modification (e.g., one or a plurality of nucleic acid modifications);
(iv) a detector probe; and
(v) 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
(b) carrying out an amplification reaction on the reaction mixture using the locus-
specific primer and the allele-specific primer to form an amplicon; and
(c) detecting the amplicon by detecting a change in a detectable property of the
detector probe, thereby detecting the first allelic variant of the target gene in the
nucleic acid sample.
In some embodiments, the nucleic acid modification(s) in the allele-specific primer
is/are located at the allele-specific nucleotide portion, at the 5'-end of the allele-specific
primer, and/or at the 3'-end of the allele-specific primer. In certain embodiments, the allele-
specific primer comprises two or more non-consecutive nucleic acid modifications. n some
embodiments, the nucleic acid modification(s) in the allele-specific primer is/are selected
from the group consisting of locked nucleic acids (L NA), peptide nucleic acids (PNA),
threose nucleic acids ( NA ), zip nucleic acids (ZNA). triazole nucleic acids (Tz A ), and
combination s thereof.
In other embodiments, the nucleic acid modification(s) in the allele-specific blocker
probe is/are located at the allele-specific nucleotide portion and/or at an internal position in
the allele-specific blocker probe. In certain embodiments, the allele-specific blocker probe
comprises two or more non-consecutive nucleic acid modifications. In some instances, the
nucleic acid modification(s) in the allele-specific blocker probe is/are selected from the group
consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids
(TNA). zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
In some embodiments, the non-extendable blocker moiety comprises a modification
to t e 3' -end of the allele-specific blocker probe which prevents t e addition of further bases
to the 3 -end by a polymerase. In particular embodiments, the non- extendable blocker
moiety is selected from the group consisting of an optionally substituted C I- ai y diol, an
optionally substituted C -C 4 alkenyl diol, an optionally substituted C alkynyi diol, and
- C 2
combinations thereof. In a preferred embodiment, the non-extendable blocker moiety
comprises a 3'-hexanedioi modification to the allele-specific blocker probe.
In certain embodiments, the non-extendable blocker moiety does not comprise or
include a minor groove binder (MGB). In certain oilier embodiments, the non-extendable
blocker moiety does not comprise or include a PO group. In further embodiments, the non-
extendable blocker moiety consists essentially of or consists of an optionally substituted Cj-
C 4 alky! diol ( .g . a 3'~hexanediol modification), an optionally substituted C 2-C 2 alkenyl
diol or an optionally substituted C2-C24 aikynyl dio .
In some embodiments, the detector probe comprises a TaqMan probe. In certain
embodiments, the nucleic acid sample is selected from the group consisting of blood, serum,
plasma, fine needle aspirate, tumor tissue, and combinations thereof. In other embodiments,
the first allelic variant is a mutant allele and the second allelic variant is the wild-type allele.
In particular embodiments, the method reduces the background signal of the second allelic
variant during the amplification reaction.
In certain embodiments, the first allelic variant of the target gene can be quantitated
by determining the threshold cycle or Ct value in which a change in the detectable property of
the detector probe first becomes detectable above a background level. In some embodiments,
a standard curve for the first allelic variant can be generated by performing the method of the
invention on a serial dilution of nucleic acid sample from a cell line positive for the allelic
variant. In some instances, the first allelic variant is quantitated by comparing the Ct value
obtained for the sample with Ct values from the standard curve. In particular embodiments,
the standard curve is used to determine one or more Ct values (e.g., relative to the starting
amount of nucleic acid in the positive control sample), line slope values, and/or line intercept
values obtained by performing the method of the invention on a serial dilution of nucleic acid
sample from a cell line positive for the allelic variant. In other embodiments, the amount
and/or percent (e.g., percent mutation) of the first allelic variant present in the sample is
calculated based upon at least one, two, three, or four of the following: starting amount of
nucleic acid in the sample (e.g., DNA per reaction, which can be expressed as a logio value in
ng or any other unit of weight); Ct value; line slope value (e.g., from the standard curve); line
intercept value (e.g., from the standard curve); and combinations thereof.
In another aspect, the present invention provides a reaction mixture comprising:
(a) a nucleic acid molecule;
(b) an allele-specific primer, wherein an allele-specific nucleotide portion of the
allele-specific primer is complementary to a first allelic variant of a target sequence,
and wherein the allele-specific primer comprises at least one nucleic acid
modification (e.g., one or a plurality of nucleic acid modifications);
(c) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising a second allelic variant, wherein the allele-specific blocker
probe comprises a non-extendable blocker moiety and at least one nucleic acid
modification (e.g., one or a plurality of nucleic acid modifications);
(d) a detector probe; and
(e) 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.
In some embodiments, the nucleic acid modification(s) in the allele-specific primer
is/are located at the allele -specific nucleotide portion, at the 5'-end of the allele-specific
primer, and/or at the 3'-end of the allele-specific primer. In certain embodiments, the allele-
specific primer comprises two or more non-consecutive nucleic acid modifications. In some
embodiments, the nucleic acid modification(s) in the allele-specific primer is/are selected
from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA),
threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and
combinations thereof.
In other embodiments, the nucleic acid modification(s) in the allele-specific blocker
probe is/are located at the a ele -speeific nucleotide portion and/or at an internal position in
the allele-specific blocker probe. In certain embodiments, the allele-specific blocker probe
comprises two or more non-consecutive nucleic acid modifications. In some instances, the
nucleic acid modification(s) in the allele-specific blocker probe is/are selected from the group
consisting of locked nucleic acids (LNA), peptide micieic acids (PNA), threose nucleic acids
(TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
In some embodiments, the non-extendable blocker moiety comprises a modification
to the 3'-end of the allele-specific blocker probe which prevents the addition of further bases
to the '-end by a polymerase. In particular embodiments, the non-extendable blocker
moiety is selected from the group consisting of an optionally substituted C - a ky diol, an
optionally substituted C2-C24 aikenyl diol, an optionally substituted C2-C24 a kyny diol, and
combinations thereof. In a preferred embodiment, the no -extend able blocker moiety
comprises a 3'-hexanediol modification to the allele-specific blocker probe.
In certain embodiments, the non-extendable blocker moiety does not comprise or
include a minor groove binder (MGB) In certain other embodiments, the non-extendable
blocker moiety does not comprise or include a . PO4 group. In further embodiments, the non-
extendable blocker moiety consists essentially of or consists of an optionally substituted C -
2 aikyl dio (e.g., a 3'-hexanediol modification), an optionally substituted C2-C24 aikenyl
diol, or an optionally substituted V C24 alkynyl diol
In some embodiments, the detector probe comprises a TaqMan probe. In certain
embodiments, the nucleic acid molecule is obtained from a sample selected from the group
consisting of blood, serum, plasma, fine needle aspirate, tumor tissue, and combinations
thereof. n other embodiments, the first allelic variant is a mutant allele and the second allelic
variant is the wild-type allele.
In yet another aspect, the present invention provides a composition comprising:
(a) 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,
and wherein the allele-specific primer comprises at least one nucleic acid
modification (e.g., one or a plurality of nucleic acid modifications); and
(b) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising a second allelic variant, wherein the allele-specific blocker
probe comprises a non-extendable blocker moiety and at least one nucleic acid
modification (e.g., one or a plurality of nucleic acid modifications).
In some embodiments, the composition further comprises: (c) a detector probe;
and/or (d) a locus-specific primer that is complementary to a region of the target sequence
that is 3' from the first allelic variant and on the opposite strand.
In some embodiments, the nucleic acid modification(s) in the allele-specific primer
is/are located at the allele-specific nucleotide portion, at the 5'-end of the allele-specific
primer, and/or at the 3'-end of the allele-specific primer. In certain embodiments, the allele-
specific primer comprises two or more non-consecutive nucleic acid modifications. In some
embodiments, the nucleic acid modification(s) in the allele-specific primer is/are selected
from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA),
threose nucleic acids ( NA ), zip xvucieic acids (ZNA), triazole nucleic acids (Tz A), and
cornb a io s thereof.
In other embodiments, the nucleic acid modification(s) in the allele-specific blocker
probe is/are located at the allele-specific nucleotide portion and/or at an internal position in
the allele-specific blocker probe. In certain embodiments, the allele-specific blocker probe
comprises two or more non-consecutive nucleic acid modifications. In some instances, the
nucleic acid modification(s) in the allele-specific blocker probe is/are selected from the group
consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), ihreose nucleic acids
(TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
In some embodiments, the non-extendable blocker moiety comprises a modification
to the 3' -end of the allele-specific blocker probe which prevents the addition of further bases
to the 3'-end by a polymerase. In particular embodiments, the non-extendable blocker
moiety is selected from the group consisting of an optionally substituted Ci-C a y diol, an
optionally substituted C -C 4 aikeny! diol, an optionally substituted C ¾- ¾4 alkynyl diol, and
combinations thereof. In a preferred embodiment, the non-extendable blocker moiety
comprises a 3'-hexanediol modification to the allele-specific blocker probe.
In certain embodiments, the non-extendable blocker moiety does not comprise or
include a minor groove binder (MGB). n certain other embodiments, the non-extendable
blocker moiety does not comprise or include a P group. In further embodiments, the on-
extendable blocker moiety consists essentially of or consists of an optionally substituted CV
C a ky diol (e.g., a 3"-hexanediol modification), an optionally substituted C alkenyl
-C24
diol, or an optionally substituted C ?~C alkynyl diol.
In further embodiments, the first allelic variant is a mutant allele and the second
allelic variant is the wild-type allele.
In another aspect, the present invention provides a kit comprising two or more
containers comprising the following components independently distributed in one of the two
or more containers:
(a) 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,
and wherein the allele-specific primer comprises at least one nucleic acid
modification (e.g., one or a plurality of nucleic acid modifications); and
(b) an allele-specific blocker probe that is complementary to a region of the target
sequence comprising a second allelic variant, wherein the allele-specific blocker
probe comprises a non-extendable blocker moiety and at least one nucleic acid
modification (e.g., one or a plurality of nucleic acid modifications).
In some embodiments, the kit further comprises: (c) a detector probe; and/or (d) a
locus-specific primer that is complementary to a region of the target sequence that is 3' from
the first allelic variant and on the opposite strand.
In some embodiments, the nucleic acid modification(s) in the allele-specific primer
is/are located at the aileie-specific nucleotide portion, at the 5'-end of the allele-specific
primer, and/or at the 3'-end of the allele-specific primer. In certain embodiments, the allele-
specific primer comprises two or more non-consecuiive nucleic acid modifications. In some
embodiments, the nucleic acid modification(s) in the allele-specific primer is/are selected
from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA),
threose nucleic acids (T A), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and
combinations thereof
In other embodiments, the nucleic acid modification(s) in the allele-specific blocker
probe is/are located at the allele-specific nucleotide portion and/or at an internal position in
the allele-specific blocker probe. In certain embodiments, the allele-specific blocker probe
comprises two or more non-consecutive nucleic acid modifications. In some instances, the
nucleic acid modification(s) in the allele-specific blocker probe is/are selected from the group
consisting of locked nucleic acids (LN.A), peptide nucleic acids (PNA), tlireose nucleic acids
T A), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
In some embodiments, the non-extendable blocker moiety comprises a modification
to the 3'-end of the allele-specific blocker probe which prevents the addition of further bases
to the '-end by a polymerase. In particular embodiments, the non-extendable blocker
moiety is selected from the group consisting of an optionally substituted C C a ky diol, an
optionally substituted C -C ? aikenyl diol, an optionally substituted C -C . alkynyl diol, and
combinations thereof. In a preferred embodiment, the non-extendable blocker moiety
comprises a 3'-hexanediol modification to t e allele-specific blocker probe.
n certain embodiments the non-extendable blocker moiety does not comprise or
include a . minor groove binder (MGB). In certain other embodiments, the non-extendable
blocker moiety does not comprise or include a P0 group. In further embodiments, the non-
extendable blocker moiety consists essentially of or consists of an optionally substituted Ci-
alkyl dio (e.g., a S'-hexanediol modification), an optionally substituted C - aikenyl
diol, or an optionally substituted C2-C24 alkynyl diol
In so e embodiments, the first allelic variant is a mutant allele and the second
allelic variant is the wild-type allele. In other embodiments, the kit further comprises
instructions for use of the allele-specific primer and the allele-specific blocker probe for
detecting or quantitating the first allelic variant of the target sequence in a nucleic acid
sample suspected of having the second allelic variant of the target sequence.
V. Examples
The present invention will be described in greater detail by way of specific
examples. The following examples are offered for illustrative purposes, and are not intended
to limit the invention in any manner. Those of skill in the art will readily recognize a variety
of noncritical parameters which can be changed or modified to yield essentially the same
results.
Example 1. Somatic Mutation Genotyping Assay Methodology.
Figure 1 depicts one embodiment of the somatic mutation detection assays of the
present invention. An allele-specific primer and an allele-specific blocker probe are used for
each single nucleotide polymorphism (SNP) to be analyzed in a sample such as a blood or
fine needle aspirate (FNA) sample. The allele-specific primer can comprise a locked nucleic
acid (LNA) at the 3'-end that is specific for a variant (e.g., mutant) allele, whereas the allele-
specific blocker probe can comprise a hexanediol blocker moiety at the 3'-end and a LNA at
a position between about 5-15 nucleotides 5' of the blocker moiety (e.g., in the middle of the
oligonucleotide sequence) that is specific for the wild-type allele.
The assay methods of the present invention can be performed on an ABI 7900HT
Real Time PCR Instrument, although any type of real time PCR instrument known to one of
skill in the art can be used. Exemplary reaction conditions include the following: Stage 1:
95.0°C for 10:00 min; Stage 2 : Repeats: 40, 95.0°C for 0:20 min and 60.0°C for 0:45 min.
As depicted in Figure 1, hybridization of the allele-specific blocker probe (e.g.,
"Blocker LNA Hexanediol Oligonucleotide") to the wild-type allele prevents amplification of
the wild-type allele, whereas hybridization of the allele-specific primer (e.g., "Allele specific
LNA primer") to the mutant allele enables the mutant allele to be selectively amplified with
high sensitivity and low background. In particular embodiments, the use of LNA nucleosides
improves the signal to noise ratio and substantially reduces the wild-type background signal.
Example 2. Exemplary Somatic Mutation Genotyping Assays for Detection of SNPs.
This examples illustrates that the use of allele-specific primers and allele-specific
blocker probes containing modified bases improves the discrimination of allelic variants.
In particular, Figure 2 shows that the use of an allele-specific primer comprising a
LNA modification at the 3'-end ("+A" in "G12S ASP-LNA") and an allele-specific blocker
probe comprising a LNA modification in the middle of the oligonucleotide sequence ("+G"
in "G12S blocker-LNA") and a 3'-hexanediol modification ("C6" in "G12S blocker-LNA")
improves the discrimination of allelic variants at the KRAS G12S polymorphic site. Allele-
specific real time PCR performed using allele-specific primers and blocker probes without
LNA modifications incorrectly detected the presence of the KRAS G12S mutant allele in all
8 of the cell lines negative for the mutant allele (i.e., H1975, H1993, U87MG, A375, PC3,
A43 INS, #28, and #29 cell lines). In contrast, allele-specific real time PCR performed using
allele-specific primers and blocker probes of the invention comprising LNA and hexanediol
modifications correctly identified these cell lines as negative for the KRAS G12S mutation.
Although the allele-specific primer and blocker probe without LNA modifications identified
the KRAS G12S mutation in the KRAS G12S positive A549 cell line, the ACt value was 9
(compare the "Ct" values for #29 versus A549), which was significantly lower than the ACt
value of 12 observed with the LNA a d hexanediol-modified primers and probes described
herein. Without being bound to any particular theory, an increase in ACt values indicates an
improvement in the discrimination of allelic variants during PGR. In addition, the use of the
modified primers and probes of the invention substantially reduces the wild-type background
signal, even in samples containing an abundance of the wild-type allele.
Similarly, Figure 3 shows that the use of an allele-specific primer comprising a
LNA modification at the 3'-end ("+C" in "G12R ASP-LNA") and an allele-specific blocker
probe comprising a LNA modification in the middle of the oligonucleotide sequence ("+G"
in "G12R blocker-LNA") and a 3'-hexanediol modification ("C6" in "G12R blocker-LNA")
improves the discrimination of allelic variants at the KRAS G12R polymorphic site. Allele-
specific real time PCT performed using allele-specific primers and blocker probes without
LNA modifications incorrectly detected the presence of the KRAS G12R mutant allele in
several of the cell lines negative for the mutant allele. In contrast, allele-specific real time
PCT performed using allele-specific primers and blocker probes of the invention comprising
LNA and hexanediol modifications correctly identified all of these cell lines as negative for
the KRAS G12R mutation.
Likewise, Figure 4 shows that the use of an allele-specific primer comprising a
LNA modification at the 3'-end ("+G" in "H1047R ASP-LNA") and an allele-specific
blocker probe comprising a LNA modification in the middle of the oligonucleotide sequence
("+A" in "H1047R blocker-LNA") and a 3'-hexanediol modification ("C6" in "H1047R
blocker-LNA") improves the discrimination of allelic variants at the PIK3CA H1047R
polymorphic site. The allele-specific primer and blocker probe without LNA modifications
identified the PIK3CA H1047R mutation in the PIK3CA H1047R positive KPL4 cell line.
However, the use of the LNA and hexanediol-modified primers and probes of the present
invention substantially increased ACt values based upon a comparison of ACt values of about
4-6 for PCR without LNA modifications to ACt values of greater than 10 for PGR with LNA
modifications, wherein ACt values were calculated by subtracting the Ct values of the KPL4
cell line from the Ct values of any of the H1975, H1993, U87MG, A375, PC3, A431NS, #28,
or #29 cell lines. As discussed herein, an increase in ACt values indicates an improvement in
the discrimination of allelic variants during PCR. In addition, the use of the modified primers
and probes of the invention substantially reduces the wild-type background signal, even in
samples containing an abundance of the wild-type allele.
Figures 5 and 6 illustrate examples of improved allelic variant discrimination at the
EGFR T790M and EGFR L858R polymorphic sites, respectively, using the LNA-modified
allele-specific primers and probes of the present invention. Allele-specific real time PCR
performed using allele-specific primers and blocker probes without LNA modifications
incorrectly detected the presence of both EGFR mutations in all 7 of the cell lines negative
for the mutant allele (i.e., H1993, U87MG, A375, PC3, #29, #31, and #32 cell lines). In
contrast, allele-specific real time PCR performed using allele-specific primers and blocker
probes of the invention comprising LNA and hexanediol modifications correctly identified all
7 of these cell lines as negative for both EGFR mutations, and correctly identified the EGFR
T790M and EGFR L858R positive H1975 cell line as containing both mutant alleles.
Figure 7 illustrates the effect of an abundant amount of wild-type DNA from whole
blood on the interference of detecting a mutant allele of interest such as the PIK3CA H1047R
variant allele in H1047R-positive KPL4 cells. Figure 8 illustrates the effect of an abundant
amount of wild-type DNA from whole blood on the interference of detecting a mutant allele
of interest such as the KRAS G12R variant allele in G12R-positive PSNl cells. As shown in
Figure 8, the G12R signal is still detectable with as low as 100 G12R-positive PSNl cells in
the spiked whole blood sample, which represents 0.01% of the whole blood count.
Example 3. Screening Colorectal Cancer Samples With Somatic Mutation Genotyping
Assay Methodology.
This examples illustrates the screening of colorectal cancer (CRC) tissue samples
for the presence of the PIK3CA H1047R variant allele using the allele-specific primers and
allele-specific blocker probes of the present invention containing modified bases including
LNA modifications.
Figure 9 shows that screening of 150 CRC tissue samples indicated that there was
no interference observed from the negative samples and that detection of a weak signal can
be validated by titration. The H1047R-positive samples #532 and #528 were validated by
titration, while the H1047R-negative samples #94, #95, and #96 had undetectable levels of
the H1047R variant allele at all concentrations tested.
In sum, this example demonstrates that the somatic mutation genotyping assays of
the present invention are highly sensitive, very specific, highly selective, and robust and can
be used to test clinical samples to detect and/or quantitate allelic variants in genes such as
KRAS, PIK3CA, and EGFR.
Example 4. Comparison of Somatic Mutation Genotyping Assay Methodology with
Scorpion and BEAMing Assays.
This examples illustrates a comparison between the somatic mutation genotyping
assay of the present invention and the Scorpion assay from Qiagen or the BEAMing assay
from Inostics on KRAS G12A detection in whole blood spiked with varying amounts of
SW1 116 (G12A-positive) cells.
Figure 10 shows that the DxS/Qiagen Scorpion assay can only detect 1000 cells in
the mixture of whole blood spiked with a serial dilution of SW1 116 (G12A-positive) cells.
The sensitivity is lost by 100 cells. In contrast, with the use of the genotyping assay of the
present invention ("Inventive Assay"), the G12A signal is still detectable with as low as 10 to
100 of the SW1 116-positive cells in the whole blood mixture. 100 cells represent 0.01% of
the whole blood count. Even at 1000 cells, the Ct curve was not tight for the DxS/Qiagen
Scorpion assay.
Figure 11 shows a serial dilution of KPL4 (H1047R), A549 (G12S), and PS 1
(G12R) cells spiked in whole blood (WB) at 10, 50, 100, 250 and 500 cells. Notably, the
Inostics BEAMing assay made the incorrect call and identified 14 out of 15 mutant samples
as wild-type samples. In fact, the Inostics assay only detected the mutation when 1000 cells
were present in whole blood, and displayed no sensitivity at 500 cells or less. In contrast,
Figure 12 shows that the somatic mutation genotyping assay of the present invention
("Inventive Assay") had a detectable signal as low as 50 to 100 positive cells in the WB
mixture. 100 cells represent 0.01% of the whole blood count.
In sum, this example demonstrates that the somatic mutation genotyping assay of
the present invention dramatically improves the allelic PCR assay compared to the Scorpion
assay from Qiagen and the BEAMing assay from Inostics. In particular, a weak signal can be
validated by titration and the assays of the invention can detect as low as 0.01% of cells with
a mutant allele in a background of whole blood with abundant levels of the wild-type allele.
As such, the methodology described herein is superior to the Scorpion and BEAMing assays
in samples such as whole blood.
Example 5. Somatic Mutation Genotyping Assay Methodology Using Nucleic Acid
Modifications.
This example illustrates multiple embodiments of the somatic mutation detection
assays of the present invention. For this example, the KRAS G12A assay was chosen as an
exemplary assay for illustrating the methods of the invention. In particular, the experiments
described herein address the following components of the assay: (1) allele-specific primers
with and without LNA; (2) 3' end modification on blocker probes; (3) blocker probes with
different Tm; (4) blocker probes with and without LNA; and (5) the combination of allele-
specific primers and blocker probes with LNA and without LNA.
The methods described in this example are based on real time allele-specific PCR in
combination with a locus-specific primer, an allele-specific primer, a blocker probe, and a
detector probe. The methods were designed to be highly selective and sensitive in
differentiating and quantitating the presence of a mutant allele from a wild type allele. In
particular, the exemplary assay was perfomed using GTXpress™ Master Mix from Applied
Biosystems (Foster City, CA). For the real-time PCR reaction, the following cycling
condition was used: Stage 1: 95.0°C for 10:00 min, and Stage 2 : Repeats: 40 times, 95.0°C
for 0:20 min and 60.0°C for 0:45 min.
Experiment # 1 shows that the KRAS G12A assay of the present invention
comprising an allele-specific primer with LNA successfully amplified the mutant allele with
high selectivity and sensitivity. In this embodiment of the present invention, the variant
nucleotide (T) of the allele-specific primer was a locked nucleic acid (LNA) and was located
at the 3'-end of the primer. The blocker probe had a LNA and a hexanediol modification at
the 3' end. The detector probe was a Taqman probe (Life Technologies). The results of the
real-time PCR assay show that the blocker probe hybridized to the wild-type allele and
blocked its amplification (Figure 13). In addition, the mutant allele was selectively amplified
with high sensitivity by the assay (Figure 13).
Experiment #2 shows that the SNP genotyping assay of the present invention has
improved selectivity and sensitivity for allelic discrimination when the allele-specific primer
comprises a locked nucleic acid (LNA). Figure 14 shows that a KRAS G12A assay without
LNA on the allele-specific primer and blocker probe generated amplification products from
the positive control (mutant allele) and negative control (wild-type allele) samples. This
embodiment of the present invention did not show sufficient selectivity for the allelic
variants. Yet, a KRAS G12A assay LNA modifications on the allele-specific primer and
blocker probe was able to selectively amplify the positive control sample and not the negative
control. Figure 14 shows that the assay with the LNA at the variant nucleotide (T) amplified
the mutant allele with high sensitivity.
Experiment #3 shows that the presence of two LNAs on a allele-specific primer in
the SNP genotyping assay improved the performance of the assay. In this embodiment of the
present invention, the design of two allele-specific primers were compared. One primer had a
LNA at the variant nucleotide (T). The other primer had a second LNA (A) located 5' of the
variant nucleotide. The assay comprising the allele-specific primer with 2 strategically
placed LNAs showed better amplification and a lower Ct value (Figure 15). LNA can be
placed at the 3'end of the allele-specific primer and at 2, 3, 4, 5 or 6 bases from the 3' end.
LNA can be placed at the 5'end and the 3' end of the allele-specific primer.
Experiment #4 shows that consecutive LNAs on an allele-specific primer of the
present invention did not generate amplification products in the assay. Figure 16 depicts a
allele-specific primer with consecutive LNAs (GAT) where T is the variant nucleotide and
shows that it failed to amplify the KRAS G12A mutant allele. Figure 16 also depicts a allele-
specific primer with six consecutive LNAs (T of the variant nucleotide and 5 LNAs upstream
of the T that are complementary to the mutant allele). This primer failed to detect the mutant
allele in the sample and generated no amplification products in the assay.
This example shows the allele-specific primer, blocker probe and detector probe of
the present invention can comprise modified base(s), such as locked nucleic acid (LNA),
peptide nucleic acid (PNA), a-L-threose nucleic acid (TNA), zip nucleic acid (ZNA) and
triazole DNA (TzDNA). Figure 17 shows an exemplary LNA molecule and other modified
LNAs. Figure 18 shows an exemplary PNA-DNA duplex (left) and an exemplary allele-
specific primer with PNA (T in Figure 18; right). Figure 19 shows an exemplary TNA
containing oligonucleotides (left) and an exemplary allele-specific primer with TNA (T in
Figure 19; right). Figure 20 shows an exemplary ZNA oligonucleotide (left) and an
exemplary ZNA modified allele-specific primer (right; T in Figure 20). Figure 2 1 shows an
exemplary TzDNA molecule (left) and an exemplary allele-specific primer with TzDNA
(right; T in Figure 21).
Experiment #5 shows that a blocker probe with a hexanediol modification at the 3'
end improves the selectivity of the assay of the present invention. When the assay was
performed without a blocker probe, both wild-type and mutant alleles were amplified
similarly and were unable to be discriminated (part I, Figure 22). In the embodiment of the
present invention with a blocker probe, the blocker hexanediol probe hybridized to the wild-
type allele of KRAS G12A SNP and hindered real-time PCR amplification (part II, Figure
22). In this assay the mutant allele was selectively amplified (part III, Figure 22).
Experiment #6 shows that the KRAS G12A assay of the present invention
comprising an allele-specific primer with LNA, a blocker probe with hexanediol, Taqman
probe and reverse primer generated an amplification product specific to the mutant allele
(positive control) and not to the wild-type allele. Part I, Figure 23 shows that the mutant
allele-specific primer amplified both the wild-type and mutant alleles of the KRAS G12A
SNP. Part II, Figure 23 shows that the mutant allele-specific primer with a single LNA at the
variant nucleotide located at the 3' end of the primer amplified the mutant allele and not the
wild-type.
Experiment #7 demonstrates a blocker probe with a hexanediol (C6) modification at
the 3'end of the oligonucleotide performed better than a probe with a 3'-phosphate group
(P0 ) in a KRAS G12A assay for the present invention comprising an allele-specific primer
with LNA at the variant nucleotide. Figure 24 shows that the assay with the blocker
hexanediol probe performed better with a lower Ct than the assay with the blocker PO probe
(Ct of 28.4 vs. Ct of 31.2). The flexibility of the hydrophobicity of the carbon chain allows
the hexanediol blocker to hybridize well without being sterically hindered. Although the
phosphate groups at the 3' end of the blocker probe also can bind the wild-type allele, the
efficiency of binding may be diminished by the bulkiness and the ionic nature of the
phosphate group.
Experiment #8 demonstrates that a blocker probe with LNA performed better in the
assay of the present invention compared to a blocker probe without LNA. Figure 25
illustrates that a blocker LNA probe had a lower Ct value than a blocker probe without LNA.
The methods of the present invention comprising a blocker probe with LNA and hexanediol
modification can produce efficient and selective amplification of allelic variants, thereby
improving allelic discrimination.
Experiment #9 shows that LNAs in the mutant allele-specific primer and the blocker
probe of the present invention resulted in highly specific amplification of the mutant allele of
the KRAS G12A SNP. Figure 26 shows that the assay with the LNA primer and LNA
blocker amplified the mutant allele (positive control) and not the wild-type allele (negative
control).
Experiment #10 shows that LNA can be strategically placed on the blocker probe
sequence to improve the performance of the SNP assay of the present invention. Figure 27A
shows the components of the assays tested in the experiment. As depicted in Figure 27B,
Blocker. l.LNA and BlockerALNA have the same blocker sequence, but the locations of the
LNA modifications are different. Blocker. l.LNA has LNAs at the 5' end and at the allelic
variant nucleotide, while BlockerALNA has LNAs at the 5' end and at the 3'end of the
sequence. The assay with Blocker. l.LNA displayed better allelic discrimination and a lower
Ct compared to the assay with BlockerALNA (Figure 27C).
Figure 28A shows the influence of Tm of the blocker probe on the performance of
the assay. Blocker. l.LNA has a Tm of 59.9°C. BlockerALNA has a Tm of 68°C. Figure
28B also shows the difference in Tm and Ct value between a blocker without LNA and one
with two LNAs. In assays of the present invention a blocker probe with LNA and a higher
Tm has better specificity for the allelic variant, better sensitivity, and efficient inhibition of
the wild-type variant.
Experiment # 11 shows that consecutive LNAs on a blocker probe of the present
invention completely block amplification of the mutant variant, thereby significantly
decreasing the performance of the assay. Figure 29 shows that the placement of 6
consecutive LNAs (cytosine (C) complementary to the wild-type variant (G) and 5 bases up
complementary to the wild type allele) on the blocker probe completely arrested
amplification during PCR cycling.
Figure 30 shows that the ACt values can be used to determine the feasibility of the
assay and its selectivity. The higher ACt value obtained with the LNA-containing primers
and probes indicate the increased feasibility and selectivity of the assay of the present
invention. Figure 31 shows how the AACt values are calculated from the Ct values from
various somatic mutation genotyping assays. Assay A of the figure that was designed with an
LNA-containing primer and blocker performed better than the other assays that contained
primers and probes with consecutive LNAs.
In sum, this example shows that the use of nucleic acid modifications such as
modified bases (e.g., LNA) in allele-specific primers and blocker probes improves the
performance of the genotyping assay of the present invention. Likewise, the selectivity and
sensitivity for allelic variant discrimination increases.
Example 6. Quantitation of the Percentage of Allelic Variant Present in the Unknown
Sample Using Somatic Mutation Genotyping Assays.
This example illustrates the use of the allele-specific primers, allele-specific blocker
probes, and detector probes containing modified bases to quantitate the allelic variants for
E545K of the PIK3CA gene, G12D of the KRAS gene, E746-A750 deletion of the EGFR
gene, or V600E of the BRAF gene. This example also illustrates that the methods of the
invention are highly selective in differentiating and quantitating the mutant variant from the
wild-type variant. Additionally, the example shows that the assays of the present invention
are linear and can be used to determine quantitative information of the allelic variant.
Somatic mutation genotyping assays were performed using methods described
herein. Allele-specific primers and allele-specific blocker probes of the present invention
containing modified bases, nucleic acid analogs, and/or blocker moieties were used to detect
for the presence of the various allelic variants of the PIK3CA, EGFR, KRAS and BRAF
genes. Table 1 lists the mutations on the aforementioned genes.
Table 1.
In order to quantitate the amount of an allelic variant present in a given sample, a
standard curve was generated. The standard curve was made for each mutation from a cell
line positive for that specific mutation. Because the standard curve is linear, it can be used to
quantitate the allelic variant in an unknown sample. Table 1 shows the allelic variants and
the positive cell lines used to create the standard curve. DNA from the cell lines were
extracted using Qiagen's DNeasy Blood & Tissue Kit. A series of dilutions of DNA (e.g.,
100, 10, 1, 0.1 and 0.01 ng) from each positive cell line was made to create an allele-specific
standard curve. In this example standard curves for E545K of PIK3CA, G12D of KRAS,
E746-A750 deletion of EGFR, and V600E of BRAF were made.
An allele-specific calculator (e.g., mathematical analysis) was established from the
standard curve generated and on the assumption that the positive cell line has a percent
mutation of 100%. For some of the cell lines the % mutation was determined by using an
allele-specific primer that preferentially hybridizes to the wild-type allele.
To determine the amount of mutation in an unknown sample, the sample was
assayed using the methods of the present invention. Then, the Ct value for the allelic variant
of the sample was analyzed using the percent mutation calculator to determine the amount
and percent of the variant present in the sample.
Figure 32A shows that the standard curve for the PIK3CA E545K allelic variant and
the MCF 7 cell line. It was created using methods described herein. The standard curve plot
of Ct value versus DNA amount was linear in logio scale. Figure 32B shows amplification
curves for two unknown samples from patients with colorectal cancer (Samples A and B) and
the positive control (MCF 7 cell line) generated using the genotyping assay. Figure 32C
shows the amount and the percentage (percent mutation) of the mutant variant E545K present
in the samples. The amount and the percent (percent mutation) of the allelic variant present
in the samples was determined using the calculator (Figure 32C). In particular, the percent
mutation was calculated from the amount of mutation with respect to the starting amount of
DNA in the sample. It was determined that Sample A has a percent mutation of 15% for the
PIK3CA E545K variant, relative to the positive control. Sample B has a percent mutation of
7.3% for the same variant, relative to the positive control. Notably, the mutant allele was
detected in 20 ng of DNA from Sample B.
Figure 33 illustrates the use of the KRAS G12D assay of the present invention to
quantify the percentage of the mutant variant present in an unknown sample. The standard
curve plot was linear in log scale and was used to quantitate the amount and percent
mutation of the unknown samples. Figure 33A shows the amplification plot and the standard
curve for the KRAS G12D genotyping assay and the LS 174T cell line. Figure 33B shows
that amplification plots for two unknown samples from patients with pancreatic cancer
(Samples A and B) and a positive control (LS 174T cell line) that were generated using
methods of the present invention. Figure 33C shows the amount of DNA in Sample A
expressing the mutant variant was calculated to be 3.25 ng or 9.6%, relative to the positive
control.
Figure 34 illustrates the use of the EGFR E746-A750 deletion EGF assay of the
present invention to quantify the percentage of the mutant variant present in an unknown
sample. Figure 34A shows the amplification plot and standard curve for the E746-A750
deletion of the EGFR gene for the H1650 cell line. Figure 34B shows that the amplification
plot for an unknown sample (Sample A) from a patient with lung cancer and a positive
control (HI 650 cell line) that were generated using methods of the present invention. Figure
34C shows the amount of DNA in Sample A expressing the EGFR deletion variant was
calculated to be 3.25 ng or 9.6%, relative to the positive control.
Figure 35 illustrates the use of the V600E BRAF assay of the present invention to
quantify the percentage of the allelic variant present in an unknown sample. Figure 35A
shows the amplification plot and standard curve for the BRAF V600E allelic variant for the
HT 29 cell line. Figure 35B shows that the amplification plot for an unknown sample
(Sample A) from a patient with lung cancer and a positive control (HI 650 cell line) that were
generated using methods of the present invention. Figure 35C shows the amount of DNA in
Sample A expressing the V600E variant of BRAF was calculated to be 0.18 ng or 1.2%,
relative to the positive control.
This example shows that the assays of the present invention (e.g., PIK3CA E545K,
KRAS G12D, EGFR E746-A750 deletion, and BRAF V600E genotyping assays) are linear in
logio scale and can be used to determine quantitative information about an allelic variant in a
patient tissue sample.
Example 7. Determining Tumor Genetic Heterogeneity.
This example illustrates a correlation between the level of a cancer biomarker and
the percent mutation for a particular allelic variant of an oncogenic gene. In gastric tumor
samples, a high level of cytokeratins (CK), detected using a Collaborative Enzyme Enhanced
Reactive (CEER) immunoassay, corresponded to a high percent mutation for the KRAS
G13D allelic variant. In pancreatic tumor samples, a high CK level correlated to a percent
mutation of 100% for the KRAS G12D allelic variant. This example illustrates that the tumor
content of a tissue sample can be assessed using the genotyping assay of the present invention
and a CEER immunoassay (also known as COPIA; see, e.g., PCT Patent Publication Nos.
and , the disclosures of which are herein incorporated by
reference in their entirety for all purposes).
Tumor tissue samples are often a mixture of tumor and non-tumor cells (e.g., blood
or adjacent non-tumor tissue). Tumor cells in a solid cancer tissue sample can be assessed by
hematoxylin and eosin (H&E) staining. Figure 36 shows H&E stained frozen sections of a
non-small cell lung cancer (NSCLC) tumor sample. Figure 36A shows a section with a high
percentage of tumor cells (the white arrow indicates tumor cells). Figure 36B shows a
section composed of a mixture of tumor cells (white arrow), stroma with blood vessels (black
arrow), inflammatory cells (e.g., lymphocytes; red arrow); and a lung alveolus filled with
macrophages (green arrow).
Cytokeratins were used as a biomarker to detect tumor cells. High CK levels were
observed in tumor samples, while lower levels were detected in adjacent normal tissue. We
evaluated the levels of CK and the percent mutation of KRAS, EGFR, and PIK3CA allelic
variants in various gastric and pancreatic tumor samples to determine whether higher CK
levels correlates to a higher percent mutation for a particular mutant allele.
Figure 37A illustrates that in gastric tumor samples both a high CK level and a low
percent mutation for the EGFR T790M, KRAS G12V, KRAS Q61H, or PIK3CA E545K
variant indicate that not all of the tumor cells are likely to carry the SNP. Figure 37A also
shows that a high CK level with a high KRAS G13D percent mutation (e.g., 90%) indicated
that most of the tumor cells in the sample are likely to carry the mutation. Figure 37B shows
that a high level of CK correlates with a high percent mutation (100%) for the KRAS G12D
variant in a pancreatic tumor sample.
In sum, this example shows that the genotyping assays of the present invention can
be used to calculate the percent mutation of a patient's tumor sample. This example further
illustrates that the percent mutation of a specific allelic variant of an oncogenic gene can be
correlated to the expression level of a cancer biomarker.
Example 8. Sensitivity of the Somatic Mutation Genotyping Assay in Discriminating
Tumor Cells in Whole Blood Samples.
In this example, the sensitivity of the somatic mutation genotyping assays of the
present invention were determined by performing assays for the G12A or G12S mutation of
KRAS on whole blood samples spiked with tumor cells carrying the mutation. Allele-
specific primers and allele-specific blocker probes of the present invention containing
modified bases such as LNA modifications were used to detect for the presence of the KRAS
G12A or G12S variant allele. This example also compares the sensitivity of the somatic
mutation genotyping assays of the present invention and Life Technologies' castPCR
Mutation Assay.
The sensitivity of the KRAS G12A assay of the present invention (e.g., "Inventive
Somatic Mutation Genotyping Assay") was tested by determining the minimum amount of
positive tumor cells (e.g., SW1 16 cell line) needed to detect the KRAS mutation in a whole
blood sample. The test samples comprised of whole blood spiked with cells of the SW1 16
cell line (e.g., either 100,000; 10,000; 1,000; 500; 250; 100; 50; 10 or 0 tumor cells per
sample). Genomic DNA from the test samples were extracted using Qiagen's DNeasy Blood
& Tissue Kit. The assays of the present invention were performed using the methods
indicated herein. Figure 38A illustrates the amplification curves generated from the KRAS
G12A assay of the present invention. Figure 38B illustrates the amplification curves from
Life Technologies' castPCR™ Mutation Assays performed on the same test samples.
As shown in Figure 38C, the genotyping assay of the present invention detected the
G12A mutation when the test sample contained as few as 250 positive tumor cells. By
comparison, a minimum of 1,000 tumor cells in the sample were needed for the castPCR™
assay. The results show that the assay of the present invention has greater sensitivity than the
castPCR™ assay for detecting the G12A KRAS mutation in whole blood samples.
To evaluate the KRAS G12S assay of the present invention, whole blood samples
were spiked with cells of the A549 cell line (e.g., either 100,000; 10,000; 1,000; 500; 250;
100; 50; 10; or 0 positive tumor cells in the test sample of whole blood). Using Qiagen's
DNeasy Blood & Tissue Kit, genomic DNA of the test samples were extracted. The presence
of the KRAS G12S variant was detected using the assay of the present invention and Life
Technologies' castPCR™ assay. The sensitivity of the assays was compared. Figure 39A
shows the amplification curves of the test samples using the assay of the present invention.
Figure 39B shows the amplification curves for Life Technologies' castPCR™ Assay.
The assays of the present invention detected the G12S mutation in samples with as
few as 100 tumor cells (Figure 39C). CastPCR™ was 10-fold less sensitive; the assay
detected the mutation in test samples containing 1,000 or more positive tumor cells. Figure
39C illustrates that the genotyping assay of the present invention has greater sensitivity than
castPCR™ for the detection of the G12S KRAS mutation in whole blood samples.
In sum, this example demonstrates that the somatic mutation genotyping assays of
the present dramatically improve the allelic PCR assay compared to the castPCR™ assay
from Life Technologies.
Example 9. Exemplary Somatic Mutation Genotyping Assays for Detection of Allelic
Variants and Determining the Percentage of the Variant in a Sample.
This example illustrates the methods of the present invention for detecting an
oncogenic single nucleotide polymorphism (SNP) in a sample and quantitating the percent
mutation of the SNP in the sample. This example also illustrates the screening of samples
(e.g., cancer cell lines and tissue from patients with cancer) for the presence of rare (e.g.,
mutant) variant alleles of using the allele-specific primers and allele-specific blocker probes
of the present invention containing modified bases. In particular, the allele-specific primer
comprises a locked nucleic acid (LNA) at the 3'-end that is specific for the variant allele and
the allele-specific blocker probe comprises a hexanediol blocker moiety at the 3'-end and a
LNA at a position in the middle of the oligonucleotide sequence that is specific for the wild-
type allele. In this example, the allelic variants detected include PIK3CA E542K, E545D,
E545K and H1047R; EGFR T790M, L858R and E746 deletion; KRAS G12C, G12R, G12S,
G12D, G12A, G12V, G13C, G13D, and Q61H; and BRAF V600E. This example also shows
that the methods of the invention can be used with various samples from cancers and tumors
such as breast cancer, colorectal cancer, lung tumor, gastric tumor, liver tumor, colon tumor,
and pancreatic tumor; cell lines (e.g., colorectal cancer cell line) and xenograft tissue.
The presence of various SNPs was determined and the percentage of the detected
variant in the sample was quantitated using methods of the present invention, such as allele-
specific real time PCR assays, establishing standard curves from assays performed on cell
lines expressing the variant, and creating a percent mutation calculator for each variant.
Briefly, to establish a standard curve for an allelic variant, SNP detection assays of the
invention were performed using serial dilutions of cell lines positive for the allelic variant.
The results of the assays were used to make a standard curve, and then a percent mutation
calculator for the allelic variant was created that can predict the amount of the allelic variant
present in a sample based on data from the SNP detection assay obtained for the sample.
Figure 40 shows the results obtained by using the methods of the present invention
to detect (e.g., presence or absence) and/or quantitate (e.g., percent mutation) the following
SNPs in breast cancer samples: PIK3CA E542K, E545D, E545K, H1047R; EGFR T790M,
L858R; KRAS G12A, G12C, G12D, G12R, G12S, G12V, and G13D; and BRAF V600E. It
was determined that the PIK3CA H1047R SNP was expressed at different percentages in the
samples. For instance, test # 1 expressed H1047R allele at 12%, while test #33 expressed the
same SNP at 41%. Test #2 expressed the mutant variant at 1%. Test #34 expressed another
PIK3CA SNP (e.g., E545K) at 100%, which predicts that all cells in the test sample have the
E545K variant.
Figure 4 1 shows that PIK3CA SNPs (E542K, E545D, E545K and H1047R) were
also detected and quantitated in another set of breast cancer samples. 45 breast cancer
samples were screened for the PIK3CA E542K, E545D, E545K and H1047R allelic variants.
The percent mutation of the variant was quantitated using methods described above and in
Example 6. The results show that sample #744 expressed E542K at a very low percentage
(e.g., 0.13%) and that sample #743 expressed H1047R at a high percentage of 100%. Other
samples that expressed the E542K variant were sample #762 at 2% and sample #767 at
3.55%. Other samples that expressed the H1047R variant were samples #746 with 89%
mutation, #775 with 51.8% mutation, #740 with 6.8% mutation and #769 with 5.9%
mutation.
Figure 42 shows that lung tumor samples can be screened for SNPs using methods
of the present invention. In this embodiment, the presence and percent mutation of various
SNPs were determined in 25 lung tumor samples. The SNPs included PIK3CA E542K,
E545D, E545K and H1047R; EGFR T790M, L858R and E746 deletion; KRAS G12C, G12R,
G12S, G12D, G12A, G12V, G13C, G13D, and Q61H; and BRAF V600E. Samples #352-
355 all expressed PIK3CA E545K at 100% which indicates that all cells in these samples
have the mutant variant. Samples #371-375 and 381 all expressed EGFR L848R at 100%.
The EGFR E746 deletion variant was detected in two samples (#164 and #381) at very low
rates (0. 1% and 0.2%, respectively). The only sample in the set that expressed the BRAF
V600E variant was sample #213 which had a mutation percentage of 0.2%.
Figure 43 illustrates the results obtained from using the methods of the present
invention on an additional 32 human lung tumor samples. The SNPs included PIK3CA
E542K, E545D, E545K and H1047R; EGFR T790M, L858R and E746 deletion; KRAS
G12C, G12R, G12S, G12D, G12A, G12V, and G13D; and BRAF V600E. Sample #9 of this
set expressed the KRAS mutations G12S at 0.3%, G12V at 2% and G13D at 0.3%. Samples
#3 and 12 expressed the PIK3CA E545K variant at 100%. Sample #25 expressed PI3KCA
E545D at 0.2% and sample #6 expressed PIK3CA H1047R at 0.06%. Samples #13 and 19
had KRAS G12C at 100%, while sample #22 had the same variant at only 2%. Sample #22
also expressed the BRAF V600E variant at 54%. The KRAS G12V variant was present in
sample #14 at a percentage of 100% and in sample #15 at 0.1%. The BRAF V600E variant
was detected in samples #4 and 3 1 at 47% and 34%, respectively.
Figure 44 shows that gastric tumor samples can be screened using methods of the
present invention. The SNPs included PIK3CA E542K, E545D, E545K and H1047R; EGFR
T790M, L858R and E746 deletion; KRAS G12C, G12R, G12S, G12D, G12A, G12V, G13D,
and Q61H; and BRAF V600E. In this embodiment, each assay was run with 40 ng of sample
(e.g., DNA). Of the 17 samples assayed, sample #233 expressed four KRAS mutations, such
as G12R at 0.001%, G12V at 2.3%, G13D at a "low" percentage, and Q61H at 0.5%. The
KRAS G12C variant was detected at "low" percentage in sample #241. The PIK3CA E542K
allele was detected in sample #253 at 0.6% and the EGFR T790M allele was detected in
sample #223 at 0.2%. Compared to other tumor samples such as breast cancer and lung
tumor, the percent mutation of the SNPs in the gastric tumor samples tested was not higher
than at 100% for any of the allelic variants screened.
Figure 45 shows the results obtained from using the methods of the invention with
xenograft samples. The SNPs included PIK3CA E542K, E545D, E545K and H1047R;
EGFR T790M, L858R and E746 deletion; KRAS G12C, G12R, G12S, G12D, G12A, G12V,
G13C, G13D, and Q61H; and BRAF V600E. The EGFR E746 deletion was present in
samples #585-588 and predicted to be in 100% of the cells in the sample. The PIK3CA
H1047R allele was detected in samples #581-584 at a percentage mutation of 3.4%, 1.2%,
1% and 1.8%, respectively.
Figure 46 illustrates that KRAS, BRAF and PIK3CA allelic variants can be detected
and quantitated in colorectal cancer samples using the methods of the present invention. The
SNPs included PIK3CA E542K, E545D, E545K and H1047R; KRAS G12C, G12R, G12S,
G12D, G12A, G12V, and G13D; and BRAF V600E. The data shows that sample #121
expressed the KRAS G13D variant at 6% and PIK3CA E545K at 58%. Sample #130
expressed the KRAS G13V variant at 54% and PIK3CA E542K at 5%.
Figure 47 also illustrates that KRAS, BRAF and PIK3CA allelic variants can be
detected and quantitated in additional colorectal cancer samples using the methods of the
invention. The data shows that sample #147 expressed the KRAS G12D and PIK3CA
variants at 100%, indicating that all cells in the sample are predicted to express the variants.
Sample #149 expressed the KRAS G13D variant at 24% and PIK3CA H1047R at 0.1%.
Sample #163 expressed KRAS G12D at 34% and PIK3CA E542K at 3%.
Figure 48 illustrates that liver tumor and colon tumor tissues from patients with
colorectal cancer can be screened for KRAS, BRAF and PIK3CA allelic variants using the
methods of the present invention. The results shows that some of the samples had a plurality
of SNPs. For instance, Samples #207 and #208 expressed KRAS G12S and PIK3CA E545K.
Using methods of the present invention, it was also determined that sample #207 expressed
G12S at 22% and sample #208 expressed the same variant at 63%. Sample #207 expressed
the E545K variant at 40% and sample #208 expressed the same variant at 79%. Sample #215
had BRAF V600E at 1% and PIK3 CA E545K at 5%. Sample #216 had BRAF V600E at 9%
and PIK3CA E545K at 23%. Sample #217 expressed KRAS G12V at 2% and PIK3CA 545K
at 5%. Sample #217 had KRAS G12V at 4% and PIK3CA 545K at 9%.
Figure 49 illustrates that samples from patients with pancreatic cancer can be
screened for SNPs and the percent mutation can be determined according to methods of the
present invention. In this embodiment, fine needle aspirate samples were from obtained from
patients and screened using SNP genotyping assays described herein. In the pancreatic
cancer samples tested, various KRAS mutations were detected, but PIK3CA (e.g., E542K,
E545D, E545K, H1047R), EGFR (e.g., T790M, L858R) and BRAF (e.g., V600E) mutations
were not detected. Sample #28 expressed the KRAS G12C variant. The KRAS G12V
variant was present in #19, 21, 26, 27 and 35, at percentage mutations of 63%, 0.2%, 4%, 1%
and 3%, respectively. Samples #9 and 11 expressed the KRAS G12D variant at 100%. This
variant was also expressed in samples #2, 6, 12, 23, 24, 37, 39 and 40 at 1.1%, 29%, 7.4%,
%, 5%, 32%, 5.4% and 0.9%, respectively. The SNPs screened were not detected in the
other pancreatic tumor samples in the set.
In sum, this example demonstrates that the somatic genotyping assays of the present
invention can be used to detect and/or quantitate allelic variants in genes such as KRAS,
PIK3CA, EGFR and BRAF. The examples shows that the methods of the present invention
can be used to detect a plurality of allelic variants in cancer and tumor tissue samples from
patients. Furthermore, the percentage of the allelic variant in the sample can be determined.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, one of skill in the art will
appreciate that certain changes and modifications may be practiced within the scope of the
appended claims. In addition, each reference provided herein is incorporated by reference in
its entirety to the same extent as if each reference was individually incorporated by reference.
Claims (52)
1. A method for detecting or quantitating a first allelic variant of a target sequence in a nucleic acid sample suspected of having at least a second allelic variant of the target sequence, said method comprising: (a) forming a reaction mixture by combining: (i) the nucleic acid sample; (ii) an allele-specific primer, wherein an allele-specific nucleotide portion of the allele- specific primer is complementary to the first allelic variant of the target sequence, and wherein the allele-specific primer comprises at least one nucleic acid modification; (iii) an allele-specific blocker probe that is complementary to a region of the target sequence comprising the second allelic variant, wherein the allele-specific blocker probe comprises a non-extendable, 3’-hexanediol, blocker moiety and at least one nucleic acid modification; (iv) a detector probe; and (v) a locus-specific primer that is complementary to a region of the target sequence, wherein the region of the target sequence complementary to the locus-specific primer 3’ from the first allelic variant; and on the opposite strand; and (b) carrying out an amplification reaction on the reaction mixture using the locus-specific primer and the allele-specific primer to form an amplicon; and (c) detecting the amplicon by detecting a change in a detectable property of the detector probe, thereby detecting the first allelic variant of the target gene in the nucleic acid sample.
2. The method of claim 1, wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the allele-specific nucleotide portion.
3. The method of claim 1 or claim 2, wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the 5’-end and/or 3’-end of the allele-specific primer.
4. The method of any one of claims 1 to 3, wherein the allele-specific primer comprises two or more non-consecutive nucleic acid modifications.
5. The method of any one of claims 1 to 4, wherein the nucleic acid modification in the allele-specific primer is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
6. The method of any one of claims 1 to 5, wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at the allele-specific nucleotide portion.
7. The method of any one of claims 1 to 6, wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at an internal position in the allele-specific blocker probe.
8. The method of any one of claims 1 to 7, wherein the allele-specific blocker probe comprises two or more non-consecutive nucleic acid modifications.
9. The method of any one of claims 1 to 8, wherein the nucleic acid modification in the allele-specific blocker probe is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
10. The method of any one of claims 1 to 9, wherein the detector probe comprises a hydrolysis probe.
11. The method of any one of claims 1 to 10, wherein the nucleic acid sample is selected from the group consisting of blood, serum, plasma, fine needle aspirate, tumor tissue, and combinations thereof.
12. The method of any one of claims 1 to 11, wherein the first allelic variant is a mutant allele and the second allelic variant is the wild-type allele.
13. The method of any one of claims 1 to 12, wherein the method reduces the background signal of the second allelic variant during the amplification reaction.
14. A composition comprising: (a) 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, and wherein the allele-specific primer comprises at least one nucleic acid modification; and (b) an allele-specific blocker probe that is complementary to a region of the target sequence comprising a second allelic variant, wherein the allele-specific blocker probe comprises a non-extendable, 3’-hexanediol, blocker moiety and at least one nucleic acid modification.
15. The composition of claim 14, further comprising: (c) a detector probe; and/or (d) a locus-specific primer that is complementary to a region of the target sequence, wherein the region of the target sequence complementary to the locus-specific primer is: 3’ from the first allelic variant; and on the opposite strand.
16. The composition of claim 14 or claim 15, wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the allele-specific nucleotide portion.
17. The composition of any one of claims 14 to 16, wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the 5’-end and/or 3’-end of the allele-specific primer.
18. The composition of any one of claims 14 to 17, wherein the allele-specific primer comprises two or more non-consecutive nucleic acid modifications.
19. The composition of any one of claims 14 to 18, wherein the nucleic acid modification in the allele-specific primer is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
20. The composition of any one of claims 14 to 19, wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at the allele-specific nucleotide portion.
21. The composition of any one of claims 14 to 20, wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at an internal position in the allele-specific blocker probe.
22. The composition of any one of claims 14 to 21, wherein the allele-specific blocker probe comprises two or more non-consecutive nucleic acid modifications.
23. The composition of any one of claims 14 to 22, wherein the nucleic acid modification in the allele-specific blocker probe is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
24. The composition of any one of claims 14 to 23, wherein the first allelic variant is a mutant allele and the second allelic variant is the wild-type allele.
25. A reaction mixture comprising the composition of claim 15 and further comprising: (e) a nucleic acid molecule.
26. The reaction mixture of claim 25, wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the allele-specific nucleotide portion.
27. The reaction mixture of claim 25 or claim 26, wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the 5’-end and/or 3’-end of the allele-specific primer.
28. The reaction mixture of any one of claims 25 to 27, wherein the allele-specific primer comprises two or more non-consecutive nucleic acid modifications.
29. The reaction mixture of any one of claims 25 to 28, wherein the nucleic acid modification in the allele-specific primer is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
30. The reaction mixture of any one of claims 25 to 29, wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at the allele- specific nucleotide portion.
31. The reaction mixture of any one of claims 25 to 30, wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at an internal position in the allele-specific blocker probe.
32. The reaction mixture of any one of claims 25 to 31, wherein the allele-specific blocker probe comprises two or more non-consecutive nucleic acid modifications.
33. The reaction mixture of any one of claims 25 to 32, wherein the nucleic acid modification in the allele-specific blocker probe is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
34. The reaction mixture of any one of claims 25 to 33, wherein the detector probe comprises a hydrolysis probe.
35. The reaction mixture of any one of claims 25 to 34, wherein the nucleic acid molecule is obtained from a sample selected from the group consisting of blood, serum, plasma, fine needle aspirate, tumor tissue, and combinations thereof.
36. The reaction mixture of any one of claims 25 to 35, wherein the first allelic variant is a mutant allele and the second allelic variant is the wild-type allele.
37. A kit comprising two or more containers comprising the following components independently distributed in one of the two or more containers: (a) 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, and wherein the allele-specific primer comprises at least one nucleic acid modification; and (b) an allele-specific blocker probe that is complementary to a region of the target sequence comprising a second allelic variant, wherein the allele-specific blocker probe comprises a non-extendable, 3’-hexanediol, blocker moiety and at least one nucleic acid modification.
38. The kit of claim 37, further comprising: (c) a detector probe; and/or (d) a locus-specific primer that is complementary to a region of the target sequence, wherein the region of the target sequence complementary to the locus-specific primer is: 3’ from the first allelic variant; and on the opposite strand.
39. The kit of claim 37 or claim 38, wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the allele-specific nucleotide portion.
40. The kit of any one of claims 37 to 39, wherein one of the at least one nucleic acid modification in the allele-specific primer is located at the 5’-end and/or 3’-end of the allele-specific primer.
41. The kit of any one of claims 37 to 40, wherein the allele-specific primer comprises two or more non-consecutive nucleic acid modifications.
42. The kit of any one of claims 37 to 41, wherein the nucleic acid modification in the allele- specific primer is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
43. The kit of any one of claims 37 to 42, wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at the allele-specific nucleotide portion.
44. The kit of any one of claims 37 to 43, wherein one of the at least one nucleic acid modification in the allele-specific blocker probe is located at an internal position in the allele-specific blocker probe.
45. The kit of any one of claims 37 to 44, wherein the allele-specific blocker probe comprises two or more non-consecutive nucleic acid modifications.
46. The kit of any one of claims 37 to 45, wherein the nucleic acid modification in the allele- specific blocker probe is selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), threose nucleic acids (TNA), zip nucleic acids (ZNA), triazole nucleic acids (TzNA), and combinations thereof.
47. The kit of any one of claims 37 to 46, wherein the first allelic variant is a mutant allele and the second allelic variant is the wild-type allele.
48. The kit of any one of claims 37 to 47, further comprising instructions for use of the allele- specific primer and the allele-specific blocker probe for detecting or quantitating the first allelic variant of the target sequence in a nucleic acid sample suspected of having the second allelic variant of the target sequence.
49. The method of claim 1, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
50. The composition of claim 14, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
51. The reaction mixture of claim 25, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
52. The kit of claim 37, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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US201161525137P | 2011-08-18 | 2011-08-18 | |
US61/525,137 | 2011-08-18 | ||
US201261588151P | 2012-01-18 | 2012-01-18 | |
US61/588,151 | 2012-01-18 | ||
PCT/US2012/051442 WO2013026027A1 (en) | 2011-08-18 | 2012-08-17 | Compositions and methods for detecting allelic variants |
Publications (2)
Publication Number | Publication Date |
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NZ620654A NZ620654A (en) | 2016-02-26 |
NZ620654B2 true NZ620654B2 (en) | 2016-05-27 |
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