U.S. patent application number 15/361280 was filed with the patent office on 2017-05-25 for methods for variant detection.
The applicant listed for this patent is Integrated DNA Technologies, Inc.. Invention is credited to Mark Aaron BEHLKE, Kristin BELTZ, Caifu CHEN, Joseph DOBOSY, Garrett RETTIG, Scott ROSE, Pak Wah TSANG.
Application Number | 20170145486 15/361280 |
Document ID | / |
Family ID | 57590833 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170145486 |
Kind Code |
A1 |
CHEN; Caifu ; et
al. |
May 25, 2017 |
METHODS FOR VARIANT DETECTION
Abstract
The invention can be used to provide a more efficient and less
error-prone method of detecting variants in DNA, such as SNPs and
indels. The invention also provides a method for performing
inexpensive multiplex assays.
Inventors: |
CHEN; Caifu; (Palo Alto,
CA) ; DOBOSY; Joseph; (Coralville, IA) ;
TSANG; Pak Wah; (Fremont, CA) ; BEHLKE; Mark
Aaron; (Coralville, IA) ; ROSE; Scott;
(Coralville, IA) ; BELTZ; Kristin; (Cedar Rapids,
IA) ; RETTIG; Garrett; (Coralville, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Integrated DNA Technologies, Inc. |
Coralville |
IA |
US |
|
|
Family ID: |
57590833 |
Appl. No.: |
15/361280 |
Filed: |
November 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62339317 |
May 20, 2016 |
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62259913 |
Nov 25, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6858 20130101;
C12Q 1/6858 20130101; C12Q 2525/131 20130101; C12Q 2525/121
20130101; C12Q 2525/186 20130101; C12Q 1/6827 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-9. (canceled)
10: A method of detecting a variation in a target DNA sequence, the
method comprising: (a) providing a reaction mixture comprising: (i)
an oligonucleotide primer having a cleavage domain positioned 5' of
a blocking group and 3' of a position of variation, the blocking
group linked at or near the end of the 3'-end of the
oligonucleotide primer wherein the blocking group prevents primer
extension and/or inhibits the primer from serving as a template for
DNA synthesis, (ii) a sample nucleic acid that may or may not have
the target sequence, and where the target sequence may or may not
have the variation, (iii) a cleaving enzyme, and (iv) a polymerase;
(b) hybridizing the primer to the target DNA sequence to form a
double-stranded substrate; (c) cleaving the hybridized primer, if
the primer is complementary at the variation, with the cleaving
enzyme at a point within or adjacent to the cleavage domain to
remove the blocking group from the primer; and (d) extending the
primer with the polymerase.
11: The method of claim 10, wherein the cleaving enzyme is a hot
start cleaving enzyme that is thermostable and has reduced activity
at lower temperatures.
12: The method of claim 10, wherein the cleaving enzyme is a
chemically modified hot start cleaving enzyme that is thermostable
and has reduced activity at lower temperatures.
13: The method of claim 12, wherein the hot start cleaving enzyme
is a chemically modified Pyrococcus abyssi RNase H2.
14: The method of claim 10, wherein the cleaving enzyme is a hot
start cleaving enzyme that is reversibly inactivated through
interaction with an antibody at lower temperatures.
15: The method of claim 10, wherein the cleaving domain is
comprised of at least one RNA base, and the cleaving enzyme cleaves
between the position complementary to the variation and the RNA
base.
16: The method of claim 10, wherein the cleaving domain is
comprised of one or more 2'-modified nucleosides, and the cleaving
enzyme cleaves between the position complementary to the variation
and the one or more modified nucleosides.
17: The method of claim 16, wherein the one or more modified
nucleosides are 2'-fluoronucleosides.
18: The method of claim 10, wherein the polymerase is a
high-discrimination polymerase.
19: The method of claim 10, wherein the polymerase is a mutant
H784Q Taq polymerase.
20: The method of claim 19, wherein the mutant H784Q Taq polymerase
is reversibly inactivated via chemical, aptamer, or antibody
modification.
21: The method of claim 10, wherein the primer contains a 5' tail
sequence that comprises a universal primer sequence and optionally
a universal probe sequence, wherein the tail is non-complementary
to the target DNA sequence.
22-53. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/339,317, filed May 20, 2016, and also claims the
benefit of U.S. Provisional Application No. 62/259,913, filed Nov.
25, 2015, the disclosures of both of which are hereby incorporated
by reference in their entireties.
FIELD OF THE INVENTION
[0002] The invention can be used to provide a more efficient and
less error-prone method of detecting variants in DNA, such as
single nucleotide polymorphisms (SNPs), multi-nucleotide
polymorphisms (MNPs), and indels. The invention also provides a
method for performing inexpensive multi-color assays, and provides
methods for visualizing multiple allele results in a
two-dimensional plot.
BACKGROUND OF THE INVENTION
[0003] RNase H2-dependent PCR (rhPCR) (see U.S. Patent Application
Publication No. US 2009/0325169 A1, incorporated by reference
herein in its entirety) and standard allele-specific PCR (ASPCR)
can both be utilized for mutation detection. In ASPCR, the DNA
polymerase performs the mismatch discrimination by detection of a
mismatch at or near the 3' end of the primer. While ASPCR is
sometimes successful in mismatch detection, the discrimination can
be limited, due to the low mismatch detection ability of wild-type
DNA polymerases.
[0004] In contrast with ASPCR, the mismatch sensitivity of the
RNase H2 enzyme in rhPCR allows for both sensitive detection of DNA
mutations, and elimination of primer-dimer artifacts from the
reaction. When attempting to detect DNA mutations with rhPCR,
however, placement of the mismatch within the primer is important.
The nearer to the cleavable RNA the mismatch is located, the more
discrimination is observed from the RNase H2 enzyme, and the
greater the discrimination of the resulting rhPCR assay. Given the
fact that most common wild-type DNA polymerases such as Taq often
display low levels of mismatch detection, the polymerase cannot be
solely relied upon to perform this discrimination after RNase H2
cleavage. Coupled with the repeated interrogation desired from
every cycle of standard rhPCR, placing the mismatch anywhere other
than immediately opposite the RNA is undesirable when utilizing
these polymerases.
BRIEF SUMMARY OF THE INVENTION
[0005] The disclosure provides assays making use of high
discrimination polymerase mutants or other high mismatch
discrimination polymerases to create a new assay design that can
utilize mismatches located 5' of the RNA.
[0006] The invention can be used to provide a more efficient and
less error-prone method of detecting mutations in DNA, such as SNPs
and indels. The invention also provides a method for performing
inexpensive multi-color assays.
[0007] These and other advantages of the invention, as well as
additional inventive features, will be apparent from the
description of the invention provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a diagram showing two primer designs utilized in
this invention. Part a) is a blocked-cleavable primer designed so
that the SNP of interest is 5' of the RNA base when hybridized to a
template. The RNase H2 cleaves, leaving a 3' interrogating base,
which is determined to be either a match or a mismatch by the
highly discriminative DNA polymerase. Thermal cycling allows for
this process to continue. Part b) illustrates the RNase H2 cleavage
and SNP detection are identical to a), but the primer also includes
a 5' "tail" domain that includes a binding site for a probe and a
universal forward primer. After 1-10 cycles of discrimination with
the RNase H2 and the polymerase, the highly concentrated universal
forward primer comes to dominate the amplification, degrading the
probe when it amplifies. This cycle is repeated 25-50.times.,
generating the output signal. This primer design may be
multiplexed, allowing for one-tube multi-color assay designs.
[0009] FIGS. 2A and 2B are end-point fluorescence plots from the
assay described in Example 1. FAM and HEX fluorescence values are
plotted onto the X and Y axis. FIG. 2A is a "Universal" SNP assay
for rs351855 performed with WT Taq polymerase. FIG. 2B is a
"Universal" SNP assay for rs351855 performed with mutant H784Q Taq
polymerase, demonstrating greatly enhanced discrimination between
each of the allelic variants as observed by the greater separation
of the clusters in the mutant Taq case. In both cases, the no
template controls (NTCs) (squares) are near the (0,0) coordinates,
as desired. Allele 1 samples are shown as circles, allele 2 samples
as diamonds, and heterozygotes as triangles. Each reaction was
performed in triplicate.
[0010] FIGS. 3A and 3B are allelic discrimination plots with
genotyping calls for rs4655751. The reaction plate was cycled
immediately after reaction setup (A) or held at room temperature on
the benchtop for 48 hours prior to cycling (B). Diamonds: no
template controls (NTCs); squares: allele 1 samples; circles:
allele 2 samples; triangles: heterozygotes. Genotypes are tightly
clustered and have good angle separation, indicating excellent
allelic specificity. Each sample was assigned the correct
genotyping call, and no change in performance was observed over the
48 hour hold period.
[0011] FIGS. 4A and 4B illustrate a side-by-side comparison of
Allelic Discrimination Plots of gene CCR2, rs1799865 from a TaqMan
based assay versus rhPCR. Diamonds: no template controls (NTCs);
squares: allele 1 samples; circles: allele 2 samples; triangles:
heterozygotes. The rhPCR Genotyping Assay (FIG. 4B) achieved higher
fluorescence signal compared to a traditional 5'-nuclease
genotyping assay (FIG. 4A) while showing concordant results.
[0012] FIGS. 5A and 5B are Allelic Discrimination plots of
tri-allelic SNP, CYP2C8 (rs72558195), using an rhPCR genotyping
single tube multiplex assay on the QuantStudio.TM. 7 Flex platform
(Thermo Fisher). In FIG. 5A, diamonds: no template controls (NTCs);
squares: allele G (allele 1) samples; circles: allele A (allele 2)
samples; triangles: heterozygotes. In FIG. 5B, diamonds: no
template controls (NTCs); squares: allele G (allele 1) samples;
circles: allele C (allele 3) samples; triangles: heterozygotes.
[0013] FIG. 6 shows the Tri-allelic Allelic Discrimination 360plot
of CYP2C8 rs72558195, using rhPCR genotyping assay with 3
allele-specific primers multiplexed in a single reaction.
[0014] FIG. 7 is an allelic discrimination plot illustrating the
ability of the rhPCR assay to perform quantitative genotyping.
[0015] FIGS. 8A and 8B illustrate genotyping results and detection
of allelic copy number variation that is possible with the present
invention. gDNA samples were tested using varying copy numbers and
varying reference genotypes. In FIG. 8A, diamonds: no template
controls (NTCs); squares: allele G samples; circles: allele C
samples; and triangles: heterozygotes. The resulting data
correlates with the test input.
[0016] FIG. 9 is a schematic representation of multiplex rhPCR.
[0017] FIG. 10 is the resulting tape station image indicating the
effectiveness of the multiplex rhPCR methods in reducing primer
dimers and increasing desired amplicon yield.
[0018] FIG. 11 graphically represents the effectiveness of the
rhPrimers in the percent of mapped reads and on-target reads.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The invention pertains to a methods of single-nucleotide
polymorphism (SNP) discrimination utilizing blocked-cleavable rhPCR
primers (see U.S. Patent Application Publication No. US
2009/0325169 A1, incorporated by reference herein in its entirety)
and a DNA polymerase with high levels of mismatch discrimination.
In one embodiment, the mismatch is placed at a location other than
opposite the RNA base. In these situations, the majority of the
discrimination comes not from the RNase H2, but from the high
discrimination polymerase. The use of blocked-cleavable primers
with RNase H2 acts to reduce or eliminate primer-dimers and provide
some increased amount of SNP or indel (insertion/deletion)
discrimination (FIG. 1a).
[0020] For the purposes of this invention, high discrimination is
defined as any amount of discrimination over the average
discrimination of WT Thermus aquaticus (Taq) polymerase. Examples
include KlenTaq.RTM. DNA polymerase (Wayne Barnes), and mutant
polymerases described in U.S. Patent Application Publication No. US
2015/0191707 (incorporated by reference herein in its entirety)
such as H784M, H784S, H784A and H784Q mutants.
[0021] In a further embodiment a universal detection sequence(s) is
added to the 5'-end of the blocked-cleavable primers. The detection
sequence includes a binding site for a probe, and a binding site
for a universal amplification primer. The primer binding site is
positioned at or near the 5'-end of the final oligonucleotide and
the probe binding site is positioned internally between the
universal primer site and the SNP-detection primer domain. Use of
more than one such chimeric probe in a detection reaction wherein
distinct probe binding sites are employed allows primers to be
multiplexed and further allows for multiple color detection of SNPs
or other genomic features. Blocked-cleavable rhPCR primers reduce
or eliminate primer-dimers. Primer-dimers are a major problem for
use of "universal" primer designs in SNP detection assays, and that
limits their utility (FIG. 1b). Combining a universal
amplification/detection domain with a SNP primer domain in
blocked-cleavable primer format overcomes this difficulty.
[0022] Previously, the best preferred embodiment for rhPCR SNP
discrimination employed blocked-cleavable primers having the
mismatch (SNP site) positioned opposite the single RNA base
(cleavage site). While this works for many SNP targets, there are
base match/mismatch pairings where sufficient discrimination is not
obtained for robust base calling. Moreover, due to the high level
of differential SNP discrimination observed with rhPCR, end-point
detection can be difficult, especially with heterozygous target
DNAs. In the proposed method, the RNA base is identical in both
discriminating primers, eliminating this issue.
[0023] In one embodiment of the invention, the method involves the
use of blocked-cleavable primers wherein the mismatch is placed 1-2
bases 5' of the RNA. In a further embodiment, the method involves
the use of blocked-cleavable primers with three or more DNA bases
3' of an RNA residue, and the primers are designed such that the
mismatch is placed immediately 5' of the RNA.
[0024] Following cleavage by RNase H2, the remaining primer has a
DNA residue positioned at the 3'-end exactly at the SNP site,
effectively creating an ASPCR primer. In this configuration, a
high-specificity DNA polymerase can discriminate between match and
mismatch with the template strand (FIGS. 1a and b). Native DNA
polymerases, such as Taq DNA polymerase, will show some level of
discrimination in this primer configuration, and if the level of
discrimination achieved is not sufficient for robust SNP calling in
a high throughput assay format then the use of polymerases with
improved template discrimination can be used. In one embodiment,
mutant DNA polymerases, such as those disclosed in U.S. Patent
Application Publication No. US 2015/0191707 (incorporated by
reference herein in its entirety) or any other polymerase designed
or optimized to improve template discrimination can be used. When
using polymerases with increased mismatch discrimination, the final
level of match/mismatch discrimination achieved will be additive
with contributions from both the ASPCR primer polymerase
interaction and from the rhPCR primer/RNaseH2 interaction. Further,
the use of blocked-cleavable primers reduces risk of primer-dimer
formation, which produces false-positive signals, making the
overall reaction more robust and having higher sensitivity and
higher specificity. The relative contributions of each component of
the assay may vary with use of different polymerases, different
blocking groups on the 3'-end of the primer and different RNase H2
enzymes.
[0025] In another embodiment, the invention may utilize a "tail"
domain added to the 5' end of the primer, containing a universal
forward primer binding site sequence and optionally a universal
probe sequence. This tail would not be complementary to the
template of interest, and when a probe is used, the tail would
allow for inexpensive fluorescent signal detection, which could be
multiplexed to allow for multiple color signal detection in qPCR
(FIG. 1b). In one embodiment, 1-10 cycles of initial cycling and
discrimination occurs from both the RNase H2 and the DNA
polymerase. After this initial pre-cycling, a highly concentrated
and non-discriminatory universal forward primer comes to dominate
the amplification, degrading the probe and generating the
fluorescent signal when the DNA amplifies. This cycle is repeated
25-50.times., allowing for robust detection. This assay design is
prone to issues with primer-dimers, and the presence of the
blocked-cleavable domain in the primers will suppress or eliminate
these issues.
[0026] In another embodiment, a forward primer is optionally used
with a reverse primer, and a tail domain is added to the 5' end of
one or both of a forward and reverse primer set. The tail domain
comprises a universal forward primer binding site. The primers can
be used to hybridize and amplify a target such as a genomic sample
of interest. The primers would add universal priming sites to the
target, and further cycles of amplification can be performed using
universal primers that contain adapter sequences that enable
further processing of the sample, such as the addition of P5/P7
flow cell binding sites and associated index or barcoding sequences
useful in adapters for next-generation sequencing (see FIG. 9). In
a further embodiment a high fidelity polymerase is used, which will
further lower the rate of base misincorporation into the extended
product and increase the accuracy of the methods of the
invention.
[0027] In a further embodiment, the tailed primers detailed above
could be used to detect editing events for genome editing
technology. For example, CRISPR/Cas9 is a revolutionary strategy in
genome editing that enables generation of targeted, double-stranded
breaks (DSBs) in genomic DNA. Methods to achieve DSBs by
CRISPR/Cas9--which is a bacterial immune defense system comprised
of an endonuclease that is targeted to double-stranded DNA by a
guide RNA--are being widely used in gene disruption, gene knockout,
gene insertion, etc. In mammalian cells, the endonuclease activity
is followed by an endogenous repair process that leads to some
frequency of insertions/deletions/substitutions in wild-type DNA at
the target locus which gives the resultant genome editing.
[0028] RNase H-cleavable primers have been designed to flank edited
loci in order to 1) generate locus-specific amplicons with
universal tails, and 2) be subsequently amplified with indexed
P5/P7 universal primers for next-generation sequencing. In pilot
experiments, this strategy resulted in reliable, locus-specific
amplification which captures CRISPR/Cas9 editing events in a
high-throughput and reproducible manner. The key finding is that
the overall targeted editing by this NGS-based method was
determined to be 95%; whereas, previous enzymatic strategies
suggested overall editing from the same samples was approximately
55% at the intended target site. Further, primers were designed to
amplify off-target locations of genomic editing based on in silico
predictions by internal bioinformatics tools.
[0029] These assays would be pooled for amplification of a single
genomic DNA sample in order to capture the on-target as well as
>100 potential sites for off-target genome editing mediated by
sequence homology to the guide RNA. The results from this
experiment would allow for 1) identification of CRISPR/Cas9
off-target sites and provide an assay for comparing strategies to
reduce those effects, 2) improved design of the CRISPR/Cas9
off-target prediction algorithm, and 3) improved design of primer
sets.
[0030] As noted in U.S. Patent Application Publication No. US
2009/0325169 (incorporated by reference herein in its entirety),
RNase H2 can cleave at positions containing one or more RNA bases,
at 2'-modified nucleosides such as 2'-fluoronucleosides. The
primers can also contain nuclease resistant linkages such as
phosphorothioate, phosphorodithioate, or methylphosphonate.
[0031] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
document, including definitions, will control. Preferred methods
and materials are described below, although methods and materials
similar or equivalent to those described herein can be used in
practice or testing of the present invention.
[0032] "Complement" or "complementary" as used herein means a
nucleic acid, and can mean Watson-Crick (e.g., A-T/U and C-G) or
Hoogsteen base pairing between nucleotides or nucleotide analogs of
nucleic acid molecules.
[0033] "Fluorophore" or "fluorescent label" refers to compounds
with a fluorescent emission maximum between about 350 and 900
nm.
[0034] "Hybridization" as used herein, refers to the formation of a
duplex structure by two single-stranded nucleic acids due to
complementary base pairing. Hybridization can occur between fully
complementary nucleic acid strands or between "substantially
complementary" nucleic acid strands that contain minor regions of
mismatch. "Identical" sequences refers to sequences of the exact
same sequence or sequences similar enough to act in the same manner
for the purpose of signal generation or hybridizing to
complementary nucleic acid sequences. "Primer dimers" refers to the
hybridization of two oligonucleotide primers. "Stringent
hybridization conditions" as used herein means conditions under
which hybridization of fully complementary nucleic acid strands is
strongly preferred. Under stringent hybridization conditions, a
first nucleic acid sequence (for example, a primer) will hybridize
to a second nucleic acid sequence (for example, a target sequence),
such as in a complex mixture of nucleic acids. Stringent conditions
are sequence-dependent and will be different in different
circumstances. Stringent conditions can be selected to be about
5-10.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength pH. The Tm can be the
temperature (under defined ionic strength, pH, and nucleic
concentration) at which 50% of an oligonucleotide complementary to
a target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions can be those in
which the salt concentration is less than about 1.0 M sodium ion,
such as about 0.01-1.0 M sodium ion concentration (or other salts)
at pH 7.0 to 8.3 and the temperature is at least about 30.degree.
C. for short probes (e.g., about 10-50 nucleotides) and at least
about 60.degree. C. for long probes (e.g., greater than about 50
nucleotides). Stringent conditions can also be achieved with the
addition of destabilizing agents such as formamide. For selective
or specific hybridization, a positive signal can be at least 2 to
10 times background hybridization. Exemplary stringent
hybridization conditions include the following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0035] The terms "nucleic acid," "oligonucleotide," or
"polynucleotide," as used herein, refer to at least two nucleotides
covalently linked together. The depiction of a single strand also
defines the sequence of the complementary strand. Thus, a nucleic
acid also encompasses the complementary strand of a depicted single
strand. Many variants of a nucleic acid can be used for the same
purpose as a given nucleic acid. Thus, a nucleic acid also
encompasses substantially identical nucleic acids and complements
thereof. A single strand provides a probe that can hybridize to a
target sequence under stringent hybridization conditions. Thus, a
nucleic acid also encompasses a probe that hybridizes under
stringent hybridization conditions.
[0036] Nucleic acids can be single stranded or double stranded, or
can contain portions of both double stranded and single stranded
sequences. The nucleic acid can be DNA, both genomic and cDNA, RNA,
or a hybrid, where the nucleic acid can contain combinations of
deoxyribo- and ribonucleotides, and combinations of bases including
uracil, adenine, thymine, cytosine, guanine, inosine, xanthine
hypoxanthine, isocytosine and isoguanine. Nucleic acids can be
obtained by chemical synthesis methods or by recombinant methods. A
particular nucleic acid sequence can encompass conservatively
modified variants thereof (e.g., codon substitutions), alleles,
orthologs, single nucleotide polymorphisms (SNPs), and
complementary sequences as well as the sequence explicitly
indicated.
[0037] "Polymerase Chain Reaction (PCR)" refers to the enzymatic
reaction in which DNA fragments are synthesized and amplified from
a substrate DNA in vitro. The reaction typically involves the use
of two synthetic oligonucleotide primers, which are complementary
to nucleotide sequences in the substrate DNA which are separated by
a short distance of a few hundred to a few thousand base pairs, and
the use of a thermostable DNA polymerase. The chain reaction
consists of a series of 10 to 40 cycles. In each cycle, the
substrate DNA is first denatured at high temperature. After cooling
down, synthetic primers which are present in vast excess, hybridize
to the substrate DNA to form double-stranded structures along
complementary nucleotide sequences. The primer-substrate DNA
complexes will then serve as initiation sites for a DNA synthesis
reaction catalyzed by a DNA polymerase, resulting in the synthesis
of a new DNA strand complementary to the substrate DNA strand. The
synthesis process is repeated with each additional cycle, creating
an amplified product of the substrate DNA.
[0038] "Primer," as used herein, refers to an oligonucleotide
capable of acting as a point of initiation for DNA synthesis under
suitable conditions. Suitable conditions include those in which
hybridization of the oligonucleotide to a template nucleic acid
occurs, and synthesis or amplification of the target sequence
occurs, in the presence of four different nucleoside triphosphates
and an agent for extension (e.g., a DNA polymerase) in an
appropriate buffer and at a suitable temperature.
[0039] "Probe" and "fluorescent generation probe" are synonymous
and refer to either a) a sequence-specific oligonucleotide having
an attached fluorophore and/or a quencher, and optionally a minor
groove binder or b) a DNA binding reagent, such as, but not limited
to, SYBR.RTM. Green dye.
[0040] "Quencher" refers to a molecule or part of a compound, which
is capable of reducing the emission from a fluorescent donor when
attached to or in proximity to the donor. Quenching may occur by
any of several mechanisms including fluorescence resonance energy
transfer, photo-induced electron transfer, paramagnetic enhancement
of intersystem crossing, Dexter exchange coupling, and exciton
coupling such as the formation of dark complexes.
[0041] The term "RNase H PCR (rhPCR)" refers to a PCR reaction
which utilizes "blocked" oligonucleotide primers and an RNase H
enzyme. "Blocked" primers contain at least one chemical moiety
(such as, but not limited to, a ribonucleic acid residue) bound to
the primer or other oligonucleotide, such that hybridization of the
blocked primer to the template nucleic acid occurs, without
amplification of the nucleic acid by the DNA polymerase. Once the
blocked primer hybridizes to the template or target nucleic acid,
the chemical moiety is removed by cleavage by an RNase H enzyme,
which is activated at a high temperature (e.g., 50.degree. C. or
greater). Following RNase H cleavage, amplification of the target
DNA can occur.
[0042] In one embodiment, the 3' end of a blocked primer can
comprise the moiety rDDDDMx, wherein relative to the target nucleic
acid sequence, "r" is an RNA residue, "D" is a complementary DNA
residue, "M" is a mismatched DNA residue, and "x" is a C3 spacer. A
C3 spacer is a short 3-carbon chain attached to the terminal 3'
hydroxyl group of the oligonucleotide, which further inhibits the
DNA polymerase from binding before cleavage of the RNA residue.
[0043] The methods described herein can be performed using any
suitable RNase H enzyme that is derived or obtained from any
organism. Typically, RNase H-dependent PCR reactions are performed
using an RNase H enzyme obtained or derived from the
hyperthermophilic archaeon Pyrococcus abyssi (P.a.), such as RNase
H2. Thus, in one embodiment, the RNase H enzyme employed in the
methods described herein desirably is obtained or derived from
Pyrococcus abyssi, preferably an RNase H2 obtained or derived from
Pyrococcus abyssi. In other embodiments, the RNase H enzyme
employed in the methods described herein can be obtained or derived
from other species, for example, Pyrococcus furiosis, Pyrococcus
horikoshii, Thermococcus kodakarensis, or Thermococcus
litoralis.
[0044] The following examples further illustrate the invention but
should not be construed as in any way limiting its scope.
Example 1
[0045] This example demonstrates an enhanced rhPCR assay that
utilizes a highly discriminatory DNA polymerase and RNase H2 for
discrimination
[0046] To demonstrate the utility of these new assay designs,
rhPrimers and standard allele-specific primers were designed
against rs113488022, the V600E mutation in the human BRAF gene.
These primers were tested in PCR and rhPCR with WT or H784Q mutant
Taq polymerase. Primers utilized in these assays were as shown in
Table 1 (SEQ ID NOs: 1-7).
TABLE-US-00001 TABLE 1 Sequence of oligonucleotides employed in SNP
discrimination assay described in Example 1. Name Sequence SEQ ID
NO. Forward non- GCTGTGATTTTGGT SEQ ID NO. 1 discriminating
CTAGCTACAG primer Forward Allele GCTGTGATTTTGGT SEQ ID NO. 2 1 ASP1
ASPCR CTAGCTACAGT primer Forward Allele GCTGTGATTTTGGT SEQ ID NO. 3
2 ASP2 ASPCR CTAGCTACAGA primer Probe FAM-TCCCATCAG- SEQ ID NO. 4
ZEN-TTTGAACAGT TGTCTGGA-IBFQ rs113488022 GCTGTGATTTTGGT SEQ ID NO.
5 Allele 1 CTAGCTACAGTgAA Forward ASP1 ATG-x rhPrimer rs113488022
GCTGTGATTTTGGT SEQ ID NO. 6 Allele 2 CTAGCTACAGAgAA Forward ASP2
ATG-x rhPrimer Reverse GCCCTCAATTCTTA SEQ ID NO. 7 rhPrimer
CCATCCACAAAaTG GAA-x Nucleic acid sequences are shown 5'-3'. DNA is
uppercase, RNA is lowercase. Location of potential mismatch is
underlined. ZEN = internal ZEN .TM. quencher (IDT, Coralville, IA),
FAM = 6-carboxyfluorescein, IBFQ = Iowa Black .RTM. FQ
(fluorescence quencher, IDT, Coralville, IA), and x = C3
propanediol spacer block
[0047] 10 .mu.L reaction volumes were used in these assays. To
perform the reaction, 5 .mu.L of 2.times. Integrated DNA
Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix
(containing dNTPs, H784Q mutant or WT Taq DNA polymerase,
stabilizers, and MgCl.sub.2) was combined with 200 nM (2 pmol) of
either of the allelic primers. 200 nM (2 pmol) of the probe, as
well as 200 nM (5 pmol) of the reverse primer were also added.
Additionally, 2.5 mU (5.25 fmol/0.53 nM) of P.a. RNase H2 and 1000
copies of synthetic gBlock.TM. (Integrated DNA Technologies,
Coralville, Iowa) template (1000 copies Allele 1, 500 copies allele
1+500 copies allele 2 (heterozygote), or 1000 copies Allele 2 (for
gBlock.TM. sequences, see Table 2, SEQ ID NOs: 8-9) were added to
the reaction mix. The reaction was thermocycled on a Bio-Rad.TM.
CFX384.TM. Real-time system. Cycling conditions were as follows:
953:00-(950:10-650:30).times.65 cycles. Each reaction was performed
in triplicate.
TABLE-US-00002 TABLE 2 Synthetic gBlock templates for Example 1
assay Name Sequence SEQ ID NO. rs113488022 AAAAAATAAGAACACTGATTTTT
SEQ ID NO. 8 gBlock GTGAATACTGGGAACTATGAAAA Template 1
TACTATAGTTGAGACCTTCAATG ACTTTCTAGTAACTCAGCAGCAT
CTCAGGGCCAAAAATTTAATCAG TGGAAAAATAGCCTCAATTCTTA
CCATCCACAAAATGGATCCAGAC AACTGTTCAAACTGATGGGACCC
ACTCCATCGAGATTTCACTGTAG CTAGACCAAAATCACCTATTTTT
ACTGTGAGGTCTTCATGAAGAAA TATATCTGAGGTGTAGTAAGTAA
AGGAAAACAGTAGATCTCATTTT CCTATCAGAGCAAGCATTATGAA
GAGTTTAGGTAAGAGATCTAATT TCTATAATTCTGTAATATAATAT
TCTTTAAAACATAGTACTTCATC TTTCCTCTTA rs113488022
AAAAAATAAGAACACTGATTTTT SEQ ID NO. 9 gBlock GTGAATACTGGGAACTATGAAAA
Template 2 TACTATAGTTGAGACCTTCAATG ACTTTCTAGTAACTCAGCAGCAT
CTCAGGGCCAAAAATTTAATCAG TGGAAAAATAGCCTCAATTCTTA
CCATCCACAAAATGGATCCAGAC AACTGTTCAAACTGATGGGACCC
ACTCCATCGAGATTTCTCTGTAG CTAGACCAAAATCACCTATTTTT
ACTGTGAGGTCTTCATGAAGAAA TATATCTGAGGTGTAGTAAGTAA
AGGAAAACAGTAGATCTCATTTT CCTATCAGAGCAAGCATTATGAA
GAGTTTAGGTAAGAGATCTAATT TCTATAATTCTGTAATATAATAT
TCTTTAAAACATAGTACTTCATC TTTCCTCTTA Nucleic acid sequences are shown
5'-3'. Location of SNPs are shown bold and underlined.
[0048] Cq Results of the experiment are shown in Table 3. This data
shows that the mismatch discrimination of the assay system
increases with rhPCR over ASPCR with WT Taq polymerase, and that
the discrimination is enhanced by the use of the H784Q Taq
polymerase.
TABLE-US-00003 TABLE 3 Resulting Cq values WT Taq H784Q Allele 1
Het Allele 2 NTC Allele 1 Het Allele 2 NTC Non discrmin 29.3 29.3
29.4 >65 30.6 30.6 30.8 >65 ASP1 ASPCR 30.2 30.2 31.4 >65
29.2 32.5 40.3 >65 ASP2 ASPCR 36.7 30.5 29.4 >65 44.2 31.7
30.8 >65 ASP1 rhPCR 30.9 32.1 38.2 >65 31.9 31.4 49.2 >65
ASP2 rhPCR 39.3 31.0 30.8 >65 43.4 33.9 32.5 >65 All numbers
in this table represent Cq values obtained from the CFX384 .TM.
instrument (Bio-Rad .TM., Hercules, CA).
Example 2
[0049] The following example demonstrates an enhanced rhPCR assay
that utilizes a highly discriminatory DNA polymerase and RNase H2
for discrimination.
[0050] In order to demonstrate that this new assay design could
function, rhPrimers and standard allele-specific primers were
designed against rs113488022, the V600E mutation in the human BRAF
gene. These primers were tested in PCR and rhPCR with H784Q mutant
Taq polymerase. Primers utilized in these assays were as shown in
Table 4 (SEQ ID NOs: 1, 4 and 10-12).
TABLE-US-00004 TABLE 4 Sequence of oligonucleotides employed in SNP
discrimination assay described in Example 2 Name Sequence SEQ ID
NO. Forward non- GCTGTGATTTTGGT SEQ ID NO. 1 discrimin CTAGCTACAG
primer Probe FAM-TCCCATCAG- SEQ ID NO. 4 ZEN-TTTGAACAGT
TGTCTGGA-IBFQ rs113488022 GCTGTGATTTTGGT SEQ ID NO. 10 Allele 1
Forward CTAGCTACAGTgAx dxxd rhPrimer xTG rs113488022 GCTGTGATTTTGGT
SEQ ID NO. 11 Allele 2 Forward CTAGCTACAGAgAx dxxd rhPrimer xTG
Reverse rhPrimer GCCCTCAATTCTTA SEQ ID NO. 12 CCATCCACAAAaTG GAA-x
Nucleic acid sequences are shown 5'-3'. DNA is uppercase, RNA is
lowercase. Location of potential mismatch is underlined. ZEN =
internal Zen .TM. fluorescent quencher (IDT, Coralville, IA). FAM =
6-carboxyfluorescein, IBFQ = Iowa Black FQ (fluorescence quencher),
and x = C3 propanediol spacer.
[0051] 10 .mu.L reaction volumes were used in these assays. To
perform the reaction, 5 .mu.L of 2.times. Integrated DNA
Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix
(containing dNTPs, H784Q mutant DNA polymerase, stabilizers, and
MgCl.sub.2) was combined with 200 nM (2 pmol) of either of the
allelic primers. 200 nM (2 pmol) of the probe, as well as 200 nM (5
pmol) of the reverse primer were also added. Additionally, 7.5 mU
(15.75 fmol/1.58 nM), 50 mU (105 fmol/10.5 nM) or 200 mU (420
fmol/42 nM) of P.a. RNase H2 and 5e4 copies of synthetic gBlock.TM.
(Integrated DNA Technologies, Coralville, Iowa) template (1e5
copies Allele 1, 5e4 copies allele 1+5e4 copies allele 2
(heterozygote), or 1e5 copies Allele 2 (for gBlock.TM. sequences,
see Table 2, SEQ ID NOs: 8-9) were added to the reaction mix. The
reaction was thermocycled on a Bio-Rad.TM. CFX384.TM. Real-time
system. Cycling conditions were as follows:
95.sup.3:00-(95.sup.0:10-65.sup.0:30).times.65 cycles. Each
reaction was performed in triplicate.
[0052] Cq Results of the experiment are shown in Table 5. This data
shows that the mismatch discrimination of the assay system
increases with rhPCR over ASPCR with WT Taq polymerase, and that
the discrimination is enhanced by the use of the H784Q Taq
polymerase.
TABLE-US-00005 TABLE 5 Resulting Cq values Averages Allele 1 Het
Allele 2 NTC .DELTA.Cq Unblocked 7.5 mU 21.9 22.3 22.1 >75 50 mU
22.7 22.5 22.7 >75 200 mU 21.8 21.8 21.9 >75 AgAxxTG 7.5 mU
43.7 25.6 24.6 >75 19.1 50 mU 50.3 24.5 23.5 >75 26.8 200 mU
48.5 25.2 24.1 >75 24.4 TgAxxTG 7.5 mU 25.1 26.3 42.5 >75
17.4 50 mU 24.2 25.4 41.0 >75 16.9 200 mU 22.9 23.8 37.2 >75
14.3 All numbers in this table represent Cq values obtained from
the CFX384 .TM. instrument (Bio-Rad .TM., Hercules, CA).
[0053] The delta Cq values were significantly higher than the ones
obtained with the Gen 1 versions of these primers, indicating that
there is an advantage to this primer design, as seen before in
rhPCR.
Example 3
[0054] The following example illustrates the heightened reliability
of universal assays using a DNA polymerase with a high mismatch
discrimination.
[0055] To demonstrate that the disclosed assays can function in a
universal format and that they are significantly improved with a
polymerase with high mismatch discrimination, "universal" assay
primers were designed against rs351855, the G338R mutation in the
human FGFR4 gene. This "universal" assay design has numerous
advantages, including the ability to multiplex the allele-specific
rhPrimers and obtain multiple-color readouts. Primers utilized in
this assay were as shown in Table 6 (SEQ ID NOs: 13-18).
TABLE-US-00006 TABLE 6 Sequences of oligonucleotides employed in
"universal" SNP discrimination assay Name Sequence SEQ ID NO.
Universal CGCCGCGTATAGTCCCGCGTAAA SEQ ID NO. 13 Forward primer
Probe 1 FAM-C+CATC+A+C+CGTG+ SEQ ID NO. 14 (FAM) CT-IBFQ Probe 2
HEX-CAATC+C+C+CGAG+ SEQ ID NO. 15 (HEX) CT-IBFQ rs351855
GCCCATGTCCCAGCGAACCA SEQ ID NO. 16 Allele 1 TCACCGTGCTAGCCCTCGAT
Forward ACAGCCCgGCCAC-x primer rs351855 GCCCATGTCCCAGCGAACAA SEQ ID
NO. 17 Allele 2 TCCCCGAGCTGCCCTCGATA Forward CAGCCTgGCCAC-x primer
Reverse GCGGCCAGGTATACGGACAT SEQ ID NO. 18 primer cATCCA-x Nucleic
acid sequences are shown 5'-3'. DNA is uppercase, RNA is lowercase.
Location of potential mismatch is underlined. LNA residues are
designated with a carboxyfluorescein, HEX =
6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein, IBFQ = Iowa Black
FQ (fluorescence quencher), and x = C3 propanediol spacer
block.
[0056] 10 .mu.L reaction volumes were used in these assays. To
perform the reaction, 5 .mu.L of 2.times. Integrated DNA
Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix
(containing dNTPs, mutant or WT Taq DNA polymerase, stabilizers,
and MgCl.sub.2) was combined with 50 nM (500 fmol) of each of the
two allelic primers. 250 nM (2.5 pmol) of each of the two probes,
as well as 500 nM (5 pmol) of the Universal Forward primer and 500
nM (5 pmol) of the reverse primer were also added. Additionally,
2.5 mU (5.25 fmol/0.53 nM) of P.a. RNase H2 and 1000 copies of
synthetic gBlock.TM. (Integrated DNA Technologies, Coralville,
Iowa) template (1000 copies Allele 1, 500 copies allele 1+500
copies allele 2 (heterozygote), or 1000 copies Allele 2 (for
gBlock.TM. sequences, see Table 7, SEQ ID NOs: 19-20) were added to
the reaction mix. The reaction was thermocycled on a Bio-Rad.TM.
CFX384.TM. Real-time system. Cycling conditions were as follows:
95.sup.3:00-(95.sup.0:10-60.sup.0:30).times.3
cycles-(95.sup.0:10-65.sup.0:30).times.65 cycles. Each reaction was
performed in triplicate. Fluorescence reads were taken after a
total of 50 cycles were completed. Fluorescence values were plotted
on the FAM and HEX axis, and results are shown in FIGS. 2a and
2b.
TABLE-US-00007 TABLE 7 Synthetic gBlock templates for Example 3
Name Sequence SEQ ID NO. rs351855 GTTGGGAGCTGGGAGGGACTGAG SEQ ID
NO. 19 gBlock TTAGGGTGCACGGGGCGGCCAGT Template 1
CTCACCACTGACCAGTTTGTCTG TCTGTGTGTGTCCATGTGCGAGG
GCAGAGGAGGACCCCACATGGAC CGCAGCAGCGCCCGAGGCCAGGT
ATACGGACATCATCCTGTACGCG TCGGGCTCCCTGGCCTTGGCTGT
GCTCCTGCTGCTGGCCGGGCTGT ATCGAGGGCAGGCGCTCCACGGC
CGGCACCCCCGCCCGCCCGCCAC TGTGCAGAAGCTCTCCCGCTTCC
CTCTGGCCCGACAGGTACTGGGC GCATCCCCCACCTCACATGTGAC
AGCCTGACTCCAGCAGGCAGAAC CAAGTCTCCCACTTTGCAGTTCT
CCCTGGAGTCAGGCTCTTCCGGC AAGTCAAGCT rs351855 GTTGGGAGCTGGGAGGGACTGAG
SEQ ID NO. 20 gBlock TTAGGGTGCACGGGGCGGCCAGT Template 2
CTCACCACTGACCAGTTTGTCTG TCTGTGTGTGTCCATGTGCGAGG
GCAGAGGAGGACCCCACATGGAC CGCAGCAGCGCCCGAGGCCAGGT
ATACGGACATCATCCTGTACGCG TCGGGCTCCCTGGCCTTGGCTGT
GCTCCTGCTGCTGGCCAGGCTGT ATCGAGGGCAGGCGCTCCACGGC
CGGCACCCCCGCCCGCCCGCCAC TGTGCAGAAGCTCTCCCGCTTCC
CTCTGGCCCGACAGGTACTGGGC GCATCCCCCACCTCACATGTGAC
AGCCTGACTCCAGCAGGCAGAAC CAAGTCTCCCACTTTGCAGTTCT
CCCTGGAGTCAGGCTCTTCCGGC AAGTCAAGCT Nucleic acid sequences are shown
5'-3'. Location of SNPs are shown bold and underlined.
[0057] The results illustrate that the mismatch discrimination
between homozygotes is sufficient with both polymerases, although
the resulting data using the WT Taq demonstrate that it is more
difficult to make an allelic call. Importantly, however, the WT Taq
polymerase cannot efficiently discriminate heterozygotes from
homozygotes, and places them too close to the allele 1 and 2
signals (FIG. 2a). In contrast, the signal from the heterozygotes
in the assays utilizing the mutant Taq polymerase are easily
distinguishable from the homozygotes (FIG. 2b).
[0058] The importance of the mutant Taq can be further understood
when examining the Cq values from this example (Table 8). The data
show that not only does the H784Q Taq mutant increase mismatch
discrimination dramatically, but the Cqs of the NTCs decrease from
the low-to-mid 50s, to greater than the number tested in the assay
(>65). From this experiment, it is shown that allele identity
can be determined from Cq values as well as end-point
fluorescence.
TABLE-US-00008 TABLE 8 Cq and delta Cq data for the experiment
described in Example 3 WT Taq H784Q Template FAM HEX Delta Cq FAM
HEX Delta Cq Allele 1 32.9 31.3 -1.6 37.5 56.8 19.3 Allele 1 31.9
31.1 -0.8 36.2 51.4 15.2 Allele 1 31.8 31.0 -0.9 36.6 54.3 17.8
Heterozygote 33.0 29.4 -3.5 38.7 37.8 -0.9 Heterozygote 32.8 29.7
-3.1 38.7 38.2 -0.5 Heterozygote 33.2 30.0 -3.3 39.9 39.1 -0.8
Allele 2 35.1 29.1 6.0 50.6 36.5 14.1 Allele 2 34.7 29.3 5.4 52.1
36.6 15.5 Allele 2 34.5 29.0 5.5 50.7 36.1 14.6 NTC 51.8 56.1 --
>65 >65 -- NTC 52.8 56.1 -- >65 >65 -- NTC 52.1 50.7 --
>65 >65 -- All numbers in this table represent Cq and delta
Cq values values obtained from the CFX384 instrument (Bio-Rad .TM.,
Hercules, CA).
Example 4
[0059] The following example illustrates the detection of rare
allelic variants with the assay designs of the present invention.
To demonstrate the utility of these new assay designs to detect
rare allelic variants, previously described second generation
rhPrimers (rdxxdm) were utilized against rs113488022, the V600E
mutation in the human BRAF gene (see Table 4; SEQ ID NOs: 1, 4 and
10-12).
[0060] 10 .mu.L reaction volumes were used in these assays. To
perform the reaction, 5 .mu.L of 2.times. Integrated DNA
Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix
(containing dNTPs, H784Q mutant or WT Taq DNA polymerase,
stabilizers, and MgCl.sub.2) was combined with 200 nM (2 pmol) of
either of the allelic primers, or the non-discriminatory forward
primer. 200 nM (2 pmol) of the probe, as well as 200 nM (5 pmol) of
the reverse primer were also added. Additionally, 50 mU (105
fmol/10.5 nM) of P.a. RNase H2 and 50,000 copies of synthetic
gBlock.TM. (Integrated DNA Technologies, Coralville, Iowa) match
template, was combined with either 0, 50, or 500 copies of the
opposite allele (for gBlock.TM. sequences, see Table 6, SEQ ID NOs:
16-17) were added to the reaction mix. The reaction was
thermocycled on a Bio-Rad.TM. CFX384.TM. Real-time system. Cycling
conditions were as follows:
95.sup.3:00-(95.sup.0:10-60.sup.0:30).times.65 cycles. Each
reaction was performed in triplicate.
[0061] Data for the WT polymerase is shown in Table 9, and for the
H784Q mutant Taq polymerase in Table 10. One of the advantages of
this system for rare allele detection over "conventional" rhPCR is
the ability to utilize a single amount of RNase H2 for both
alleles. This advantage halves the potential requirement for
determining the enzyme amount required for cleavage.
TABLE-US-00009 TABLE 9 Average Cq and delta Cq values for the rare
allele experiment with the WT Taq polymerase described in Example
4. Back-ground 50,000 50,000 50,000 0 0 0 Target 500 50 0 500 50 0
SEQ Non-discrimin 22.9 23.1 22.8 30.4 34.2 >65 ID No. 1 SEQ . .
. TgAxxTG 31.6 34.5 36.1 31.6 36.0 >65 ID NO. 10 SEQ . . .
AgAxxTG 29.1 29.1 29.9 30.8 34.4 >65 ID NO. 11 All numbers in
this table represent Cq and delta Cq values values obtained from
the CFX384 instrument (Bio-Rad .TM., Hercules, CA). DNA is
uppercase, RNA is lowercase. Location of potential mismatch is
underlined. x = internal C3 propanediol spacer block.
TABLE-US-00010 TABLE 10 Average Cq and delta Cq values for the rare
allele experiment with the H784Q mutant Taq polymerase described in
Example 4. Back-ground 50,000 50,000 50,000 0 0 0 Target 500 50 0
500 50 0 SEQ Non-discrimin 22.7 23.2 23.2 31.5 34.2 >65 ID No. 1
SEQ . . . TgAxxTG 33.8 36.6 47.3 33.1 36.4 >65 ID NO. 10 SEQ . .
. AgAxxTG 32.1 35.2 38.8 32.0 35.5 >65 ID NO. 11 All numbers in
this table represent Cq and delta Cq values obtained from the
CFX384 instrument (Bio-Rad .TM., Hercules, CA). DNA is uppercase,
RNA is lowercase. Location of potential mismatch is underlined. x =
internal C3 propanediol spacer block.
[0062] The data clearly shows that the H784Q DNA polymerase allows
for detection of 50 copies of target in a 50,000 copies of
background DNA (a 1:1000 discrimination level) for the mutant A
allele of rs113488022, with a delta Cq of over 11 cycles. While
only slightly more than 3 cycles was observed for the T allele in
this assay, this was a significant improvement over the WT Taq
polymerase, which did not show any discrimination for the T allele,
and only a delta of 3 cycles for the A allele.
Example 5
[0063] This example demonstrates successful allelic discrimination
with the use of a universal rhPCR genotyping assay and Integrated
DNA Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master
mix, and the robust stability of the reaction components. To
demonstrate the robust nature of the assay design and mixture
components, universal primers were designed against rs4657751, a
SNP located on the human Chromosome 1 (See Table 11, SEQ ID NOs:
14, 21-25).
[0064] Identical universal rhPCR genotyping reactions were set up
in two white Hard-Shell.RTM. 384-well skirted PCR plates (Bio-Rad,
Hercules, Calif.) on the Bio-Rad CFX384 Touch.TM. Real-Time PCR
Detection System with 10 .mu.L final volume. Each well contained
the rhPCR assay primers (150 nM of rs4657751 Allele Specific Primer
1 (SEQ ID NO: 23), 150 nM of rs4657751 Allele Specific Primer 2
(SEQ ID NO: 24), and 500 nM rs4657751 Locus Specific Primer (SEQ ID
NO: 25). Reactions contained universal reporter oligos at the
following concentrations: 250 nM of universal FAM probe (SEQ ID NO:
14), 450 nM of universal Yakima Yellow.RTM. (SEQ ID NO: 22) probe,
and 1000 nM of universal forward primer (SEQ ID NO: 21), and 5
.mu.L of 2.times. Integrated DNA Technologies (IDT) (Coralville,
Iowa) rhPCR genotyping master mix (containing dNTPs, a mutant H784Q
Taq polymerase (see Behlke, et al. U.S. 2015/0191707), chemically
modified Pyrococcus abyssi RNase H2 (See Walder et al.
UA20130288245A1), stabilizers, and MgCl.sub.2).
[0065] gBlocks.RTM. Gene Fragments (Integrated DNA Technologies,
Inc., Coralville, Iowa) containing either allele of the rs4657751
SNP were utilized as the source of template DNA (See Table 12, SEQ
ID NOs: 26 and 27). Each well contained template representing one
of three possible genotypes: allele 1 homozygote (1000 copies
rs4657751 Allele 1 gBlock.RTM. template (SEQ ID NO: 26)), allele 2
homozygote (1000 copies rs4657751 Allele 2 gBlock.RTM. template
(SEQ ID NO: 27)), or heterozygote (mix of 500 copies of rs4657751
Allele 1 gBlock.RTM. template (SEQ ID NO: 26) and 500 copies of
rs4657751 Allele 2 gBlock.RTM. template (SEQ ID NO: 27)). Template
or water for the no template control (NTC) reactions were added
into three replicate wells of two individual plates. The reactions
underwent the following cycling protocol: 95.degree. C. for 10
minutes, then 45 cycles of 95.degree. C. for 10 seconds and
60.degree. C. for 45 seconds.
TABLE-US-00011 TABLE 11 Sequences of oligonucleotides used in
Example 5 Name Sequence SEQ ID NO. Universal CGGCCCATGTCCCAGCGAA
SEQ ID NO. 21 Forward primer Probe 1 FAM-C+CATC+A+C+CGTG+CT-IBFQ
SEQ ID NO. 14 (FAM) Probe 2 Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ ID NO.
22 (Yakima Yellow) rs4657751 GCCCATGTCCCAGCGAACCATCACCGT SEQ ID NO.
23 Allele 1 GCTACTTCCCACACCCTCATATCuTGT Forward TA-x primer
rs4657751 GCCCATGTCCCAGCGAACAATCCCCGA SEQ ID NO. 24 Allele 2
GCTCTTACTTCCCACACCCTCATATAu Forward TGTTA-x primer rs4657751
GCGCTAAGTAAACATTCCTGATTGCAa SEQ ID NO. 25 Reverse CTTAT-x primer
Nucleic acid sequences are shown 5'-3'. DNA is uppercase, RNA is
lowercase. Location of potential mismatch is underlined. LNA
residues are designated with a carboxyfluorescein, Yak = Yakima
Yellow
(3-(5,6,4',7'-tetrachloro-5'-methyl-3',6'-dipivaloylfluorescein-2-yl)),
IBFQ = Iowa Black FQ (fluorescence quencher), and x = C3
propanediol spacer block.
TABLE-US-00012 TABLE 12 Synthetic gBlock .RTM. templates used in
Example 5. Name Sequence SEQ ID NO. rs4657751
GATTTTTTTTTTTTGGCATTTCT SEQ ID NO. 26 Allele 1
TCTTAGATTTCTATCTCCTAACA gBlock TAGGATCACTTATTTGTGAAATT template
ATTTGTATACCTTTTTTATGGAG TGATGATGTGATACAAATTCTAT
CCTTAAGGATATAAGAACATCTT TTCTTTATATTAGGATTTTTCTG
GACCCATGAGTTACATGCTTACT TCCCACACCCTCATATCTTGTTT
AAATTTGTAGAATTAAATTCATA GGTAATTATTTCTGAAACTTCTT
CCCTGTGTGAGCAATCTAAATAA TTATTACAATGCCTTAAGTTGCA
ATCAGGAATGTTTACTTAGCACA GACTTTTTTCCCCACTACTGCAC
TCAAAGGATAACAGATATATGGC AAATCTAACCATATTCTTTGTCC
TTTGTCCATGTTGCGGAGGGAAG CTCATCAGTGGGGCCACGAGCTG
AGTGCGTCCTGTCACTCCACTCC CATGTCCCTTGGGAAGGTCTGAG ACTAGGG rs4657751
GATTTTTTTTTTTTGGCATTTCT SEQ ID NO. 27 Allele 2
TCTTAGATTTCTATCTCCTAACA gBlock TAGGATCACTTATTTGTGAAATT template
ATTTGTATACCTTTTTTATGGAG TGATGATGTGATACAAATTCTAT
CCTTAAGGATATAAGAACATCTT TTCTTTATATTAGGATTTTTCTG
GACCCATGAGTTACATGCTTACT TCCCACACCCTCATATATTGTTT
AAATTTGTAGAATTAAATTCATA GGTAATTATTTCTGAAACTTCTT
CCCTGTGTGAGCAATCTAAATAA TTATTACAATGCCTTAAGTTGCA
ATCAGGAATGTTTACTTAGCACA GACTTTTTTCCCCACTACTGCAC
TCAAAGGATAACAGATATATGGC AAATCTAACCATATTCTTTGTCC
TTTGTCCATGTTGCGGAGGGAAG CTCATCAGTGGGGCCACGAGCTG
AGTGCGTCCTGTCACTCCACTCC CATGTCCCTTGGGAAGGTCTGAG ACTAGGG Nucleic
acid sequences are shown 5'-3'. DNA is uppercase. The location of
the SNP is underlined.
[0066] One reaction plate was cycled immediately (0 hr benchtop
hold) and one reaction plate was held at room temperature for 2
days (48 hr benchtop hold) to demonstrate reaction stability over
time. Allelic discrimination analysis was performed using Bio-Rad
CFX Manager 3.1 software (Bio-Rad, Hercules, Calif.). FAM and
Yakima Yellow fluorophores were detected in each well. For both
fluorophores the baseline cycles were set to begin at cycle 10 and
end at cycle 25. Fluorescence signal (RFU) in each well at the end
of 45 cycles was used to generate an allelic discrimination plot
and genotypes were determined with auto-call features of the
analysis software. Identical performance was obtained with the
immediate run (FIG. 3A) and 48 hour hold plate (FIG. 3B),
demonstrating robust stability of the reaction components. Each
sample is assigned the correct genotyping call and samples of the
same genotype are tightly clustered together. The heterozygote
cluster is separated from both of the homozygous clusters by an
approximate 45 degree angle, indicating excellent allelic
specificity of the universal rhPCR genotyping assays and master
mix.
Example 6
[0067] The following example compares the performance of the
genotyping methods of the present invention versus traditional 5'
nuclease genotyping assay methods (Taqman.TM.).
[0068] The rs1799865 SNP in the CCR2 gene was selected, and rhPCR
genotyping primers as well as an rs1799865 5' nuclease assay
(Thermo-Fisher (Waltham, Mass.)), were designed and obtained.
Sequences for the rs1799865 rhPCR genomic SNP assay are shown in
Table 14 (SEQ ID NOs: 14, 21, 22, and 28-30). Thermo-Fisher 5'
nuclease primer/probe (Taqman.TM.) sequences are not published, and
therefore are not included in this document.
[0069] Reactions were performed in 10 .mu.L volumes, containing 10
ng Coriell genomic DNA (Camden, N.J.), 250 nM of universal FAM
probe (SEQ ID NO: 14), 450 nM of universal Yakima Yellow.RTM. (SEQ
ID NO: 22) probe, 1000 nM of universal forward primer (SEQ ID NO:
21), 150 nM of the two allele-specific forward primers (SEQ ID NOs:
28 and 29), 500 nM of the reverse primer (SEQ ID NO: 30), and 5
.mu.L of 2.times. Integrated DNA Technologies (IDT) (Coralville,
Iowa) rhPCR genotyping master mix (containing dNTPs, a mutant H784Q
Taq polymerase (see Behlke, et al. U.S. 2015/0191707), chemically
modified Pyrococcus abyssi RNase H2 (See Walder et al.
UA20130288245A1), stabilizers, and MgCl.sub.2).
[0070] PCR was performed on Life Technologies (Carlsbad, Calif.)
QuantStudio.TM. 7 Flex real-time PCR instrument using the following
cycling conditions: 10 mins at 95.degree. C. followed by 50 cycles
of 95.degree. C. for 10 seconds and 60.degree. C. for 45 seconds.
End-point analysis of each of the plates was performed after 45
cycles with the QuantStudio.TM. Real-Time PCR Software v1.3
(Carlsbad, Calif.).
TABLE-US-00013 TABLE 14 Sequences of oligonucleotides used for the
rs1799865 genotyping assay in Example 6. Name Sequence SEQ ID NO.
Universal CGGCCCATGTCCCAGCGAA SEQ ID NO. 21 Forward primer Probe 1
FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ ID NO. 14 (FAM) Probe 2
Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ ID NO. 22 (Yakima Yellow) rs1799865
GCCCATGTCCCAGCGAACCATCACCGT SEQ ID NO. 28 Allele 1
GCTTTCTCTTCTGGACTCCCTATAATa Forward TTGTG-x primer rs1799865
GCCCATGTCCCAGCGAACAATCCCCGA SEQ ID NO. 29 Allele 2
GCTTTCTCTTCTGGACTCCCTATAACa Forward TTGTG-x primer rs1799865
GCGGATTGATGCAGCAGTGAgTCAT SEQ ID NO. 30 Reverse G-x primer Nucleic
acid sequences are shown 5'-3'. DNA is uppercase, RNA is lowercase.
LNA residues are designated with a +. Location of potential
mismatch is underlined. FAM = 6-carboxyfluorescein, Yak = Yakima
Yellow
(3-(5,6,4',7'-tetrachloro-5'-methyl-3',6'-dipivaloylfluorescein-2-yl)),
IBFQ = Iowa Black FQ (fluorescence quencher), and x = C3
propanediol spacer block.
[0071] FIGS. 4A and 4B show a side-by-side comparison of the
resulting allelic discrimination plots. The rhPCR Genotyping Assay
(FIG. 4B) achieved higher fluorescence signal compared to a
traditional 5'-nuclease genotyping assay (FIG. 4A) while showing
concordant results. The higher signal and minimal non-specific
amplification from NTC in the rhPCR assay allow better cluster
separation and accurate genotype calls.
Example 7
[0072] The following example illustrates the present methods
allowing for detection and analysis of tri-allelic SNP. The
rs72558195 SNP is present in the CYP2C8 gene, and has three
potential genotypes. This SNP was selected for analysis with the
rhPCR genotyping system.
[0073] Conventional workflow of interrogating tri-allelic SNP, as
illustrated in FIGS. 5A and 5B, involves running a pair of assays
using the same samples, manual calling, and comparing the paired
assay result to obtain the true genotype of samples.
[0074] To demonstrate that such a system can function with the
universal rhPCR genotyping system, reactions were set up in a white
Hard-Shell.RTM. 384-well skirted PCR plates (Bio-Rad, Hercules,
Calif.) on the Life Technologies (Carlsbad, Calif.) QuantStudio.TM.
7 Flex real-time PCR with 10 .mu.L final volume. Each well
contained the rhPCR assay primers (See Table 16, SEQ ID NOs: 14,
21, 22, 31-33). Specifically, 150 nM of rs72558195 G:A Allele
Specific Primer 1 (SEQ ID NO: 31) and 150 nM of rs72558195 G:A
Allele Specific Primer 2 (SEQ ID NO: 32), or 150 nM of rs72558195
G:A Allele Specific Primer 1 (SEQ ID NO: 31) and 150 nM of
rs72558195 G:C Allele Specific Primer 3 (SEQ ID NO: 33) as well as
500 nM rs72558195 Locus Specific Primer (SEQ ID NO: 34) were
included in the reactions.
[0075] Reactions contained universal reporter oligos at the
following concentrations: 250 nM of universal FAM probe (SEQ ID NO:
14), 450 nM of universal Yakima Yellow.RTM. (SEQ ID NO: 22) probe,
and 1000 nM of universal forward primer (SEQ ID NO: 21), 50 nM ROX
internal standard, and 5 .mu.L of 2.times. Integrated DNA
Technologies (IDT) (Coralville, Iowa) rhPCR genotyping master mix
(containing dNTPs, a mutant H784Q Taq polymerase (see Behlke, et
al. U.S. 2015/0191707), chemically modified Pyrococcus abyssi RNase
H2 (See Walder et al. UA20130288245A1), stabilizers, and
MgCl.sub.2).
TABLE-US-00014 TABLE 16 Sequences of oligonucleotides used for the
rs72558195 genotyping assay in Example 7. Name Sequence SEQ ID NO.
Universal CGGCCCATGTCCCAGCGAA SEQ ID NO. 21 Forward primer Probe 1
FAM-C+CATC+A+C+CGTG+CT- SEQ ID NO. 14 (FAM) IBFQ Probe 2
Yak-CAATC+C+C+CGAG+CT- SEQ ID NO. 22 (Yakima IBFQ Yellow)
rs72558195: GCCCATGTCCCAGCGAACCATCA SEQ ID NO. 31 G:A Allele
CCGTGCTCTCCGTTGTTTTCCAG 1 Forward AAACgATTTC-x primer rs72558195:
GCCCATGTCCCAGCGAACAATCC SEQ ID NO. 32 G:A Allele
CCGAGCTCTCCGTTGTTTTCCAG 2 Forward AAATgATTTC-x primer rs72558195:
GCCCATGTCCCAGCGAACAATCC SEQ ID NO. 33 G:C Allele
CCGAGCTCTCCGTTGTTTTCCAG 3 Forward AAAGgATTTC-x primer rs1135840
GCAACCAAGTCTTCCCTACAACc SEQ ID NO. 34 Reverse TTGAT-x primer
Nucleic acid sequences are shown 5'-3'. DNA is uppercase, RNA is
lowercase. LNA residues are designated with a +. Location of
potential mismatch in underlined. FAM = 6-carboxyfluorescein, Yak =
Yakima Yellow
(3-(5,6,4',7'-tetrachloro-5'-methyl-3',6'-dipivaloylfluorescein-2-yl)),
IBFQ = Iowa Black FQ (fluorescence quencher), and x = C3
propanediol spacer block.
[0076] gBlocks.RTM. Gene Fragments (Integrated DNA Technologies,
Inc., Coralville, Iowa) containing alleles of the rs72558195 SNP
were utilized as the source of template DNA (See Table 17, SEQ ID
NOs: 35, 36 and 37). Each well contained template representing one
of six possible genotypes: allele 1 homozygote (1000 copies
rs72558195 Allele 1 gBlock.RTM. template (SEQ ID NO: 35)), allele 2
homozygote (1000 copies rs72558195 Allele 2 gBlock.RTM. template
(SEQ ID NO: 36)), allele 3 homozygote (1000 copies rs72558195
Allele 2 gBlock.RTM. template (SEQ ID NO: 37)), heterozygote (mix
of 500 copies of rs72558195 Allele 1 gBlock.RTM. template (SEQ ID
NO: 35) and 500 copies of rs72558195 Allele 2 gBlock.RTM. template
(SEQ ID NO: 36). heterozygote (mix of 500 copies of rs72558195
Allele 1 gBlock.RTM. template (SEQ ID NO: 35) and 500 copies of
rs72558195 Allele 3 gBlock.RTM. template (SEQ ID NO: 37)). Template
or water for the no template control (NTC) reactions were added
into three replicate wells of two individual plates. The reactions
underwent the following cycling protocol: 95.degree. C. for 10
minutes, then 45 cycles of 95.degree. C. for 10 seconds and
60.degree. C. for 45 seconds.
TABLE-US-00015 TABLE 17 gBlock .RTM. sequences used in Example 7
Name Sequence SEQ ID NO. rs72558195 ACATCATTTTTATTGTATAA SEQ ID NO.
35 Allele 1 AAGCATTTTAGTATCAATTT gBlock TCTCATTTTTAAACCAAGTC
template TTCCCTACAACCTTGAATAA ATGGTTTCCAAGGAAAATAA
AATCTTGGCCTTACCTGGAT CCATGGGGAGTTCAGAATCC TGAAGTTTTCATTGAATCTT
TTCATCAGGGTGAGAAAATT CTGATCTTTATAATCAAATC GTTTCTGGAAAACAACGGAG
CAGATCACATTGCAGGGAGC ACAGCCCAGGATGAAAGTGG GATCACAGGGTGAAGCTAAA
GATTTAAAAATTTTTAAAAA AATTATTAAAAAATAAATAT TTAAAAGATTTGCATTTGTT
AAGACATAAAGGAAATTTAG AAATTTTAAACAATATCTTA CAAATTCCCCATGTGTCCAA A
rs72558195 ACATCATTTTTATTGTATAA SEQ ID NO. 36 Allele 2
AAGCATTTTAGTATCAATTT gBlock TCTCATTTTTAAACCAAGTC template
TTCCCTACAACCTTGAATAA ATGGTTTCCAAGGAAAATAA AATCTTGGCCTTACCTGGAT
CCATGGGGAGTTCAGAATCC TGAAGTTTTCATTGAATCTT TTCATCAGGGTGAGAAAATT
CTGATCTTTATAATCAAATC ATTTCTGGAAAACAACGGAG CAGATCACATTGCAGGGAGC
ACAGCCCAGGATGAAAGTGG GATCACAGGGTGAAGCTAAA GATTTAAAAATTTTTAAAAA
AATTATTAAAAAATAAATAT TTAAAAGATTTGCATTTGTT AAGACATAAAGGAAATTTAG
AAATTTTAAACAATATCTTA CAAATTCCCCATGTGTCCAA A rs72558195
ACATCATTTTTATTGTATAA SEQ ID NO. 37 Allele 3 AAGCATTTTAGTATCAATTT
gBlock TCTCATTTTTAAACCAAGTC template TTCCCTACAACCTTGAATAA
ATGGTTTCCAAGGAAAATAA AATCTTGGCCTTACCTGGAT CCATGGGGAGTTCAGAATCC
TGAAGTTTTCATTGAATCTT TTCATCAGGGTGAGAAAATT CTGATCTTTATAATCAAATC
CTTTCTGGAAAACAACGGAG CAGATCACATTGCAGGGAGC ACAGCCCAGGATGAAAGTGG
GATCACAGGGTGAAGCTAAA GATTTAAAAATTTTTAAAAA AATTATTAAAAAATAAATAT
TTAAAAGATTTGCATTTGTT AAGACATAAAGGAAATTTAG AAATTTTAAACAATATCTTA
CAAATTCCCCATGTGTCCAA A Nucleic acid sequences are shown 5'-3'. DNA
is uppercase. The location of the SNP is underlined.
[0077] The results are shown in FIGS. 5A and 5B. From this, it is
clear that the universal rhPCR genotyping system can be used to
characterize multi-allelic genotypes.
[0078] A Tri-allelic AD 360plot was designed for illustrating
allelic discrimination. Fluorescence signal (.DELTA.Rn) from the
last PCR cycle of each dye was normalized across the three dyes
from the same well. Angle and distance of data point from the
origin is calculated using formula below:
Angle=tan.sup.-1(.DELTA.RnDye.sub.1/.DELTA.RnDye.sub.2).times.120/90
Distance from origin= {square root over
((.DELTA.RnDye.sub.1).sup.2+(.DELTA.RnDye.sub.2).sup.2)}
[0079] FIG. 5B shows the Tri-allelic Allelic Discrimination 360plot
of rs72558195, using rhPCR genotyping assay with 3 allele-specific
primers multiplexed in a single reaction. By collecting
fluorescence signal from all assays, six genotypes could be
detected in a single reaction. The distance of data points from
origin indicated the signal strength of dyes and the wide angle
separation between data clusters indicated specificity of multiplex
assay. NTC in the center of the plot indicated no primer dimers or
non-specific amplification. The specificity of multiplex assay is
achieved by the selectivity of RNase H2 and the mutant Taq DNA
polymerase as used in the previous examples. This AD 360plot will
also enable auto-calling capability by genotyping software.
[0080] A 360plot could be implemented for tetra-allelic,
penta-allelic or hexa-allelic visualization. Therefore,
visualization is possible for positions that could have multiple
bases as well as potential deletions. The distance from origin
remains unchanged for each calculation, and the angle formulas
would be:
tetra-allelic (4 alleles):
Angle=tan.sup.-1(.DELTA.RnDye.sub.1/.DELTA.RnDye.sub.2)
penta-allelic (5 alleles):
Angle=tan.sup.-1(.DELTA.RnDye.sub.1/.DELTA.RnDye.sub.2).times.72/90
hexa-allelic (6 alleles):
Angle=tan.sup.-1(.DELTA.RnDye.sub.1/.DELTA.RnDye.sub.2).times.60/90
Example 8
[0081] The following example illustrates the capability of the
methods of the present invention to provide quantitative SNP
genotyping, allowing for determination of the copy numbers of
different alleles. To demonstrate this, an assay was designed
against rs1135840, a SNP in the human CYP2D6 gene. This gene can be
present in multiple copies, and the number of copies with the
rs1135840 SNP appears to affect drug metabolism (rapid metabolism
of the drug Debrisoquine).
[0082] To demonstrate that the assay system can detect small
differences in allele rations, a standard curve for analysis was
created. Two gBlock.TM. (IDT, Coralville, Iowa) gene fragments were
synthesized (Allele 1 and Allele 2, representing the two allelic
variants (G>C) of the rs1135840 SNP) and then mixed at different
ratios. Reactions were performed in 10 .mu.L volumes, containing a
total of 1500 copies of template at the ratios shown (10:0, 9:1,
8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10), 250 nM of
universal FAM probe (SEQ ID NO: 14), 450 nM of universal Yakima
Yellow.RTM. (SEQ ID NO: 22) probe, 1000 nM of universal forward
primer (SEQ ID NO: 21), 150 nM of the two allele-specific forward
primers, 500 nM of the reverse primer, and 5 .mu.L of 2.times.
Integrated DNA Technologies (IDT) (Coralville, Iowa) rhPCR
genotyping master mix (containing dNTPs, a mutant H784Q Taq
polymerase (see Behlke, et al. U.S. 2015/0191707), chemically
modified Pyrococcus abyssi RNase H2 (See Walder et al.
UA20130288245A1), stabilizers, and MgCl.sub.2).
[0083] PCR was performed on Life Technologies (Carlsbad, Calif.)
QuantStudio.TM. 7 Flex real-time PCR instrument using the following
cycling conditions: 10 mins at 95.degree. C. followed by 45 cycles
of 95.degree. C. for 10 seconds and 60.degree. C. for 45 seconds.
End-point analysis of each of the plates was performed after 45
cycles with software provided by the respective companies (Bio-Rad
CFX Manager 3.1 software (Bio-Rad, Hercules, Calif.) and
QuantStudio.TM. Real-Time PCR Software v1.3 (Carlsbad,
Calif.)).
TABLE-US-00016 TABLE 16 Oligonucleotide sequences used in Example
8. Name Sequence SEQ ID NO. Universal CGGCCCATGTCCCAGCGAA SEQ ID
NO. 21 Forward primer Probe 1 FAM-C+CATC+A+C+CGTG+ SEQ ID NO. 14
(FAM) CT-IBFQ Probe 2 Yak-CAATC+C+C+CGAG+ SEQ ID NO. 22 (Yakima
CT-IBFQ Yellow) rs1135840 GCCCATGTCCCAGCGAACCA SEQ ID NO. 38 Allele
1 TCACCGTGCTGTCTTTGCTT Forward TCCTGGTGAGcCCATG-x primer rs1135840
GCCCATGTCCCAGCGAACAA SEQ ID NO. 39 Allele 2 TCCCCGAGCTGTCTTTGCTT
Forward TCCTGGTGAcCCATG-x primer rs1135840 GCGTTGGAACTACCACATTG SEQ
ID NO. 40 Reverse CTTTATuGTACT-x primer Nucleic acid sequences are
shown 5'-3'. DNA is uppercase, RNA is lowercase. LNA residues are
designated with a +. Location of potential mismatch is underlined.
FAM = 6-carboxyfluorescein, Yak = Yakima Yellow
(3-(5,6,4',7'-tetrachloro-5'-methyl-3',6'-dipivaloylfluorescein-2-yl)),
IBFQ = Iowa Black FQ (fluorescence quencher), and x = C3
propanediol spacer block.
[0084] The resulting data is illustrated in FIG. 7. The spread of
each of the sample mixes is sufficient for the determination of the
number of copies of each template.
[0085] After demonstration of the required amount of separation of
allelic quantities, it is possible to determine the number of
copies present of each allele in an experimental sample. To test
this, the previously described assay designed against rs1135840,
was utilized to test thirteen Coriell genomic DNA (Camden, N.J.)
samples with varying CYP2D6 copy numbers with varying rs1135840
genotypes. These samples have known defined copy numbers and
rs1135840 genotypes which could be verified after testing with the
universal rhPCR genotyping mix. From this, these samples can also
be categorized as being homozygotes for either allele, or
heterozygotes.
[0086] To calculate the copy number from the data, two duplex
reactions were run for each sample. Reactions were performed in 10
.mu.L volumes, containing 3 ng of one of the following genomic
DNAs: NA17123, NA17131, NA17132, NA17149, NA17104, NA17113,
NA17144, NA17213, NA17221, NA17114, NA17235, or NA17241. Each
individual assay also contained 50 nM ROX normalizer oligo, 250 nM
of universal FAM probe (SEQ ID NO: 14), 450 nM of universal Yakima
Yellow.RTM. (SEQ ID NO: 22) probe, 1000 nM of universal forward
primer (SEQ ID NO: 21), 150 nM of the two allele-specific forward
primers (SEQ ID NO: 38 and 39), 500 nM of the reverse primer (SEQ
ID NO: 40), and 5 .mu.L of 2.times. Integrated DNA Technologies
(IDT) (Coralville, Iowa) rhPCR genotyping master mix (containing
dNTPs, a mutant H784Q Taq polymerase (see Behlke, et al. U.S.
2015/0191707), chemically modified Pyrococcus abyssi RNase H2 (See
Walder et al. UA20130288245A1), stabilizers, and MgCl.sub.2).
Assays also contained a separate RNase P assay (See table 17, SEQ
ID NOs: 41-43)) for normalization of the template
concentration.
TABLE-US-00017 TABLE 17 RNase P assay sequences used in Example 8.
Name Sequence SEQ ID NO. RNase P GCGGAGGGAAGCTCATCAG SEQ ID NO. 41
Forward primer RNase P CCCTAGTCTCAGACCTTCCCAA SEQ ID NO. 42 Reverse
primer Probe 2 Yak-CCACGAGCTGAGTGCGTC SEQ ID NO. 43 (Yakima
CTGTCA-IBFQ Yellow) Nucleic acid sequences are shown 5'-3'. DNA is
uppercase. FAM = 6-carboxyfluorescein, Yak = Yakima Yellow
(3-(5,6,4',7'-tetrachloro-5'-methyl-3',6'-dipivaloylfluorescein-2-yl)),
IBFQ = Iowa Black FQ (fluorescence quencher).
[0087] Quantitative PCR was performed on Life Technologies
(Carlsbad, Calif.) QuantStudio.TM. 7 Flex real-time PCR instrument
using the following cycling conditions: 10 mins at 95.degree. C.
followed by 45 cycles of 95.degree. C. for 10 seconds and
60.degree. C. for 45 seconds. End-point analysis of each of the
plates was performed after 45 cycles with the QuantStudio.TM.
Real-Time PCR Software v1.3 (Carlsbad, Calif.) software provided by
the company.
[0088] Copy number was determined by the following method. For each
sample shown to be a homozygote, .DELTA.Cq (RNase P Cq-rs1135840
assay Cq) was calculated for each sample. For samples shown to be
heterozygotes, .DELTA.Cq was calculated for both alleles (RNase P
Cq-rs1135840 assay 1 Cq and RNase P Cq-rs1135840 assay 2 Cq). Next,
.DELTA..DELTA.Cq (.DELTA.Cq-mean .DELTA.Cq for known 2 copy control
DNA samples) was calculated for each allele. This correction
allowed for normalization against amplification differences between
the SNP assay and the RNase P assay. Finally, the following
equation was used to calculate copy number for each allele:
Copy number of allele=2*(2 (.DELTA..DELTA.Cq))
[0089] The resulting end-point data is shown in FIG. 8A and
calculated copy numbers are shown in FIG. 8B. The genotypes
determined in FIG. 8A (homozygotes allele 1, Homozygotes allele 2,
or heterozygote) all matched the known genotypes, and allowed
correct calculation of the copy number. The established reference
copy number of the individual samples is shown under each result.
In each case, the copy number determined by the assay correctly
determined the genotype and copy number of the input DNA.
Example 9
[0090] The following example demonstrates that a variation of an
rhPCR probe can be used for multiplexed rhPCR.
[0091] The assay schematic is provided in FIG. 9. In the first
round of PCR, 5' tailed target-specific rhPrimers are used. The 5'
tails upon incorporation into the amplicon contain binding sites
for a second round of PCR with different primers (blocked or
unblocked) to add application specific sequences. For example, as
depicted in FIG. 9, this system can be used for amplification
enrichment for next generation sequencing. In this case, 5' tailed
rhPCR primers contain read 1/read 2 primer sequences. The second
round of PCR adds adapter sequences such as the P5/P7 series for
Illumina.RTM. based sequencing platforms or other adaptors,
including ones containing barcodes/unique molecular identifiers.
This approach allows for adding any additional sequences onto the
amplicon necessary for input into any NGS platform type.
[0092] As illustrated in FIG. 10, two primers sets, including one
containing a 96-plex set of 5' tailed rhPrimers, and one containing
96 DNA "standard" 5' tailed PCR primers were designed using an IDT
algorithm. The two primer sets differed only in that the rhPrimers
contained an internal cleavable RNA base and a blocking group on
the 3' end. Once the blocking group was removed by RNase H2
cleavage, the primer sequences become identical.
[0093] The first round of PCR reactions contained the 96 plex at 10
nM of each blocked target specific primer, 10 ng of NA12878 human
genomic DNA (Coriell Institute for Medical Research, Camden, N.J.),
200 mU of chemically modified Pyrococcus abyssi RNase H2 (See
Walder et al. UA20130288245A1) (IDT, Coralville, Iowa) and
1.times.KAPA 2G HotStart Fast Ready Mix.TM. (Kapa Biosystems,
Wilmington, Mass.). The thermal cycling profile was 10 mins at
95.degree. C. followed by 8 cycles of 95.degree. C. for 15 seconds
and 60.degree. C. for 4 minutes, and a final 99.degree. C.
finishing step for 15 minutes. Reactions were cleaned up with a
2.times. AMPure.TM. XP beads (Beckman Coulter, Brea, Calif.).
Briefly, 100 .mu.L AMPure.TM. SPRI beads were added to each PCR
well, incubated for 5 minutes at room temperature and collected for
5 minutes at room temperature on plate magnet (DynaMag.TM.
(Thermo-Fisher, (Watherham, Mass.) 96-well plate side-magnet).
Beads were washed twice with 80% ethanol, and allowed to dry for 3
minutes at room temperature. Samples were eluted in 22 .mu.L of TE
at pH 8.0.
[0094] The second round of PCR was set up using 20 .mu.L of the
cleaned up first round PCR products, universal PCR-50F and PCR-47R
primers (See table 18, SEQ ID NOs: 44 and 45) at 2 uM and
1.times.KAPA 2G HotStart Fast Ready Mix.TM. (KAPA Biosystems,
Wilmington, Mass.). Reactions were cycled for 45 seconds at
98.degree. C. followed by 20 cycles of 98.degree. C. for 15
seconds, 60.degree. C. for 30 seconds, and 72.degree. C. for 30
seconds. A final 1 minute 72.degree. C. polishing step finished the
reaction. Samples were cleaned up again with 0.8.times. AMPure.TM.
beads. Briefly, 40 .mu.L AMPure.TM. SPRI beads were added the
second PCR wells, incubated for 5 minutes at room temperature and
collected for 5 minutes at room temperature on plate magnet
(DynaMag.TM. (Thermo-Fisher, (Watherham, Mass.) 96-well plate
side-magnet). Beads were washed twice with 80% ethanol, and allowed
to dry for 3 minutes at room temperature. Samples were eluted in 22
.mu.L of TE at pH 8.0, and 20 .mu.L was transferred to a new
tube.
[0095] 2 .mu.L of the samples were analyzed using the Agilent.RTM.
High Sensitivity D1000.TM. Screen Tape.TM. on the Agilent.RTM. 2200
Tape Station.TM. (Agilent Technologies.RTM., Santa Clara, Calif.).
Quantification was performed using the KAPA Library Quantification
Kit (KAPA Biosystems, Wilmington, Mass.) for Illumina.RTM.
Platforms, according to the manufacturer's protocol. Replicate
samples were pooled to a final concentration of 10 pM, and 1% PhiX
bacteriophage sequencing control was added. Samples were run with a
V2 300 cycle MiSeq.TM. kit on an Illumina.RTM. (San Diego, Calif.)
MiSeq.TM. platform, using standard protocols from the
manufacturer.
TABLE-US-00018 TABLE 18 Universal assay sequences used in Example
9. Name Sequence SEQ ID NO. Universal AATGATACGGCGACCAC SEQ ID NO.
44 PCR-50F CGAGATCTACACTCTTT CCCTACACGACGCTCT Universal
CAAGCAGAAGACGGCAT SEQ ID NO. 45 PCR-47R ACGAGATGGACCTATGT
GACTGGAGTTCAGACGT GTGC Nucleic acid sequences are shown 5'-3'. DNA
is uppercase.
[0096] FIG. 10 shows the results from the Agilent.RTM. Tape
Station. The primer dimer product was the most significant product
produced using standard DNA primers in the presence of DNA
template, with only a small amount of full length expected product.
In the absence of template, the primer dimer product was the major
component of the reaction. In the case of the blocked rhPCR
primers, the vast majority of the material was the desired PCR
products, with little primer dimer observed. In the absence of
template, there is no primer dimer present, contrasting with the
overwhelming abundance of primer dimer observed in the no template
lane of the unblocked DNA primers. Quantitation of the product
versus primer dimer bands show that mass ratio of product to primer
dimer for the unblocked DNA primers was 0.6. The mass ratio for the
rhPCR primers was 6.3.
[0097] FIG. 11 summarizes two key sequencing metrics. The first is
the percent of mapped reads from the sequencing data. The rhPCR
reactions gave a percentage of reads mapped to the human genome at
85%, whereas the non-blocked DNA primers on give a mapped read
percentage of less than 20. A second metric, the percentage of
on-target reads, is almost 95% when using rhPCR primers, but less
than 85% when the non-blocked primers are used in the multiplex.
These results clearly demonstrate the utility of using rhPCR in
multiplexing, where a large increase of the desired material is
seen, and a vast reduction in undesired side products is observed.
The differences mean less unwanted sequencing reads, and the depth
of coverage of desired sequences is higher.
[0098] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0099] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0100] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
45124DNAArtificial SequenceSynthetic 1gctgtgattt tggtctagct acag
24225DNAArtificial SequenceSynthetic 2gctgtgattt tggtctagct acagt
25325DNAArtificial SequenceSynthetic 3gctgtgattt tggtctagct acaga
25427DNAArtificial SequenceSynthetic 4tcccatcagt ttgaacagtt gtctgga
27531DNAArtificial SequenceSynthetic 5gctgtgattt tggtctagct
acagtgaaat g 31631DNAArtificial SequenceSynthetic 6gctgtgattt
tggtctagct acagagaaat g 31731DNAArtificial SequenceSynthetic
7gccctcaatt cttaccatcc acaaaatgga a 318401DNAArtificial
SequenceSynthetic 8aaaaaataag aacactgatt tttgtgaata ctgggaacta
tgaaaatact atagttgaga 60ccttcaatga ctttctagta actcagcagc atctcagggc
caaaaattta atcagtggaa 120aaatagcctc aattcttacc atccacaaaa
tggatccaga caactgttca aactgatggg 180acccactcca tcgagatttc
actgtagcta gaccaaaatc acctattttt actgtgaggt 240cttcatgaag
aaatatatct gaggtgtagt aagtaaagga aaacagtaga tctcattttc
300ctatcagagc aagcattatg aagagtttag gtaagagatc taatttctat
aattctgtaa 360tataatattc tttaaaacat agtacttcat ctttcctctt a
4019401DNAArtificial SequenceSynthetic 9aaaaaataag aacactgatt
tttgtgaata ctgggaacta tgaaaatact atagttgaga 60ccttcaatga ctttctagta
actcagcagc atctcagggc caaaaattta atcagtggaa 120aaatagcctc
aattcttacc atccacaaaa tggatccaga caactgttca aactgatggg
180acccactcca tcgagatttc tctgtagcta gaccaaaatc acctattttt
actgtgaggt 240cttcatgaag aaatatatct gaggtgtagt aagtaaagga
aaacagtaga tctcattttc 300ctatcagagc aagcattatg aagagtttag
gtaagagatc taatttctat aattctgtaa 360tataatattc tttaaaacat
agtacttcat ctttcctctt a 4011029DNAArtificial SequenceSynthetic
10gctgtgattt tggtctagct acagtgatg 291129DNAArtificial
SequenceSynthetic 11gctgtgattt tggtctagct acagagatg
291231DNAArtificial SequenceSynthetic 12gccctcaatt cttaccatcc
acaaaatgga a 311323DNAArtificial SequenceSynthetic 13cgccgcgtat
agtcccgcgt aaa 231413DNAArtificial SequenceSynthetic 14ccatcaccgt
gct 131513DNAArtificial SequenceSynthetic 15caatccccga gct
131653DNAArtificial SequenceSynthetic 16gcccatgtcc cagcgaacca
tcaccgtgct agccctcgat acagcccggc cac 531752DNAArtificial
SequenceSynthetic 17gcccatgtcc cagcgaacaa tccccgagct gccctcgata
cagcctggcc ac 521826DNAArtificial SequenceSynthetic 18gcggccaggt
atacggacat catcca 2619401DNAArtificial SequenceSynthetic
19gttgggagct gggagggact gagttagggt gcacggggcg gccagtctca ccactgacca
60gtttgtctgt ctgtgtgtgt ccatgtgcga gggcagagga ggaccccaca tggaccgcag
120cagcgcccga ggccaggtat acggacatca tcctgtacgc gtcgggctcc
ctggccttgg 180ctgtgctcct gctgctggcc gggctgtatc gagggcaggc
gctccacggc cggcaccccc 240gcccgcccgc cactgtgcag aagctctccc
gcttccctct ggcccgacag gtactgggcg 300catcccccac ctcacatgtg
acagcctgac tccagcaggc agaaccaagt ctcccacttt 360gcagttctcc
ctggagtcag gctcttccgg caagtcaagc t 40120401DNAArtificial
SequenceSynthetic 20gttgggagct gggagggact gagttagggt gcacggggcg
gccagtctca ccactgacca 60gtttgtctgt ctgtgtgtgt ccatgtgcga gggcagagga
ggaccccaca tggaccgcag 120cagcgcccga ggccaggtat acggacatca
tcctgtacgc gtcgggctcc ctggccttgg 180ctgtgctcct gctgctggcc
aggctgtatc gagggcaggc gctccacggc cggcaccccc 240gcccgcccgc
cactgtgcag aagctctccc gcttccctct ggcccgacag gtactgggcg
300catcccccac ctcacatgtg acagcctgac tccagcaggc agaaccaagt
ctcccacttt 360gcagttctcc ctggagtcag gctcttccgg caagtcaagc t
4012119DNAArtificial SequenceSynthetic 21cggcccatgt cccagcgaa
192213DNAArtificial SequenceSynthetic 22caatccccga gct
132356DNAArtificial SequenceSynthetic 23gcccatgtcc cagcgaacca
tcaccgtgct acttcccaca ccctcatatc utgtta 562459DNAArtificial
SequenceSynthetic 24gcccatgtcc cagcgaacaa tccccgagct cttacttccc
acaccctcat atautgtta 592532DNAArtificial SequenceSynthetic
25gcgctaagta aacattcctg attgcaactt at 3226490DNAArtificial
SequenceSynthetic 26gatttttttt ttttggcatt tcttcttaga tttctatctc
ctaacatagg atcacttatt 60tgtgaaatta tttgtatacc ttttttatgg agtgatgatg
tgatacaaat tctatcctta 120aggatataag aacatctttt ctttatatta
ggatttttct ggacccatga gttacatgct 180tacttcccac accctcatat
cttgtttaaa tttgtagaat taaattcata ggtaattatt 240tctgaaactt
cttccctgtg tgagcaatct aaataattat tacaatgcct taagttgcaa
300tcaggaatgt ttacttagca cagacttttt tccccactac tgcactcaaa
ggataacaga 360tatatggcaa atctaaccat attctttgtc ctttgtccat
gttgcggagg gaagctcatc 420agtggggcca cgagctgagt gcgtcctgtc
actccactcc catgtccctt gggaaggtct 480gagactaggg
49027490DNAArtificial SequenceSynthetic 27gatttttttt ttttggcatt
tcttcttaga tttctatctc ctaacatagg atcacttatt 60tgtgaaatta tttgtatacc
ttttttatgg agtgatgatg tgatacaaat tctatcctta 120aggatataag
aacatctttt ctttatatta ggatttttct ggacccatga gttacatgct
180tacttcccac accctcatat attgtttaaa tttgtagaat taaattcata
ggtaattatt 240tctgaaactt cttccctgtg tgagcaatct aaataattat
tacaatgcct taagttgcaa 300tcaggaatgt ttacttagca cagacttttt
tccccactac tgcactcaaa ggataacaga 360tatatggcaa atctaaccat
attctttgtc ctttgtccat gttgcggagg gaagctcatc 420agtggggcca
cgagctgagt gcgtcctgtc actccactcc catgtccctt gggaaggtct
480gagactaggg 4902859DNAArtificial SequenceSynthetic 28gcccatgtcc
cagcgaacca tcaccgtgct ttctcttctg gactccctat aatattgtg
592959DNAArtificial SequenceSynthetic 29gcccatgtcc cagcgaacaa
tccccgagct ttctcttctg gactccctat aacattgtg 593026DNAArtificial
SequenceSynthetic 30gcggattgat gcagcagtga gtcatg
263156DNAArtificial SequenceSynthetic 31gcccatgtcc cagcgaacca
tcaccgtgct ctccgttgtt ttccagaaac gatttc 563256DNAArtificial
SequenceSynthetic 32gcccatgtcc cagcgaacaa tccccgagct ctccgttgtt
ttccagaaat gatttc 563356DNAArtificial SequenceSynthetic
33gcccatgtcc cagcgaacaa tccccgagct ctccgttgtt ttccagaaag gatttc
563428DNAArtificial SequenceSynthetic 34gcaaccaagt cttccctaca
accttgat 2835401DNAArtificial SequenceSynthetic 35acatcatttt
tattgtataa aagcatttta gtatcaattt tctcattttt aaaccaagtc 60ttccctacaa
ccttgaataa atggtttcca aggaaaataa aatcttggcc ttacctggat
120ccatggggag ttcagaatcc tgaagttttc attgaatctt ttcatcaggg
tgagaaaatt 180ctgatcttta taatcaaatc gtttctggaa aacaacggag
cagatcacat tgcagggagc 240acagcccagg atgaaagtgg gatcacaggg
tgaagctaaa gatttaaaaa tttttaaaaa 300aattattaaa aaataaatat
ttaaaagatt tgcatttgtt aagacataaa ggaaatttag 360aaattttaaa
caatatctta caaattcccc atgtgtccaa a 40136401DNAArtificial
SequenceSynthetic 36acatcatttt tattgtataa aagcatttta gtatcaattt
tctcattttt aaaccaagtc 60ttccctacaa ccttgaataa atggtttcca aggaaaataa
aatcttggcc ttacctggat 120ccatggggag ttcagaatcc tgaagttttc
attgaatctt ttcatcaggg tgagaaaatt 180ctgatcttta taatcaaatc
atttctggaa aacaacggag cagatcacat tgcagggagc 240acagcccagg
atgaaagtgg gatcacaggg tgaagctaaa gatttaaaaa tttttaaaaa
300aattattaaa aaataaatat ttaaaagatt tgcatttgtt aagacataaa
ggaaatttag 360aaattttaaa caatatctta caaattcccc atgtgtccaa a
40137401DNAArtificial SequenceSynthetic 37acatcatttt tattgtataa
aagcatttta gtatcaattt tctcattttt aaaccaagtc 60ttccctacaa ccttgaataa
atggtttcca aggaaaataa aatcttggcc ttacctggat 120ccatggggag
ttcagaatcc tgaagttttc attgaatctt ttcatcaggg tgagaaaatt
180ctgatcttta taatcaaatc ctttctggaa aacaacggag cagatcacat
tgcagggagc 240acagcccagg atgaaagtgg gatcacaggg tgaagctaaa
gatttaaaaa tttttaaaaa 300aattattaaa aaataaatat ttaaaagatt
tgcatttgtt aagacataaa ggaaatttag 360aaattttaaa caatatctta
caaattcccc atgtgtccaa a 4013856DNAArtificial SequenceSynthetic
38gcccatgtcc cagcgaacca tcaccgtgct gtctttgctt tcctggtgag cccatg
563955DNAArtificial SequenceSynthetic 39gcccatgtcc cagcgaacaa
tccccgagct gtctttgctt tcctggtgac ccatg 554032DNAArtificial
SequenceSynthetic 40gcgttggaac taccacattg ctttatugta ct
324119DNAArtificial SequenceSynthetic 41gcggagggaa gctcatcag
194222DNAArtificial SequenceSynthetic 42ccctagtctc agaccttccc aa
224324DNAArtificial SequenceSynthetic 43ccacgagctg agtgcgtcct gtca
244450DNAArtificial SequenceSynthetic 44aatgatacgg cgaccaccga
gatctacact ctttccctac acgacgctct 504555DNAArtificial
SequenceSynthetic 45caagcagaag acggcatacg agatggacct atgtgactgg
agttcagacg tgtgc 55
* * * * *