U.S. patent application number 16/374752 was filed with the patent office on 2019-07-18 for methods for variant detection.
The applicant listed for this patent is Integrated DNA Technologies, Inc.. Invention is credited to Mark Aaron Behlke, Joseph Dobosy, Richard Owczarzy.
Application Number | 20190218611 16/374752 |
Document ID | / |
Family ID | 59786177 |
Filed Date | 2019-07-18 |
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United States Patent
Application |
20190218611 |
Kind Code |
A1 |
Dobosy; Joseph ; et
al. |
July 18, 2019 |
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. The invention also provides methods
for detection of DNA sequences altered after cleavage by a
targetable endonuclease, such as the CRISPR Cas9 protein from the
bacterium Streptococcus pyogenes.
Inventors: |
Dobosy; Joseph; (Coralville,
IA) ; Owczarzy; Richard; (Coralville, IA) ;
Behlke; Mark Aaron; (Coralville, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Integrated DNA Technologies, Inc. |
Coralville |
IA |
US |
|
|
Family ID: |
59786177 |
Appl. No.: |
16/374752 |
Filed: |
April 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15604204 |
May 24, 2017 |
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16374752 |
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15487401 |
Apr 13, 2017 |
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15604204 |
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15361280 |
Nov 25, 2016 |
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15487401 |
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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 2600/156 20130101;
C12Q 1/6876 20130101; C07K 2319/21 20130101; C12N 2310/20 20170501;
C12Y 207/07007 20130101; C12Q 1/6858 20130101; C12Q 1/6853
20130101; C12Y 301/00 20130101; C12N 15/11 20130101; C12Y 301/26004
20130101; C12Q 1/6858 20130101; C12Q 2521/327 20130101; C12Q
2525/121 20130101; C12Q 2525/186 20130101; C12Q 2525/125 20130101;
C12N 9/22 20130101; C12Q 2525/161 20130101 |
International
Class: |
C12Q 1/6876 20060101
C12Q001/6876; C12N 15/11 20060101 C12N015/11; C12N 9/22 20060101
C12N009/22; C12Q 1/6853 20060101 C12Q001/6853; C12Q 1/6858 20060101
C12Q001/6858 |
Claims
1. A blocked-cleavable primer for rhPCR, the primer comprising:
5'-A-B-Z-E-3' wherein A is optional and is a tail extension that is
not complementary to a target; B is a sequence domain that is
complementary to a target; Z comprises: C, a discrimination domain,
and D, a cleavage domain that, when hybridized to the target, is
cleavable by RNase H2; and E is a blocking domain that prevents
extension of the primer.
2. The primer of claim 1, wherein the discrimination domain C is
located 5' of the cleavage domain D.
3. The primer of claim 1, wherein the discrimination domain C is
located 3' of the cleavage domain D.
4. The primer of claim 1, wherein the discrimination domain C
overlaps with the cleavage domain D.
5. The primer of claim 1, wherein C comprises 1-3 RNA bases.
6. The primer of claim 1, wherein C comprises 1 RNA base.
7. The primer of claim 6, wherein the discrimination domain C
consists of the 1 RNA base.
8. The primer of claim 1, wherein the cleavage domain comprises one
or more of the following moieties: a DNA residue, an abasic
residue, a modified nucleoside, or a modified phosphate
internucleotide linkage.
9. The primer of claim 8, wherein a sequence flanking the cleavage
site contains one or more internucleoside linkages resistant to
nuclease cleavage.
10. The primer of claim 9, wherein the nuclease resistant linkage
is a phosphorothioate.
11. The primer of claim 5, wherein the 3' oxygen atom of at least
one of the RNA residues is substituted with an amino group, thiol
group, or a methylene group.
12. The primer of claim 1, wherein the blocking group is attached
to the 3'-terminal nucleotide of the primer.
13. The primer of claim 1, wherein A comprises a region that is
identical to a universal forward primer and optionally a probe
binding domain.
14-54. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 15/487,401, filed Apr. 13, 2017, which is a
continuation-in-part of U.S. application Ser. No. 15/361,280, filed
Nov. 25, 2016, which 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 all 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. The invention also provides methods for
detection of DNA sequences altered after cleavage by a targetable
endonuclease, such as the CRISPR Cas9 protein from the bacterium
Streptococcus pyogenes.
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.
[0005] There is thus a need for assays with improved mismatch
sensitivity.
[0006] In addition, there is a need for improved methods for
detection of mutations altered after cleavage by targetable
endonucleases, such as the CRISPR Cas9 protein. A commonly used
method to detect mutations introduced into genomic DNA following
repair of dsDNA cleavage events is the enzymatic mismatch cleavage
assay (EMCA). EMCA assays cleave at sites where base mismatches are
present in dsDNA. For EMCA detection of the mutations introduced
into DNA following Cas9 cleavage and repair, genomic DNA from cells
is harvested and the regions around the dsDNA cut site is amplified
by PCR using primers that flank the cut site. Typically 100-1000
base amplicons are used for this purpose. Following completion of
amplification, heteroduplexes are formed by heating the reaction
products and allowing them to re-anneal, which leads to formation
of homoduplex WT/WT, Mut/Mut or heteroduplex WT/Mut or Mut1/Mut2
variants. The dsDNAs are then subjected to cleavage by a mismatch
endonuclease (such as T7EI, Surveyor, etc.). Heteroduplexes are
cleaved and the presence of shorter fragments is detected by gel
electrophoresis, capillary electrophoresis, or any of a number of
methods known to those of skill in the art. Although such an assay
is fast and inexpensive, it often does not accurately reflect the
changes that are actually generated from the CRISPR mutagenesis
process. If the same mutation is introduced a large number of
times, Mut/Mut homodimers form, which are not detected. Further,
the mismatch endonuclease enzymes often fail to cleave single-base
events, leading to yet another class of mutations that are
undetected. Thus an EMCA assay will almost always underestimate the
extent of genome editing that occurred after Cas9 dsDNA cleavage
and repair.
[0007] An alternative method of analysis involves large scale DNA
sequencing using "Next-Gen" sequencing (NGS) methods of the
modified DNA, which is highly accurate, but is slow and costly.
Other methods are available to assess the mutation outcome
following CRISPR/Cas9 cleavage and repair. For example, Sanger
sequencing results can be analyzed using sequence trace
decomposition ("TIDE" analysis); fluorescent-labeled
primer-extension on an amplicon spanning the Cas9 cut site can be
used to map indels using Indel Detection by Amplicon Analysis
(IDAA); or high resolution melt analysis (HRM) can be applied to
PCR amplicons that span the Cas9 cut site. However, none of these
methods approaches the accuracy of NGS analysis, while all are more
costly and slower to perform than EMCA methods. Thus, improved
methods are needed to assess the frequency of mutations that arise
from genome editing experiments that are rapid and low cost.
BRIEF SUMMARY OF THE INVENTION
[0008] 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, 3' of the RNA,
overlapping with the RNA, and/or in the same position as the
RNA.
[0009] 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. The invention also provides methods
for detection of DNA sequences altered after cleavage by a
targetable endonuclease, such as the CRISPR Cas9 protein from the
bacterium Streptococcus pyogenes.
[0010] 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
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] FIGS. 5A and 5B are Allelic Discrimination plots of
tri-allelic SNP, CYP2C8 (r572558195), 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.
[0016] 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.
[0017] FIG. 7 is an allelic discrimination plot illustrating the
ability of the rhPCR assay to perform quantitative genotyping.
[0018] 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.
[0019] FIG. 9 is a schematic representation of multiplex rhPCR.
[0020] 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.
[0021] FIG. 11 graphically represents the effectiveness of the
rhPrimers in the percent of mapped reads and on-target reads.
[0022] FIG. 12 shows placement of the RNA residue relative to the
most common cleavage site for the methods of the disclosure (such
as in Example 10). The RNA is shown in lower case, while DNA
residues are shown in upper case. Cas9 cleavage site is shown with
a line through both strands of DNA. RN2=RNase H2 enzyme;
pot=polymerase.
[0023] FIGS. 13A-13B show analysis of CRISPR mutations
demonstrating that the results obtained using the qPCR methods of
the disclosure are more accurate than using the T7EI EMCA method.
FIG. 13A: the percentage of mutated templates is consistently
underestimated by T7EI EMCA (empty squares), when compared with NGS
results (grey circles) on the same samples. FIG. 13B: using the
same samples from FIG. 13A, the percentage of mutations detected is
seen to be much more accurately estimated by the qPCR methods of
the disclosure (empty squares) when compared with NGS results.
DETAILED DESCRIPTION OF THE INVENTION
[0024] 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).
[0025] 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.
[0026] 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.
[0027] 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.
[0028] Placing the mismatch at either a position 5' or 3' of the
RNA is also useful in some embodiments. For example, a mutation
located 5' of the RNA allows for the use of a highly discriminatory
DNA polymerase as a secondary selection step during PCR
amplification following primer cleavage, although this
configuration restricts the mismatch discrimination potential of
the RNase H2 to the first cleavage event, and subsequent priming
events on daughter amplicons will bind to whatever sequence was
present in the primer. Placement of the mismatch 3' of the RNA
reduces or eliminates the potential for primer conversion to the
opposite allele.
[0029] Thus, in some aspects, the disclosure provides methods and
compositions comprising mutant RNase H2 enzymes that show enhanced
mismatch discrimination during rhPCR when the mutations are
positioned 5'- or 3'-relative to the RNA residue. For example, and
without limiting the scope of the disclosed aspects and embodiments
of the disclosure, mutations located at the twelfth, thirteenth,
and 169th amino acid positions in the RNase H2 enzyme from
Pyrococcus abyssi (P.a.) have been identified that have this
unexpected and useful property. Similar mutations in RNase H2
enzymes from other species that support rhPCR may likewise show
similar improved properties relative to their WT forms.
[0030] In other embodiments, the methods of the disclosure comprise
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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] Further aspects of the disclosure pertain to detection of
DNA sequences altered after cleavage by a targetable endonuclease,
such as the CRISPR Cas9 protein from the bacterium Streptococcus
pyogenes. This protein and similar ones have successfully been used
for targeted genomic modification in the well documented Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR)
system.
[0036] 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--a bacterial immune defense system comprised of an
endonuclease that is targeted to double-stranded DNA by a guide
RNA--are 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.
[0037] 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.
[0038] 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.
[0039] Thus, in further aspects, the disclosure provides methods
that employ the above-described universal rhPCR assay system to
detect mutations generated by a targetable endonuclease such as
Cas9 or Cpf1. The rhPCR assays according to these aspects of the
disclosure utilizes a thermostable RNase H2 enzyme, and optionally
a DNA polymerase with enhanced mismatch discrimination. The RNase
H2 cleaves at the single RNA residue only when the primer
oligonucleotide is duplexed with a target nucleic acid, which
removes a 3'-blocking group and activates the primer. The DNA
polymerase uses the primer to initiate DNA synthesis and, in
multiple cycles, supports PCR. Discrimination of mutations is
achieved by the action of the RNase H2 or the combined action of
both the RNase H2 and the DNA polymerase, wherein the RNase H2 has
reduced de-blocking activity when a mismatch is present and the DNA
polymerase has reduced priming/DNA synthesis activity when a
mismatch is present. In one embodiment, the primers comprise
multiple functional domains including (from the 5'-end): a
universal primer domain, a universal probe binding domain, a
target-specific primer domain, a single RNA residue (cleavable
linkage), a short 3'-extension domain, and a 3'-blocking group that
prevents the oligonucleotide from priming DNA synthesis. Cleavage
by RNase H2 removes the RNA residue, 3'-extension domain, and
3'-blocking group.
[0040] In some embodiments, a second assay is present in the
reaction and runs as a 2-color multiplex, targeting the RNase P
gene or some other control gene. This second assay allows for
normalization to an internal control gene that was not targeted by
the CRISPR genome editing reaction. This control assay may be
performed as either a standard three-oligonucleotide 5' nuclease
assay, or as a second rhPCR-based universal assay.
[0041] In another embodiment, the primers lack the universal 5'
domain, but still retain the 3' removable blocking group. In this
alternative embodiment, a standard 5' nuclease
fluorescence-quenched probe is placed between the forward and
reverse primers. The probe is positioned within the amplicon such
that it lies outside of any region that may be altered by the
genome editing event.
[0042] In each experiment, relative position of the discriminatory
(i.e., mutation interrogating) primer on the sequence is important.
RNase H2 cleaves 5' of an RNA residue. Placement of the primer so
that the RNA residue binds two nucleotides after the most common
cleavage site is important for recognition of the mutagenized
samples. A diagram of this principle is shown in FIG. 12. In the
Wild-Type (WT) samples, amplification occurs normally, as neither
the RNase H2 nor the DNA polymerase are hindered in their
functions. If an insertion is introduced to the sequence, a
mismatch for both the RNase H2 and the DNA polymerase are produced,
allowing two independent chances to distinguish mutant from WT. The
same interrogation of the samples is achieved if a deletion is
present--both the RNase H2 and the DNA polymerase detect the
mismatches generated (FIG. 12). This double level of interrogation
allows for very precise quantification of the presence of mutated
DNA in a heterogeneous sample.
[0043] Thus, in one aspect, the disclosure provides
blocked-cleavable primers for rhPCR, the primers comprising:
5'-A-B-C-D-E-3', wherein A is optional and is a tail extension that
is not complementary to a target; B is a sequence domain that is
complementary to a target; C is a discrimination domain; D is a
cleavable domain that, when hybridized to the target, is cleavable
by RNase H2; and E is a blocking domain that prevents extension of
the primer.
[0044] In some embodiments, D is comprised of 1-3 RNA bases. In
some embodiments, D is comprised of 1 RNA base. In some
embodiments, the cleavage domain comprises one or more of the
following moieties: a DNA residue, an abasic residue, a modified
nucleoside, or a modified phosphate internucleotide linkage. In
some embodiments, a sequence flanking the cleavage site contains
one or more internucleoside linkages resistant to nuclease
cleavage. In some embodiments, the nuclease resistant linkage is a
phosphorothioate. In some embodiments, the 3' oxygen atom of at
least one of the RNA residues is substituted with an amino group,
thiol group, or a methylene group. In some embodiments, the
blocking group is attached to the 3'-terminal nucleotide of the
primer. In some embodiments, A is comprised of a region that is
identical to a universal forward primer and optionally a probe
binding domain.
[0045] In another aspect, the disclosure provides methods 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.
[0046] In some embodiments, the cleaving enzyme is a hot start
cleaving enzyme which is thermostable and has reduced activity at
lower temperatures. In some embodiments, the cleaving enzyme is a
chemically modified hot start cleaving enzyme which is thermostable
and has reduced activity at lower temperatures. In some
embodiments, the hot start cleaving enzyme is a chemically modified
Pyrococcus abyssi RNase H2. In some embodiments, the cleaving
enzyme is a hot start cleaving enzyme that is reversibly
inactivated through interaction with an antibody at lower
temperatures which is thermostable and has reduced activity at
lower temperatures. In some embodiments, 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. In some embodiments, 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. In some embodiments, the one or more
modified nucleosides are 2'-fluoronucleosides. In some embodiments,
the polymerase is a high-discrimination polymerase. In some
embodiments, the polymerase is a mutant H784Q Taq polymerase. In
some embodiments, the mutant H784Q Taq polymerase is reversibly
inactivated via chemical, aptamer or antibody modification. In some
embodiments, 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. In some embodiments, the target DNA sequence is a sample
that has been treated with a gene editing enzyme. In some
embodiments, the target DNA sequence is a sample that has been
treated with a CRISPR enzyme. In some embodiments, the target DNA
sequence is a sample that has been treated with a Cas9 or Cpf1
enzyme.
[0047] In another aspect, the disclosure provides oligonucleotide
compositions for genotyping a target sample, wherein the
composition comprises, from 5' to 3': (a) a tailing portion that is
non-complementary to the target sample but contains (5' to 3') a
first sequence that is identical to a universal forward primer and
a second sequence that is identical to a reporter probe sequence
that corresponds to an allele; (b) a region complementary to a
target; (c) an allele-specific domain; and (d) a 3' terminal region
containing a blocking domain. In some embodiments, the
allele-specific domain is capable of being cleaved by an RNase H
enzyme when hybridized to the target sample. In some embodiments,
the allele-specific domain is 5'-D-R-3', wherein D is a DNA base
that aligns with a SNP position of interest and R is an RNA base.
In some embodiments, the oligonucleotide is about 15-30 bases long.
In some embodiments, the blocking domain is comprised of 0-5 DNA
bases. In some embodiments, the blocking domain is comprised of at
least two of the flowing: DNA, RNA, 1,3-propanediol, mismatched DNA
or RNA, and a labeling moiety. In some embodiments, the
allele-specific domain is comprised of 2'-fluoro analogues.
[0048] In another aspect, the disclosure provides kits for
genotyping assays, the kits comprising: (a) a first allele
oligonucleotide comprised as in the previous aspect; (b) a second
allele oligonucleotide comprised as in the previous aspect; (c) a
locus-specific reverse primer; (d) a universal forward primer; (e)
a polymerase; (f) a first probe corresponding to a first allele;
(g) a second probe corresponding to a second allele; and (h) an
RNase H enzyme.
[0049] In another aspect, the disclosure provides methods of
visualization of multiple different fluorescent signals from
allelic amplification plots, the methods comprising: (a) using
three fluorescent signals from multiple fluorescent dye signals in
a single reaction well, subtracting a lowest fluorescence Dye.sub.3
from fluorescence signals from Dye.sub.1 and Dye.sub.2; (b)
calculating the distance of data from an origin and an angle from
one of the axis with an equation
Distance from origin = ( .DELTA. R n Dye 1 ) 2 + ( .DELTA. R n Dye
2 ) 2 ##EQU00001## Angle = tan - 1 ( .DELTA. R n Dye 1 / .DELTA. R
n Dye 2 ) .times. 120 90 ; and ##EQU00001.2##
(c) plotting on a circle plot with three axis, one for each dye or
allele, the resulting distance.
[0050] In another aspect, the disclosure provides methods of target
enrichment comprising: (a) providing a reaction mixture comprising
(i) a first oligonucleotide primer having a tail domain that is not
complementary to a target sequence, the tail domain comprising a
first universal primer sequence; 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 first
oligonucleotide primer wherein the blocking group prevents primer
extension and/or inhibits the first primer from serving as a
template for DNA synthesis, (ii) a sample nucleic acid that may or
may not have the target sequence, (iii) a cleaving enzyme and (iv)
a polymerase; (b) hybridizing the first primer to the target DNA
sequence to form a double-stranded substrate; (c) cleaving the
hybridized first primer, if the first primer is complementary to
the target, with the cleaving enzyme at a point within or adjacent
to the cleavage domain to remove the blocking group from the first
primer; and (d) extending the first primer with the polymerase.
[0051] In some embodiments, the methods further comprise a second
primer in reverse orientation to support priming and extension of
the first primer extension product. In some embodiments, the second
primer further comprises a tail domain comprising a second
universal primer sequence. In some embodiments, steps b-d are
performed 1-10 times. In some embodiments, the methods further
comprise removing unextended primers from the reaction and
hybridizing universal primers to the extension product to form a
second extension product. In some embodiments, the universal
primers further comprise tailed sequences for addition of adapter
sequences to the second extension product. In some embodiments,
sequencing is performed on the second extension product to
determine the sequence of the target. In some embodiments, the
target DNA sequence is a sample that has been treated with a gene
editing enzyme. In some embodiments, the target DNA sequence is a
sample that has been treated with a CRISPR enzyme. In some
embodiments, the target DNA sequence is a sample that has been
treated with a Cas9 or Cpf1 enzyme. In some embodiments, the
cleaving enzyme is a hot start cleaving enzyme which is
thermostable and has reduced activity at lower temperatures. In
some embodiments, the cleaving enzyme is a chemically modified hot
start cleaving enzyme which is thermostable and has reduced
activity at lower temperatures. In some embodiments, the hot start
cleaving enzyme is a chemically modified Pyrococcus abyssi RNase
H2. In some embodiments, the cleaving enzyme is a hot start
cleaving enzyme that is reversibly inactivated through interaction
with an antibody at lower temperatures which is thermostable and
has reduced activity at lower temperatures. In some embodiments,
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. In some embodiments, 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. In some
embodiments, the one or more modified nucleosides are
2'-fluoronucleosides. In some embodiments, the polymerase is a
high-discrimination polymerase. In some embodiments, the polymerase
is a mutant H784Q Taq polymerase. In some embodiment the mutant
H784Q Taq polymerase is reversibly inactivated via chemical,
aptamer or antibody modification.
[0052] In another aspect, the disclosure provides methods of
detecting variations in target DNA sequences that have been altered
with a gene editing enzyme, the methods 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.
[0053] In some embodiments, the target DNA sequence has been
treated with a CRISPR enzyme. In some embodiments, the target DNA
sequence has been treated with a Cas9 or Cpf1 enzyme. In some
embodiments, the cleaving enzyme is a hot start cleaving enzyme
which is thermostable and has reduced activity at lower
temperatures. In some embodiments, the cleaving enzyme is an RNase
H2 enzyme. In some embodiments, the cleaving enzyme is Pyrococcus
abyssi RNase H2 enzyme. In some embodiments, the cleaving enzyme is
a chemically modified hot start cleaving enzyme which is
thermostable and has reduced activity at lower temperatures. In
some embodiments, the hot start cleaving enzyme is a chemically
modified Pyrococcus abyssi RNase H2. In some embodiments, the
cleaving enzyme is a hot start cleaving enzyme that is reversibly
inactivated through interaction with an antibody at lower
temperatures.
[0054] In some embodiments, the cleavage domain comprises at least
one RNA base, and the cleaving enzyme cleaves between the position
complementary to the variation and the RNA base. In some
embodiments, the cleavage domain comprises at least one RNA base
located 3' of the position of variation, and comprises one DNA base
between the position of variation and the RNA base. In some
embodiments, there are no DNA bases between the position of
variation and the RNA base. In other embodiments, the RNA base is
located within the position of variation. In some embodiments, the
cleavage domain comprises 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. In some
embodiments, the one or more modified nucleosides are
2'-fluoronucleosides.
[0055] In some embodiments, the polymerase is a high-discrimination
polymerase. In some embodiments, the polymerase is a mutant H784Q
Taq polymerase. In some embodiments, the mutant H784Q Taq
polymerase is reversibly inactivated via chemical, aptamer, or
antibody modification. In some embodiments, 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.
[0056] In some embodiments, the methods of this aspect of the
disclosure further comprise (e) detection of an internal control
gene not targeted by the gene editing enzyme; and (f) normalization
of the results of steps (a)-(d) to the results of step (e). In some
embodiments, the internal control gene not targeted by the gene
editing enzyme is the RNase P gene. In some embodiments, the
reaction mixture further comprises a control oligonucleotide primer
specific for the internal control gene not targeted by the gene
editing enzyme, wherein the control oligonucleotide primer
comprises 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. In some embodiments,
the internal control gene not targeted by the gene editing enzyme
is detected using a three-oligonucleotide 5' nuclease assay.
[0057] In another aspect, the disclosure provides methods of target
enrichment comprising: (a) providing a reaction mixture comprising:
(i) a first oligonucleotide primer having a tail domain that is not
complementary to a target sequence, the tail domain comprising a
first universal primer sequence; 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 first
oligonucleotide primer wherein the blocking group prevents primer
extension and/or inhibits the first primer from serving as a
template for DNA synthesis; (ii) a sample nucleic acid that has
been treated with a gene editing enzyme, which may or may not have
the target sequence; (iii) a cleaving enzyme; and (iv) a
polymerase; (b) hybridizing the first primer to the target DNA
sequence to form a double-stranded substrate; (c) cleaving the
hybridized first primer, if the first primer is complementary to
the target, with the cleaving enzyme at a point within or adjacent
to the cleavage domain to remove the blocking group from the first
primer; and (d) extending the first primer with the polymerase.
[0058] In some embodiments, the target DNA sequence is a sample
that has been treated with a CRISPR enzyme. In some embodiments,
the target DNA sequence is a sample that has been treated with a
Cas9 or Cpf1 enzyme. In some embodiments, the methods further
comprise a second primer in reverse orientation to support priming
and extension of the first primer extension product. In some
embodiments, the second primer further comprises a tail domain
comprising a second universal primer sequence. In some embodiments,
steps (b)-(d) are performed 1-10 times. In some embodiments, the
methods further comprise removing unextended primers from the
reaction and hybridizing universal primers to the extension product
to form a second extension product. In some embodiments, the
universal primers further comprise tailed sequences for addition of
adapter sequences to the second extension product. In some
embodiments, sequencing is performed on the second extension
product to determine the sequence of the target.
[0059] In some embodiments, the cleaving enzyme is a hot start
cleaving enzyme which is thermostable and has reduced activity at
lower temperatures. In some embodiments, the cleaving enzyme is an
RNase H2 enzyme. In some embodiments, the cleaving enzyme is
Pyrococcus abyssi RNase H2 enzyme. In some embodiments, the
cleaving enzyme is a chemically modified hot start cleaving enzyme
which is thermostable and has reduced activity at lower
temperatures. In some embodiments, the hot start cleaving enzyme is
a chemically modified Pyrococcus abyssi RNase H2. In some
embodiments, the cleaving enzyme is a hot start cleaving enzyme
that is reversibly inactivated through interaction with an antibody
at lower temperatures which is thermostable and has reduced
activity at lower temperatures. In some embodiments, the cleavage
domain comprises at least one RNA base, and the cleaving enzyme
cleaves between the position complementary to the variation and the
RNA base.
[0060] In some embodiments, the cleavage domain comprises at least
one RNA base located 3' of the position of variation, and comprises
one DNA base between the position of variation and the RNA base. In
some embodiments, there are no DNA bases between the position of
variation and the RNA base. In other embodiments, the RNA base is
located within the position of variation. In some embodiments, the
cleavage domain comprises 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. In some
embodiments, the one or more modified nucleosides are
2'-fluoronucleosides. In some embodiments, the polymerase is a
high-discrimination polymerase. In some embodiments, the polymerase
is a mutant H784Q Taq polymerase. In some embodiments, the mutant
H784Q Taq polymerase is reversibly inactivated via chemical,
aptamer or antibody modification.
[0061] In another aspect, the disclosure provides blocked-cleavable
primers for rhPCR, comprising: 5'-A-B-C-D-E-3', wherein A is
optional and is a tail extension that is not complementary to a
target; B is a sequence domain that is complementary to a target; C
is a discrimination domain; D is a cleavage domain that, when
hybridized to the target, is cleavable by RNase H2, and which
comprises an RNA base; and E is a blocking domain that prevents
extension of the primer.
[0062] In some embodiments, D is a cleavage domain that, when
hybridized to the target, is cleavable by RNase H2, and which
comprises an RNA base that is: separated from the discrimination
domain by one base position, within the discrimination domain, or
adjacent to the discrimination domain. In some embodiments, the RNA
base is separated from the discrimination domain by one base
position. In some embodiments, the RNA base is within the
discrimination domain. In some embodiments, the RNA base is
adjacent to the discrimination domain. For example, when the RNA
base is adjacent to the discrimination domain, no intervening DNA
residue is present between the RNA base and the discrimination
domain.
[0063] In some embodiments, D comprises 1-3 RNA bases. In some
embodiments, the cleavage domain comprises one or more of the
following moieties: a DNA residue, an abasic residue, a modified
nucleoside, or a modified phosphate internucleotide linkage. In
some embodiments, a sequence flanking the cleavage site contains
one or more internucleoside linkages resistant to nuclease
cleavage. In some embodiments, the nuclease resistant linkage is a
phosphorothioate. In some embodiments, the 3' oxygen atom of at
least one of the RNA residues is substituted with an amino group,
thiol group, or a methylene group. In some embodiments, the
blocking group is attached to the 3'-terminal nucleotide of the
primer. In some embodiments, A is comprised of a region that is
identical to a universal forward primer and optionally a probe
binding domain.
[0064] In some embodiments, the discrimination domain C does not
comprise or overlap with the cleavage domain D. In some other
embodiments, the discrimination domain C comprises the cleavage
domain D. In some other embodiments, the discrimination domain C
overlaps with the cleavage domain D.
[0065] In another aspect, the disclosure provides blocked-cleavable
primers for rhPCR, the primers comprising:
5'-A-B-Z-E-3'
wherein A is optional and is a tail extension that is not
complementary to a target; B is a sequence domain that is
complementary to a target; Z comprises: C, a discrimination domain,
and D, a cleavage domain that, when hybridized to the target, is
cleavable by RNase H2; and E is a blocking domain that prevents
extension of the primer.
[0066] In some embodiments, the discrimination domain C is located
5' of the cleavage domain D. In some embodiments, the
discrimination domain C is located 3' of the cleavage domain D. In
some embodiments, the discrimination domain C overlaps with the
cleavage domain D. In some embodiments, C comprises 1-3 RNA bases.
In some embodiments, C comprises 1 RNA base. In some embodiments,
the discrimination domain C consists of the 1 RNA base. In some
embodiments, the cleavage domain comprises one or more of the
following moieties: a DNA residue, an abasic residue, a modified
nucleoside, or a modified phosphate internucleotide linkage. In
some embodiments, a sequence flanking the cleavage site contains
one or more internucleoside linkages resistant to nuclease
cleavage. In some embodiments, the nuclease resistant linkage is a
phosphorothioate. In some embodiments, the 3' oxygen atom of at
least one of the RNA residues is substituted with an amino group,
thiol group, or a methylene group. In some embodiments, the
blocking group is attached to the 3'-terminal nucleotide of the
primer. In some embodiments, A comprises a region that is identical
to a universal forward primer and optionally a probe binding
domain.
[0067] In another aspect, the disclosure provides methods of
detecting a variation in a target DNA sequence, the methods
comprising: (a) providing a reaction mixture comprising (i) an
oligonucleotide primer having a cleavage domain positioned 5' of a
blocking group, 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.
[0068] In some embodiments, the cleaving enzyme is a hot start
cleaving enzyme comprising at least one amino acid substitution as
compared to its wild type, which is thermostable and has reduced
activity at lower temperatures, and which exhibits enhanced
mismatch discrimination during rhPCR as compared to its wild type.
In some embodiments, the hot start cleaving enzyme is Pyrococcus
abyssi RNase H2 comprising (a) a G12A amino acid substitution; (b)
a P13T amino acid substitution; (c) a G169A amino acid
substitution; or (d) a combination thereof. In some embodiments,
the cleaving enzyme is chemically modified. In some embodiments,
the cleaving enzyme is reversibly inactivated through interaction
with an antibody at lower temperatures. In some embodiments, the
cleavage domain comprises at least one RNA base. In some
embodiments, the cleavage domain is located 3' of the position of
variation. In some embodiments, the cleavage domain is located 5'
of the position of variation. In some embodiments, the cleavage
domain overlaps with the position of variation. In some
embodiments, the cleavage domain comprises 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. In some embodiments, the one or more modified
nucleosides are 2'-fluoronucleosides. In some embodiments, the
polymerase is a high-discrimination polymerase. In some
embodiments, the polymerase is a mutant H784Q Taq polymerase. In
some embodiments, the mutant H784Q Taq polymerase is reversibly
inactivated via chemical, aptamer or antibody modification. In some
embodiments, 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. In some embodiments, the target DNA sequence is a sample
that has been treated with a gene editing enzyme. In some
embodiments, the target DNA sequence is a sample that has been
treated with a CRISPR enzyme. In some embodiments, the target DNA
sequence is a sample that has been treated with a Cas9 or Cpf1
enzyme.
[0069] In another aspect, the disclosure provides methods of target
enrichment comprising: (a) providing a reaction mixture comprising
(i) a first oligonucleotide primer having a tail domain that is not
complementary to a target sequence, the tail domain comprising a
first universal primer sequence; a cleavage domain positioned 5' of
a blocking group, the blocking group linked at or near the end of
the 3'-end of the first oligonucleotide primer wherein the blocking
group prevents primer extension and/or inhibits the first primer
from serving as a template for DNA synthesis, (ii) a sample nucleic
acid that may or may not have the target sequence, (iii) a cleaving
enzyme, and (iv) a polymerase; (b) hybridizing the first primer to
the target DNA sequence to form a double-stranded substrate; (c)
cleaving the hybridized first primer, if the first primer is
complementary to the target, with the cleaving enzyme at a point
within or adjacent to the cleavage domain to remove the blocking
group from the first primer; and (d)extending the first primer with
the polymerase.
[0070] In some embodiments, the methods further comprise a second
primer in reverse orientation to support priming and extension of
the first primer extension product. In some embodiments, the second
primer further comprises a tail domain comprising a second
universal primer sequence. In some embodiments, steps (b)-(d) are
performed 1-10 times. In some embodiments, the methods further
comprise removing unextended primers from the reaction and
hybridizing universal primers to the extension product to form a
second extension product. In some embodiments, the universal
primers further comprise tailed sequences for addition of adapter
sequences to the second extension product. In some embodiments,
sequencing is performed on the second extension product to
determine the sequence of the target. In some embodiments, the
target DNA sequence is a sample that has been treated with a gene
editing enzyme. In some embodiments, the target DNA sequence is a
sample that has been treated with a CRISPR enzyme. In some
embodiments, the target DNA sequence is a sample that has been
treated with a Cas9 or Cpf1 enzyme. In some embodiments, the
cleaving enzyme is a hot start cleaving enzyme comprising at least
one amino acid substitution as compared to its wild type, which is
thermostable and has reduced activity at lower temperatures, and
which exhibits enhanced mismatch discrimination during rhPCR as
compared to its wild type. In some embodiments, the hot start
cleaving enzyme is Pyrococcus abyssi RNase H2 comprising (a) a G12A
amino acid substitution; (b) a P13T amino acid substitution; or (c)
a G169A amino acid substitution; or (d) a combination thereof. In
some embodiments, the cleaving enzyme is chemically modified. In
some embodiments, the cleaving enzyme is a hot start cleaving
enzyme that is reversibly inactivated through interaction with an
antibody at lower temperatures. In some embodiments, the cleaving
domain comprises at least one RNA base. In some embodiments, the
cleavage domain is located 3' of the position of variation. In some
embodiments, the cleavage domain is located 5' of the position of
variation. In some embodiments, the cleavage domain overlaps with
the position of variation. In some embodiments, the cleaving domain
comprises 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. In some embodiments, the
one or more modified nucleosides are 2'-fluoronucleosides. In some
embodiments, the polymerase is a high-discrimination polymerase. In
some embodiments, the polymerase is a mutant H784Q Taq polymerase.
In some embodiments, the mutant H784Q Taq polymerase is reversibly
inactivated via chemical, aptamer or antibody modification.
[0071] 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.
[0072] "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.
[0073] "Fluorophore" or "fluorescent label" refers to compounds
with a fluorescent emission maximum between about 350 and 900
nm.
[0074] "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.
[0075] 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.
[0076] 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.
[0077] "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.
[0078] "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.
[0079] "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.
[0080] "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.
[0081] 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.
[0082] The term "discrimination domain" can be the same or
different as the cleavage domain. The discrimination domain is the
position of the potential mutation site, and the enzyme will only
cleave at the cleavage site if the criteria at the discrimination
domain are met. For example, in one embodiment RNase H2 will cleave
or not cleave a double-stranded target at the RNA residue (cleavage
domain), depending on whether a mutation exists at the
discrimination domain.
[0083] 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.
[0084] 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.
[0085] The following examples further illustrate the invention but
should not be construed as in any way limiting its scope.
Example 1
[0086] This example demonstrates an enhanced rhPCR assay that
utilizes a highly discriminatory DNA polymerase and RNase H2 for
discrimination
[0087] 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. SEQ ID Name Sequence
NO. Forward non- GCTGTGATTTTGGTCTAGCTACAG SEQ ID discriminating NO.
1 primer Forward Allele GCTGTGATTTTGGTCTAGCTACAGT SEQ ID 1 ASP1
ASPCR NO. 2 primer Forward Allele GCTGTGATTTTGGTCTAGCTACAGA SEQ ID
2 ASP2 ASPCR NO. 3 primer Probe FAM-TCCCATCAG-ZEN- SEQ ID
TTTGAACAGTTGTCTGGA-IBFQ NO. 4 rs113488022
GCTGTGATTTTGGTCTAGCTACAGTgAA SEQ ID Allele 1 ATG-x NO. 5 Forward
ASP1 rhPrimer rs113488022 GCTGTGATTTTGGTCTAGCTACAGAgAA SEQ ID
Allele 2 ATG-x NO. 6 Forward ASP2 rhPrimer Reverse
GCCCTCAATTCTTACCATCCACAAAaTG SEQ ID rhPrimer GAA-x NO. 7 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
[0088] 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 SEQ ID Name Sequence NO. rs113488022
AAAAAATAAGAACACTGATTTTTGTGAAT SEQ ID gBlock
ACTGGGAACTATGAAAATACTATAGTTGA NO. 8 Template 1
GACCTTCAATGACTTTCTAGTAACTCAGCA GCATCTCAGGGCCAAAAATTTAATCAGTG
GAAAAATAGCCTCAATTCTTACCATCCAC AAAATGGATCCAGACAACTGTTCAAACTG
ATGGGACCCACTCCATCGAGATTTCACTGT AGCTAGACCAAAATCACCTATTTTTACTGT
GAGGTCTTCATGAAGAAATATATCTGAGG TGTAGTAAGTAAAGGAAAACAGTAGATCT
CATTTTCCTATCAGAGCAAGCATTATGAAG AGTTTAGGTAAGAGATCTAATTTCTATAAT
TCTGTAATATAATATTCTTTAAAACATAGT ACTTCATCTTTCCTCTTA rs113488022
AAAAAATAAGAACACTGATTTTTGTGAAT SEQ ID gBlock
ACTGGGAACTATGAAAATACTATAGTTGA NO. 9 Template 2
GACCTTCAATGACTTTCTAGTAACTCAGCA GCATCTCAGGGCCAAAAATTTAATCAGTG
GAAAAATAGCCTCAATTCTTACCATCCAC AAAATGGATCCAGACAACTGTTCAAACTG
ATGGGACCCACTCCATCGAGATTTCTCTGT AGCTAGACCAAAATCACCTATTTTTACTGT
GAGGTCTTCATGAAGAAATATATCTGAGG TGTAGTAAGTAAAGGAAAACAGTAGATCT
CATTTTCCTATCAGAGCAAGCATTATGAAG AGTTTAGGTAAGAGATCTAATTTCTATAAT
TCTGTAATATAATATTCTTTAAAACATAGT ACTTCATCTTTCCTCTTA Nucleic acid
sequences are shown 5'-3'. Location of SNPs are shown bold and
underlined.
[0089] 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
[0090] All numbers in this table represent Cq values obtained from
the CFX384.TM. instrument (Bio-Rad.TM., Hercules, Calif.).
Example 2
[0091] The following example demonstrates an enhanced rhPCR assay
that utilizes a highly discriminatory DNA polymerase and RNase H2
for discrimination.
[0092] 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 SEQ ID Name Sequence
NO. Forward non- GCTGTGATTTTGGTCTAGCTACAG SEQ ID discrimin NO. 1
primer Probe FAM-TCCCATCAG-ZEN- SEQ ID TTTGAACAGTTGTCTGGA-IBFQ NO.
4 rs113488022 GCTGTGATTTTGGTCTAGCTACAGTg SEQ ID Allele 1 AxxTG NO.
10 Forward dxxd rhPrimer rs113488022 GCTGTGATTTTGGTCTAGCTACAGAg SEQ
ID Allele 2 AxxTG NO. 11 Forward dxxd rhPrimer Reverse
GCCCTCAATTCTTACCATCCACAAAa SEQ ID rhPrimer TGGAA-x NO. 12 Nucleic
acid sequences are shown 5'-3'. DNA is uppercase, RNA is lowercase.
Location of potential mismatch is underlined. Z EN = internal Zen
.TM. fluorescent quencher (IDT, Coralville, IA). FAM =
6-carboxyfluorescein, IBFQ = Iowa Black FQ (fluorescence quencher),
and x = C3 propanediol spacer.
[0093] 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.
[0094] 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).
[0095] 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
[0096] The following example illustrates the heightened reliability
of universal assays using a DNA polymerase with a high mismatch
discrimination.
[0097] 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 SEQ ID Name Sequence NO.
Universal CGCCGCGTATAGTCCCGCGTAAA SEQ ID Forward NO. 13 primer
Probe 1 FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ ID (FAM) NO. 14 Probe 2
HEX-CAATC+C+C+CGAG+CT-IBFQ SEQ ID (HEX) NO. 15 rs351855
GCCCATGTCCCAGCGAACCATCACCGT SEQ ID Allele 1
GCTAGCCCTCGATACAGCCCgGCCAC- NO. 16 Forward x primer rs351855
GCCCATGTCCCAGCGAACAATCCCCGA SEQ ID Allele 2
GCTGCCCTCGATACAGCCTgGCCAC-x NO. 17 Forward primer Reverse
GCGGCCAGGTATACGGACATcATCC SEQ ID primer A-x NO. 18 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 +. FAM = 6-carboxyfluorescein, HEX =
6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein, IBFQ = Iowa Black
FQ (fluorescence quencher), and x = C3 propanediol spacer
block.
[0098] 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 SEQ
ID Name Sequence NO. rs351855 GTTGGGAGCTGGGAGGGACTGAGTTAGGG SEQ ID
gBlock TGCACGGGGCGGCCAGTCTCACCACTGAC NO. 19 Template 1
CAGTTTGTCTGTCTGTGTGTGTCCATGTGC GAGGGCAGAGGAGGACCCCACATGGACC
GCAGCAGCGCCCGAGGCCAGGTATACGGA CATCATCCTGTACGCGTCGGGCTCCCTGGC
CTTGGCTGTGCTCCTGCTGCTGGCCGGGCT GTATCGAGGGCAGGCGCTCCACGGCCGGC
ACCCCCGCCCGCCCGCCACTGTGCAGAAG CTCTCCCGCTTCCCTCTGGCCCGACAGGTA
CTGGGCGCATCCCCCACCTCACATGTGAC AGCCTGACTCCAGCAGGCAGAACCAAGTC
TCCCACTTTGCAGTTCTCCCTGGAGTCAGG CTCTTCCGGCAAGTCAAGCT rs351855
GTTGGGAGCTGGGAGGGACTGAGTTAGGG SEQ ID gBlock
TGCACGGGGCGGCCAGTCTCACCACTGAC NO. 20 Template 2
CAGTTTGTCTGTCTGTGTGTGTCCATGTGC GAGGGCAGAGGAGGACCCCACATGGACC
GCAGCAGCGCCCGAGGCCAGGTATACGGA CATCATCCTGTACGCGTCGGGCTCCCTGGC
CTTGGCTGTGCTCCTGCTGCTGGCCAGGCT GTATCGAGGGCAGGCGCTCCACGGCCGGC
ACCCCCGCCCGCCCGCCACTGTGCAGAAG CTCTCCCGCTTCCCTCTGGCCCGACAGGTA
CTGGGCGCATCCCCCACCTCACATGTGAC AGCCTGACTCCAGCAGGCAGAACCAAGTC
TCCCACTTTGCAGTTCTCCCTGGAGTCAGG CTCTTCCGGCAAGTCAAGCT Nucleic acid
sequences are shown 5'-3'. Location of SNPs are shown bold and
underlined.
[0099] 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).
[0100] 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
[0101] 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).
[0102] 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.
[0103] 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 ID Non- 22.9 23.1 22.8 30.4 34.2 >65 No. 1 discrimin SEQ ID
...TgAxxTG 31.6 34.5 36.1 31.6 36.0 >65 NO. 10 SEQ ID ...AgAxxTG
29.1 29.1 29.9 30.8 34.4 >65 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 ID Non- 22.7 23.2 23.2 31.5 34.2 >65 No. 1
discrimin SEQ ID ...TgAxxTG 33.8 36.6 47.3 33.1 36.4 >65 NO. 10
SEQ ID ...AgAxxTG 32.1 35.2 38.8 32.0 35.5 >65 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.
[0104] 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
[0105] 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).
[0106] 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).
[0107] 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 SEQ ID Name Sequence NO. Universal CGGCCCATGTCCCAGCGAA
SEQ ID Forward NO. 21 primer Probe 1 FAM-C+CATC+A+C+CGTG+CT-IBFQ
SEQ ID (FAM) NO. 14 Probe 2 Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ ID
(Yakima NO. 22 Yellow) rs4657751 GCCCATGTCCCAGCGAACCATCACCGTGC SEQ
ID Allele 1 TACTTCCCACACCCTCATATCuTGTTA-x NO. 23 Forward primer
rs4657751 GCCCATGTCCCAGCGAACAATCCCCGAGC SEQ ID Allele 2
TCTTACTTCCCACACCCTCATATAuTGTTA-x NO. 24 Forward primer rs4657751
GCGCTAAGTAAACATTCCTGATTGCAaCTT SEQ ID Reverse AT-x NO. 25 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 +. 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.
TABLE-US-00012 TABLE 12 Synthetic gBlock .RTM. templates used in
Example 5. SEQ ID Name Sequence NO. rs4657751
GATTTTTTTTTTTTGGCATTTCTTCTTAGAT SEQ ID Allele 1
TTCTATCTCCTAACATAGGATCACTTATTT NO. 26 gBlock
GTGAAATTATTTGTATACCTTTTTTATGGA template
GTGATGATGTGATACAAATTCTATCCTTAA GGATATAAGAACATCTTTTCTTTATATTAG
GATTTTTCTGGACCCATGAGTTACATGCTT ACTTCCCACACCCTCATATCTTGTTTAAAT
TTGTAGAATTAAATTCATAGGTAATTATTT CTGAAACTTCTTCCCTGTGTGAGCAATCTA
AATAATTATTACAATGCCTTAAGTTGCAAT CAGGAATGTTTACTTAGCACAGACTTTTTT
CCCCACTACTGCACTCAAAGGATAACAGA TATATGGCAAATCTAACCATATTCTTTGTC
CTTTGTCCATGTTGCGGAGGGAAGCTCATC AGTGGGGCCACGAGCTGAGTGCGTCCTGT
CACTCCACTCCCATGTCCCTTGGGAAGGTC TGAGACTAGGG rs4657751
GATTTTTTTTTTTTGGCATTTCTTCTTAGAT SEQ ID Allele 2
TTCTATCTCCTAACATAGGATCACTTATTT NO. 27 gBlock
GTGAAATTATTTGTATACCTTTTTTATGGA template
GTGATGATGTGATACAAATTCTATCCTTAA GGATATAAGAACATCTTTTCTTTATATTAG
GATTTTTCTGGACCCATGAGTTACATGCTT ACTTCCCACACCCTCATATATTGTTTAAAT
TTGTAGAATTAAATTCATAGGTAATTATTT CTGAAACTTCTTCCCTGTGTGAGCAATCTA
AATAATTATTACAATGCCTTAAGTTGCAAT CAGGAATGTTTACTTAGCACAGACTTTTTT
CCCCACTACTGCACTCAAAGGATAACAGA TATATGGCAAATCTAACCATATTCTTTGTC
CTTTGTCCATGTTGCGGAGGGAAGCTCATC AGTGGGGCCACGAGCTGAGTGCGTCCTGT
CACTCCACTCCCATGTCCCTTGGGAAGGTC TGAGACTAGGG Nucleic acid sequences
are shown 5'-3'. DNA is uppercase. The location of the SNP is
underlined.
[0108] 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
[0109] The following example compares the performance of the
genotyping methods of the present invention versus traditional 5'
nuclease genotyping assay methods (Taqman.TM.).
[0110] 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.
[0111] 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).
[0112] 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
GCCCATGTCCCAGCGAACCATCACCG SEQ ID NO. 28 Allele 1
TGCTTTCTCTTCTGGACTCCCTATAA Forward TaTTGTG-x primer rs1799865
GCCCATGTCCCAGCGAACAATCCCCG SEQ ID NO. 29 Allele 2
AGCTTTCTCTTCTGGACTCCCTATAA Forward CaTTGTG-x primer rs1799865
GCGGATTGATGCAGCAGTGAgTCA SEQ ID NO. 30 Reverse TG-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.
[0113] 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
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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. SEQ ID Name Sequence NO.
Universal CGGCCCATGTCCCAGCGAA SEQ ID Forward primer NO. 21 Probe 1
FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ ID (FAM) NO. 14 Probe 2
Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ ID (Yakima Yellow) NO. 22
rs72558195: GCCCATGTCCCAGCGAACCATCACCGTGCTC SEQ ID G:A Allele 1
TCCGTTGTTTTCCAGAAACgATTTC-x NO. 31 Forward primer rs72558195:
GCCCATGTCCCAGCGAACAATCCCCGAGCTC SEQ ID G:A Allele 2
TCCGTTGTTTTCCAGAAATgATTTC-x NO. 32 Forward primer rs72558195:
GCCCATGTCCCAGCGAACAATCCCCGAGCTC SEQ ID G:C Allele 3
TCCGTTGTTTTCCAGAAAGgATTTC-x NO. 33 Forward primer rs1135840
GCAACCAAGTCTTCCCTACAACcTTGAT-x SEQ ID Reverse primer NO. 34 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.
[0118] 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
SEQ Name Sequence ID NO. rs72558195 ACATCATTTTTATTGTATAAAAGCATTTTA
SEQ ID Allele 1 GTATCAATTTTCTCATTTTTAAACCAAGTC NO. 35 gBlock
TTCCCTACAACCTTGAATAAATGGTTTCCA template
AGGAAAATAAAATCTTGGCCTTACCTGGA TCCATGGGGAGTTCAGAATCCTGAAGTTT
TCATTGAATCTTTTCATCAGGGTGAGAAA ATTCTGATCTTTATAATCAAATCGTTTCTG
GAAAACAACGGAGCAGATCACATTGCAG GGAGCACAGCCCAGGATGAAAGTGGGAT
CACAGGGTGAAGCTAAAGATTTAAAAATT TTTAAAAAAATTATTAAAAAATAAATATT
TAAAAGATTTGCATTTGTTAAGACATAAA GGAAATTTAGAAATTTTAAACAATATCTT
ACAAATTCCCCATGTGTCCAAA rs72558195 ACATCATTTTTATTGTATAAAAGCATTTTA
SEQ ID Allele 2 GTATCAATTTTCTCATTTTTAAACCAAGTC NO. 36 gBlock
TTCCCTACAACCTTGAATAAATGGTTTCCA template
AGGAAAATAAAATCTTGGCCTTACCTGGA TCCATGGGGAGTTCAGAATCCTGAAGTTT
TCATTGAATCTTTTCATCAGGGTGAGAAA ATTCTGATCTTTATAATCAAATCATTTCTG
GAAAACAACGGAGCAGATCACATTGCAG GGAGCACAGCCCAGGATGAAAGTGGGAT
CACAGGGTGAAGCTAAAGATTTAAAAATT TTTAAAAAAATTATTAAAAAATAAATATT
TAAAAGATTTGCATTTGTTAAGACATAAA GGAAATTTAGAAATTTTAAACAATATCTT
ACAAATTCCCCATGTGTCCAAA rs72558195 ACATCATTTTTATTGTATAAAAGCATTTTA
SEQ ID Allele 3 GTATCAATTTTCTCATTTTTAAACCAAGTC NO. 37 gBlock
TTCCCTACAACCTTGAATAAATGGTTTCCA template
AGGAAAATAAAATCTTGGCCTTACCTGGA TCCATGGGGAGTTCAGAATCCTGAAGTTT
TCATTGAATCTTTTCATCAGGGTGAGAAA ATTCTGATCTTTATAATCAAATCCTTTCTG
GAAAACAACGGAGCAGATCACATTGCAG GGAGCACAGCCCAGGATGAAAGTGGGAT
CACAGGGTGAAGCTAAAGATTTAAAAATT TTTAAAAAAATTATTAAAAAATAAATATT
TAAAAGATTTGCATTTGTTAAGACATAAA GGAAATTTAGAAATTTTAAACAATATCTT
ACAAATTCCCCATGTGTCCAAA Nucleic acid sequences are shown 5'-3'. DNA
is uppercase. The location of the SNP is underlined.
[0119] 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.
[0120] 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 - 1 ( .DELTA. R n Dye 1 / .DELTA. R n Dye 2 ) .times.
120 90 ##EQU00002## Distance from origin = ( .DELTA. R n Dye 1 ) 2
+ ( .DELTA. R n Dye 2 ) 2 ##EQU00002.2##
[0121] 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.
[0122] 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 - 1 ( .DELTA. R n Dye 1
/ .DELTA. R n Dye 2 ) ##EQU00003## penta - allelic ( 5 alleles ) :
Angle = tan - 1 ( .DELTA. R nDye 1 / .DELTA. R nDye 2 ) .times. 72
90 ##EQU00003.2## hex a - allelic ( 6 alleles ) : Angle = tan - 1 (
.DELTA. R nDye 1 / .DELTA. R nDye 2 ) .times. 60 90
##EQU00003.3##
Example 8
[0123] 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).
[0124] 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 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).
[0125] 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. SEQ Name Sequence ID NO. Universal CGGCCCATGTCCCAGCGAA SEQ ID
Forward NO. 21 primer Probe 1 FAM-C+CATC+A+C+CGTG+CT-IBFQ SEQ ID
(FAM) NO. 14 Probe 2 Yak-CAATC+C+C+CGAG+CT-IBFQ SEQ ID (Yakima NO.
22 Yellow) rs1135840 GCCCATGTCCCAGCGAACCATCACCGTGC SEQ ID Allele 1
TGTCTTTGCTTTCCTGGTGAGcCCATG-x NO. 38 Forward primer rs1135840
GCCCATGTCCCAGCGAACAATCCCCGAGC SEQ ID Allele 2
TGTCTTTGCTTTCCTGGTGAcCCATG-x NO. 39 Forward primer rs1135840
GCGTTGGAACTACCACATTGCTTTATuGTA SEQ ID Reverse CT-x NO. 40 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.
[0126] 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.
[0127] 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.
[0128] 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-CCACGAGCTGAGTGCGTCCTGTCA- SEQ ID NO. 43 (Yakima
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).
[0129] 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.
[0130] 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{circumflex over (
)}(.DELTA..DELTA.Cq))
[0131] 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
[0132] The following example demonstrates that a variation of an
rhPCR probe can be used for multiplexed rhPCR.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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 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 of TE at pH
8.0, and 20 .mu.L was transferred to a new tube.
[0137] 2 .mu.L of the samples were analyzed using the Agilent 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.)
MiSeg.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 AATGATACGGCGACCACCGAGATCTACAC
SEQ ID NO. PCR-50F TCTTTCCCTACACGACGCTCT 44 Universal
CAAGCAGAAGACGGCATACGAGATGGACC SEQ ID NO. PCR-47R
TATGTGACTGGAGTTCAGACGTGTGC 45 Nucleic acid sequences are shown
5'-3'. DNA is uppercase.
[0138] FIG. 10 shows the results from the Agilent 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.
[0139] 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.
Example 10
[0140] This example demonstrates enhanced sensitivity and accuracy
of assay systems of the disclosure as compared to standard T7
endonuclease cleavage assays.
[0141] A total of 36 sites modified with the CRISPR/Cas9 protein
system were chosen to be comparatively analyzed by T7 endonuclease,
next-generation sequencing (NGS), or a qPCR "Genie" assay system of
the disclosure.
[0142] To mutate the chosen genomic sites, a HEK293 cell line was
generated with stable expression of S pyogenes Cas9. AltR.TM. guide
RNAs were reverse transfected into the cells in 96-well plates
(40,000 cells/well) using 0.75 .mu.L RNAiMAX (Thermo) per well.
Briefly, AltR.TM. crRNAs were designed from the IDT CRISPR2.0
design engine to target exon 1 or 2 of selected human genes. Prior
to transfection, guide RNAs (crRNA/tracrRNA with AltR.TM.
chemistry) were duplexed in an equimolar ratio at 3 .mu.M final
concentration of the complex in IDT duplex buffer (Integrated DNA
Technologies, Coralville, Iowa). Complexes were heated to
95.degree. C. for 5 min and cooled to room temperature. Genomic DNA
was isolated after 48 hrs with 50 .mu.L Quick Extract buffer
(Epicentre), using standard techniques described by the
manufacturer. DNA solutions were further diluted with 100 .mu.L
water before further analysis was performed.
[0143] Analysis by T7 endonuclease digestion was done as follows.
CRISPR-Cas9-treated cells were washed with 100 .mu.L of PBS. Cells
were lysed by adding 50 .mu.L of QuickExtract.TM. DNA Extraction
Solution (Integrated DNA Technologies). Cell lysates were then
transferred to appropriate PCR tubes or plate, then vortexed and
heated in a thermal cycler at 65.degree. C. for 10 min, followed by
98.degree. C. for 5 min, after which 100 .mu.L of Nuclease-Free
Water was added to dilute the genomic DNA. The samples were then
vortexed and spun down. PCR was set up using template, primers, and
components of the Alt-R Genome Editing Detection Kit and KAPA HiFi
HotStart PCR Kit as follows. Sample: 4 (.about.40 ng) genomic DNA,
300 nM forward primer, 300 nM reverse primer, 5 (1.times.) of KAPA
HiFi Fidelity Buffer (5.times.), 1.2 mM (0.3 mM each) dNTPs, 0.5 U
KAPA HiFi Hotstart DNA Polymerase (1 U/.mu.L), for a total volume
of 25 .mu.L. Alt-R.TM. Control A: 1 .mu.L Alt-R.TM. Control A
template/primer mix, 5 (1.times.) of KAPA HiFi Fidelity Buffer
(5.times.), 1.2 mM (0.3 mM each) dNTPs, 0.5 U KAPA HiFi Hotstart
DNA Polymerase (1 U/.mu.t), for a total volume of 25 .mu.L.
Alt-R.TM. Control B: 1 .mu.L Alt-R.TM. Control B template/primer
mix, 5 .mu.L (1.times.) of KAPA HiFi Fidelity Buffer (5.times.),
1.2 mM (0.3 mM each) dNTPs, 0.5 U KAPA HiFi Hotstart DNA Polymerase
(1 U/.mu.t), for a total volume of 25 .mu.L. PCR was run using the
following conditions: denature at 95.degree. C. for 5 min; 30
cycles of: denature at 98.degree. C. for 20 sec, anneal between
64-67.degree. C. (depending on polymerase) for 15 sec, extend at
72.degree. C. for 30 sec; then extend at 72.degree. C. for 2
minutes. Heteroduplexes for T7EI digestion were formed as follows.
2 .mu.L T7EI Reaction Buffer (10.times.) and 6 .mu.L Nuclease-Free
Water was combined with 10 .mu.L experimental target or Alt-R.TM.
HPRT control from the PCR, 10 .mu.L Control A PCR component
(homoduplex control), or 5 .mu.L Control A and 5 .mu.L Control B
(heteroduplex control). The PCR products were then placed in a
thermal cycler with 95.degree. C. denaturation for 10 min, ramp
from 95-85.degree. C. at a ramp rate of -2.degree. C./sec, then
ramp from 85-25.degree. C. at a ramp rate of -0.3.degree. C./sec.
18 .mu.L of PCR heteroduplexes from the previous step were combined
with 2 .mu.L T7 endonuclease I (1 U/.mu.L), then the T7EI reaction
was incubated at 37.degree. C. for 60 min. T7EI mismatch detection
result were visualized on a Fragment Analyzer.TM. system with
Mutation Discovery Kit according to the manufacturer's instructions
(Integrated DNA Technologies). After amplification and cleavage,
amplicons were sized on the Fragment Analyzer.TM. (Advanced
Analytical, Inc, Ames, Iowa) capillary electrophoresis system.
[0144] NGS sequencing analysis of the mutated samples employed
locus-specific primers positioned approximately 75-bp flanking the
Cas9 cleavage site. Primers contained universal 5'-tails that
allowed for secondary amplification that added Illumina.TM.
TruSeq.TM. i5 and i7 adapters with sample-specific barcodes to the
amplicons. The locus-specific primers were designed as RNase
H2-cleavable primers with the 4DMX blocking modification at the
3'-end (where the 4DMX nomenclature indicates 4 DNA bases, a
mismatched DNA base and a propanediol C3-spacer 3' of the RNA
base). Using a master-mix containing a hot-start Taq polymerase and
hot-start RNaseH2, the genomic DNAs were amplified using the
following cycling conditions: 95.degree. C..sup.5:00+(95.degree.
C..sup.0:15+60.degree. C..sup.1:00).times.8 cycles+99.degree.
C..sup.15:00. Samples were purified using SPRI beads (1.5.times.
Agencourt.TM. Ampure.RTM. XP beads, Beckman Coulter) per the
manufacturer's protocol. The second PCR incorporated the Illumina
adapters and was run under the following conditions: 95.degree.
C..sup.5:00+(95.degree. C..sup.0:15+60.degree.
C..sup.0:30+72.degree. C..sup.0:30).times.18 cycles+99.degree.
C..sup.15:00.
[0145] The resultant amplicons underwent 1.times.SPRI.TM. clean-up
and were quantified via the KAPA.TM. library qPCR quantitation
(KAPA Biosystems, Wilmington, Mass.) kit per the manufacturer's
recommended protocol. In addition, amplicons were sized on the
Fragment Analyzer.TM. (Advanced Analytical, Inc., Ames, Iowa)
capillary electrophoresis system.
[0146] DNA sequencing was carried out on an Illumina.TM. MiSeq.RTM.
using a MiSeq.RTM. Nano cartridge (v2, 300 cycles). Data was
de-multiplexed via an in-house bioinformatics processing pipeline.
Analysis for specific editing events relative to a reference
amplicon was performed with CRISPResso.TM. using methods
described.
[0147] Quantitative PCR assay primers for analysis according to
methods of the disclosure were designed so that the RNA nucleotide
was located two bases after the primary Cas9 cleavage site,
allowing for maximal discrimination from both the RNase H2 enzyme
and the DNA polymerase (FIG. 12). Primers were designed to include
a proprietary universal 5' domain (UniFor-UniPro-), which has
sequence identity with both a universal forward primer, and a
universal 5' nuclease degradable probe (Table 19, Seq ID No. 1-72
and Table 20, Seq ID No. 76-147). A Taqman-based RNase P assay (Seq
ID No. 73-75) was utilized as a universal control for template
concentration normalization in all cases. All primers were
synthesized at Integrated DNA Technologies (IDT, Coralville, Iowa).
Amplification was performed with 2.5 .mu.L of the same
QuickExtract.TM. genomic DNA utilized in T7 and NGS analyses. A
wild-type (WT) control that was grown and extracted by the same
method was also analyzed for normalization purposes. Reaction
volumes were 10 .mu.L in all cases, and included the universal
forward primer, the universal probe, the interrogating primer, and
the non-interrogating reverse primer. 1.times. of a
rhPCR-genotyping master mix, containing hot-start RNase H2, a
thermophilic DNA polymerase, buffer, and dNTPs was also included.
The universal forward primer was present in all reactions at 1000
nM (10 pmol), and the universal assay probe was present at 300 nM
(3 pmol). The assay specific primers were present at 200 nM (2
pmol) for the forward (mutation interrogating) primer, while the
non-interrogating locus-specific reverse primer was present at 500
nM (5 pmol). RNase P control reactions were run with 500 nM of
forward and reverse RNase P primers, and 250 nM probe (Seq ID No.
73-75). Reactions were run on a CFX384.TM. Real-Time qPCR machine
(Bio-Rad.RTM., Hercules, Calif.). The following cycling conditions
were utilized: 95.degree. C..sup.10:00+(95.degree.
C..sup.0:15+59.degree. C..sup.0:20+72.degree. C..sup.0:30).times.55
cycles.
TABLE-US-00019 TABLE 19 Discriminatory forward primers utilized in
Example 10. Seq ID Name Sequence No. GCK-356-1
UniFor-UniPro-CCCTGGGTCCCTGGGaGAATC-x 46 GCK-356-2
UniFor-UniPro-CGAGGAGAACCACATTCTCCcAGGGT-x 47 ERBB3-33-1
UniFor-UniPro-GGGCGGCCGTGACuCACC-x 48 ERBB3-33-2
UniFor-UniPro-GAGGGAAGGGGGTGAGTcACGGCG-x 49 TTR-1257-1
UniFor-UniPro-CCTGGGAGCCATTTGCCTCuGGGTT-x 50 TTR-1257-2
UniFor-UniPro-CTTTGGCAACTTACCCAGAGGcAAATC-x 51 HAMP-253-1
UniFor-UniPro-GCACTGAGCTCCCAGAuCTGGC-x 52 HAMP-253-2
UniFor-UniPro-GCAAGCGGCCCAGATCuGGGAC-x 53 BIRC5-606-1
UniFor-UniPro-GACGACCCCATGTAAGTCTTCuCTGGG-x 54 BIRC5-606-2
UniFor-UniPro-CGAGGCTGGCCAGAGAaGACTTT-x 55 SAA 146-1 UniFor-UniPro-
56 CTTTCCCAACAAGATTATCATTTCCTTTAAaAAAAT-x SAA 146-2
UniFor-UniPro-CGCCCCAGGATAACTATTTTTTTTaAAGGT-x 57 IDO1-97-1
UniFor-UniPro-AGACACTGAGGGGCACCaGAGGT-x 58 IDO1-97-2
UniFor-UniPro-CTTGTAGTCTGCTCCTCTGGuGCCCG-x 59 IDO1-176-1
UniFor-UniPro- 60 AGTAAAGAGTACCATATTGATGAAGAAgTGGGA-x IDO1-176-2
UniFor-UniPro-GCAGAGCAAAGCCCACTTcTTCAA-x 61 CYP27A-
UniFor-UniPro-CCTTTGGTGAGGACTCCCAgATGGC-x 62 31016-1 CYP27A-
UniFor-UniPro-CCTGGGCCCCATCTGgGAGTG-x 63 31016-2 SAA 226-1
UniFor-UniPro-TCTCCTCTGATCTAGAGAGGTAAGcAGGGA-x 64 SAA 226-2
UniFor-UniPro-ACCAGGCCCGACCCTGCTuACCTG-x 65 KIF11-369-1
UniFor-UniPro-GAGAAGGGGAAGAACATCCAgGTGGA-x 66 KIF11-369-2
UniFor-UniPro-GCATCTCACCACCACCTGgATGTA-x 67 C3-1394-1
UniFor-UniPro-CTGGACAGCACTAGTTTTTTGCcTGGGT-x 68 C3-1394-2
UniFor-UniPro-CCACGACTTCCCAGGCaAAAAT-x 69 HOGA-505-1
UniFor-UniPro-CACTGCAGAGGTGGACTaTGGGT-x 70 HOGA-505-2
UniFor-UniPro-GATTCTCCTCCAGTTTCCCATAGuCCACG-x 71 EGFR-
UniFor-UniPro-CCAGAGGATGTTCAATAACTGTGAgGTGGA-x 72 123344-1 EGFR-
UniFor-UniPro-CAAATTCCCAAGGACCACCuCACAC-x 73 123344-2 ALDH2-
UniFor-UniPro-TGAAGGGGACAAGGTGAGAaCTGGA-x 74 15144-1 ALDH2-
UniFor-UniPro-CCCAAGGTAAGTCACCAGTTCuCACCA-x 75 15144-2 AGXT-140-1
UniFor-UniPro-CCATGGCCTCTCACAAGCTgCTGGA-x 76 AGXT-140-2
UniFor-UniPro-GGGGGTCACCAGCAGcTTGTC-x 77 APOC-2929-1
UniFor-UniPro-CCGTTAAGGACAAGTTCTCTGAGTuCTGGC-x 78 APOC-2929-2
UniFor-UniPro-TCAGGGTCCAAATCCCAGAACuCAGAC-x 79 Met 27554-1
UniFor-UniPro- 80 AATTTTATTTACTTCTTGACGGTCCAAAGGgAAACA-x Met
27554-2 UniFor-UniPro-GTCTGAGCATCTAGAGTTTCCCuTTGGT-x 81 SAA 88-1
UniFor-UniPro-AGGTGAGGAGCACACCAAGGAgTGATA-x 82 SAA 88-2
UniFor-UniPro- 83 GAAAACAGAGTAAGTTTTAAAAATCACTCcTTGGA-x HIF1A-293-1
UniFor-UniPro-TCGCACCCCCACCTcTGGAG-x 84 HIF1A-293-2
UniFor-UniPro-GAAGGAAAGGCAAGTCCAGAGgTGGGC-x 85 Met 27475-1
UniFor-UniPro- 86 AATTTTATTTACTTCTTGACGGTCCAAAGGgAAACA-x Met
27475-2 UniFor-UniPro-GTCTGAGCATCTAGAGTTTCCCuTTGGT-x 87 HAMP-295-1
UniFor-UniPro-CTCGCCAGCCTGACCaGTGGG-x 88 HAMP-295-2
UniFor-UniPro-GGGAAAACAGAGCCACTGGuCAGGG-x 89 GRHPR-
UniFor-UniPro-GCCTCCTCTCCGACCAcGTGGT-x 90 2234-1 GRHPR-
UniFor-UniPro-GGATCCTCTTGTCCACGTGgTCGGAC-x 91 2234-2 HAMP-88-1
UniFor-UniPro-GGCGCCACCACCTTcTTGGT-x 92 HAMP-88-2
UniFor-UniPro-GCTCTGTCTCATTTCCAAGAAgGTGGA-x 93 Met 27254-1
UniFor-UniPro-GAGCCAAAGTCCTTTCATCTGTaAAGGT-x 94 Met 27254-2
UniFor-UniPro-GAAGTTGATGAACCGGTCCTTTACaGATGT-x 95 GRHPR-
UniFor-UniPro-ACAAGAGGATCCTGGATGCTgCAGGA-x 96 2264-1 GRHPR-
UniFor-UniPro-CGCTCTAGCTCCTTGGCaGGGAA-x 97 2264-2 Serpina 279-1
UniFor-UniPro-ACTCAGTTCCACAGGTGGGAGgGAGGC-x 98 Serpina 279-2
UniFor-UniPro-CACTCTAAGCCCTGCTGTCCCaCCTGA-x 99 Myc 459-1
UniFor-UniPro-CGGGAGGCTATTCTGCCCATTuGGGAT-x 100 Myc 459-2
UniFor-UniPro-CGGGGAAGTGTCCCCAAAuGGGCT-x 101 Serpina 130-1
UniFor-UniPro-GCTGCTGCTGCCAGGAAuTCCAC-x 102 Serpina 130-2
UniFor-UniPro-CCCCTCCAACCTGGAATTcCTGGG-x 103 Myc 490-1
UniFor-UniPro-CTGCCAGGACCCGCTTCuCTGAT-x 104 Myc 490-2
UniFor-UniPro-CAAGGAGAGCCTTTCAGAGAaGCGGC-x 105 GRHPR-
UniFor-UniPro-GATGAGCCCATCCCTGCcAAGGT-x 106 2179-1 GRHPR-
UniFor-UniPro-CGCTCTAGCTCCTTGGCaGGGAA-x 107 2179-2 GYG-2851-1
UniFor-UniPro-TCGCCACCCCTCAGGuCTCAC-x 108 GYG-2851-2
UniFor-UniPro-ACCTCATGGAGTCTGAGACCuGAGGC-x 109 GYG-2793-1
UniFor-UniPro-GCCCTGGTCCTGGGAuCATCA-x 110 GYG-2793-2
UniFor-UniPro-CTGTGCTGTTTCAGAGATGATCcCAGGT-x 111 Serpina 79-1
UniFor-UniPro-CAAGAGTCCTGAGCTGAACCAAgAAGGT-x 112 Serpina 79-2
UniFor-UniPro-CGACCCCCTCCTCCTTCTTgGTTCT-x 113 Myc 538-1
UniFor-UniPro-CTGCTTAGACGCTGGATTTTTTuCGGGA-x 114 Myc 538-2
UniFor-UniPro-CTGGTTTTCCACTACCCGAAArAAAAA-x 115 GYG-2744-1
UniFor-UniPro-CTTTGTATTAAGATCAGGCCTTTGTgACACA-x 116 GYG-2744-2
UniFor-UniPro-CATCGTTTGTGGTTAGTGTCACaAAGGG-x 117 RNase P For
GCGGAGGGAAGCTCATCAG 118 RNase P Rev CCCTAGTCTCAGACCTTCCCAA 119
RNase P FAM-CCACGAGCTGAGTGCGTCCTGTCA-IBFQ 120 probe DNA is
uppercase, RNA is lowercase. FAM = 6-Fluorescein fluorescent dye
(IDT, Coralville, IA). IBFQ = Iowa Black .TM. fluorescent quencher
(IDT, Coralville, IA). UniFor-UniPro = universal forward primer,
and universal probe binding site. X = propanediol (C3) spacer
blocking group.
TABLE-US-00020 TABLE 20 Non-discriminatory reverse primers utilized
in Example 10. Seq ID Name Sequence No. GCK-356-1
GAGGAAACTGTGACTGAACCTC 121 GCK-356-2 CCAAGGCTTCTCCGCC 122
ERBB3-33-1 GAGTCCGGGGAGGGATG 123 ERBB3-33-2 CAATCCCTACTCCAGCCTC 124
TTR-1257-1 ATGTGAGCCTCTCTCTACCAA 125 TTR-1257-2
GTCCTCTGATGGTCAAAGTTCTA 126 HAMP-253-1 CACTGGTCAGGCTGGC 127
HAMP-253-2 CAAGCTCAAGACCCAGCA 128 BIRC5-606-1
CAACTCAAATCTTTTGACAACTCAG 129 BIRC5-606-2 GGAGCTGGAAGGCTGG 130 SAA
146-1 TTCAGAATGGTATGGCTGTATGC 131 SAA 146-2 CACAGATCAGGTGAGGAGCA
132 IDO1-97-1 GTTTTCCATAGCGTGTGCC 133 IDO1-97-2 GTGGTCACTGGCTGTGG
134 IDO1-176-1 TTCCCACATTTTACTGCCTTCTC 135 IDO1-176-2
CGCTATGGAAAACTCCTGGA 136 CYP27A- CAGGTCTGTGCATCAGCG 137 31016-1
CYP27A- CTTTCTGGAAGCGATACCTG 138 31016-2 SAA 226-1
CGCACAGAACTCAACATGGG 139 SAA 226-2 AATAGTTATCCTGGGGCATACAGC 140
KIF11-369-1 GCTCGGAATCCTGTCAGC 141 KIF11-369-2 CAGCCAAATTCGTCTGCG
142 C3-1394-1 GGGATGTTCCAGTCACTGTTAC 143 C3-1394-2 GGTTGGTGGCAGGGG
144 HOGA-505-1 AGGGGAAGGTGCCCAG 145 HOGA-505-2 AAGGTGGACATTGCGGG
146 EGFR- TCATAATTCCTCTGCACATAGGT 147 123344-1 EGFR-
GCCAAGGCACGAGTAACA 148 123344-2 ALDH2- CGTATAAAATAGAAGACGAATCCATCCC
149 15144-1 ALDH2- ATGGCACGATGCCGT 150 15144-2 AGXT-140-1
GGCTTGAGCAGGGCC 151 AGXT-140-2 TGGCCAAGGCCAGTG 152 APOC-2929-1
TCAGGCAGCCACGGC 153 APOC-2929-2 GTGACCGATGGCTTCAGT 154 Met 27554-1
CATACGCAGCCTGAAGTATATTAAACA 155 Met 27554-2
TAGATGCTCAGACTTTTCACACAAGA 156 SAA 88-1 TTCAGAATGGTATGGCTGTATGC 157
SAA 88-2 AGCAGGGAAGGCTCAGTATAAATAG 158 HIF1A-293-1 TAAGCGCTGGCTCCCT
159 HIF1A-293-2 CTCTAGTCTCACGAGGGGTT 160 Met 27475-1
CTCTTTTCTGTGAGAATACACTCCAG 161 Met 27475-2 TACCCCATTAAGTATGTCCATGCC
162 HAMP-295-1 TCTCCCATCCCTGCTGC 163 HAMP-295-2 CCGCTTGCCTCCTGC 164
GRHPR- CACCCAGTGTGCACCT 165 2234-1 GRHPR- GCCAAGGAGCTAGAGCGA 166
2234-2 HAMP-88-1 GAGGCGGTGGTCTGAG 167 HAMP-88-2 TGTTCCCTGTCGCTCTG
168 Met 27254-1 CTTTAGCCTTCTCACTGATATCGAATG 169 Met 27254-2
GCATATTCTCCCCACAGATAGAAGA 170 GRHPR- CCTGCCCACCCAGTG 171 2264-1
GRHPR- CTGTGAGGTGGAGCAGTG 172 2264-2 Serpina 279-1
TAGCTCCTGGGCATTTCTTCC 173 Serpina 279-2 AGCTTGAGGAGAGCAGGAAAG 174
Myc 459-1 CCTGGTTTTCCACTACCCGA 175 Myc 459-2 CACTGGAACTTACAACACCCG
176 Serpina 130-1 TTCCTGCTCTCCTCAAGCTCT 177 Serpina 130-2
GAGCTGAACCAAGAAGGAGGA 178 Myc 490-1 AGGCATTCGACTCATCTCAGC 179 Myc
490-2 TGCACTGGAACTTACAACACC 180 GRHPR- TCGGAGAGGAGGCAGAG 181 2179-1
GRHPR- TTCTCCTGAGGGCCTCC 182 2179-2 GYG-2851-1
ACAGGGAGAAGGATGTCAGAG 183 GYG-2851-2 GTCCTGGGATCATCTCTGAAAC 184
GYG-2793-1 ACAGGGAGAAGGATGTCAGAG 185 GYG-2793-2
CACTAACCACAAACGATGCCT 186 Serpina 79-1 GAATTCCTGGCAGCAGCA 187
Serpina 79-2 CTACTGCCTCCACCCGAA 188 Myc 538-1
TAGGCATTCGACTCATCTCAGC 189 Myc 538-2 TGCACTGGAACTTACAACACC 190
GYG-2744-1 GGACCAGGGCACCTTTG 191 GYG-2744-2 GGCTTTCTCCAGATAAGATACTG
192 DNA is uppercase.
[0148] Although the reverse primers were not cleaved by RNase H2 in
these assays, the results suggest that blocked-cleavable reverse
primers could also be utilized in these assays.
[0149] Analysis of the amplification data was performed using a
.DELTA..DELTA.Cq method. Briefly, the .DELTA.Cq was calculated
between each of the target Cqs and the corresponding reference
(RNase P) Cqs. A conversion was then done with the calculated
.DELTA.Cq, where .DELTA.Cq experimental was calculated as being
equal to 2A-.DELTA.Cq. .DELTA..DELTA.Cq was then calculated by
normalization against the .DELTA.Cq calculated from the WT
(un-mutated) control.
[0150] Experimental results are shown in FIGS. 13A and 13B. FIG.
13A shows the clear difference between the NGS and T7 endonuclease
cleavage data. Of the 72 assays tested, 64 of the T7EI results were
>25% discordant in their quantification of the amount of mutant
template present compared to the NGS gold standard. The error in
T7EI quantification is expected as EMCA assays usually
underestimate genome editing rates, as discussed above.
[0151] FIG. 13B shows the comparison between the NGS data and the
method of the present invention. A total of 74 assays were tested
in the new assay format. Of these, 13 failed to amplify (17%), of
which 10/13 (76%) of the failed assays showed sequence features
that impair primer function and could easily be removed from future
testing by a design algorithm (G-quadraplexes, hairpins, etc.). Of
the 61/74 assays that amplified, only 2/61 (3.3%) showed more than
25% divergence from the results obtained with the NGS experiment.
These data, combined with the data from FIG. 13A, demonstrate the
utility of the assays of the disclosure in providing a rapid,
inexpensive PCR-based method to detect CRISPR genome mutation
events and how the accuracy of this method is much superior to EMCA
assays.
Example 11
[0152] This example demonstrates the effect of certain mutations on
the activity of P.a. RNase H2 enzyme. In particular, the mutation
G12A has limited effect on mismatch discrimination when the
mismatch is located opposite the RNA, while the mutation P13T has
no effect.
[0153] Towards identifying mutant RNase H2 enzymes with the desired
properties, a collection of P.a. RNase H2 mutants were developed.
Among these, mutations G12A and P13T were tested and found to
confer increased specificity. Similar mutations in RNase H2 enzymes
from other species that support rhPCR may likewise show similar
improved properties relative to their WT forms.
[0154] 6.times.-His-tagged-mutant and WT P.a. RNase H2 proteins
were generated by standard site-directed mutagenesis (SDM)
techniques (see, e.g., Weiner M. et al., Gene, 151:119-123 (1994)).
The primers used for SDM of the P.a. RNase H2 enzyme are shown in
Table 21. The mutagenized proteins were sequence verified and
expressed in E. coli using standard methods. Purification was done
by affinity purification over a charged Ni.sup.2+ column, as
described in Dobosy et al. (2011) and U.S. Pat. No. 8,911,948 B2).
Final amino acid sequences for the mutagenized proteins are shown
in Table 22. After expression and purification, specific activity
and unit definitions were determined using previously described
techniques (U.S. Pat. No. 8,911,948 B2).
TABLE-US-00021 TABLE 21 SDM primers for mutagenesis of the RNase H2
enzymes used in Example 11. SEQ Specific Primer ID AA name NO:
changes Sequence G12A 193 G12A TGCAGGTGCAGATGAAGCTGGTCGTGCCC
Forward CAGTTATTGGTCCGCTGGTTATTG Oligo G12A 194 G12A
CAATAACCAGCGGACCAATAACTGGGGCA Reverse CGACCAGCTTCATCTGCACCTGCA
Oligo P13T 195 P13T AGGTGCAGATGAAGCTGGTCGTGGTACGG Forward
TTATTGGTCCGCTGGTTATTGTTG Oligo P13T 196 P13T
CAACAATAACCAGCGGACCAATAACCGTA Reverse CCACGACCAGCTTCATCTGCACCT
Oligo All bases are DNA. Characters shown in bold are the mutagenic
nucleotides.
TABLE-US-00022 TABLE 22 Amino acid sequences for the RNase H2
proteins in Example 11. Mut SEQ Specific ID ID AA # NO: changes
Sequence N/A 197 WT RNase H2 MKVAGADEAGRGPVIGPLVIVAAVVEEDKIR
SLTKLGVKDSKQLTPAQREKLFDEIVKVLDD YSVVIVSPQDIDGRKGSMNELEVENFVKALN
SLKVKPEVIYIDSADVKAERFAENIRSRLAYE AKVVAEHKADAKYEIVSAASILAKVIRDREIE
KLKAEYGDFGSGYPSDPRTKKWLEEWYSKH GNFPPIVRRTWDTAKKIEEKFKRAQLTLDNFL
KRFRN 1 198 G12A MKVAGADEAGRAPVIGPLVIVAAVVEEDKIR
SLTKLGVKDSKQLTPAQREKLFDEIVKVLDD YSVVIVSPQDIDGRKGSMNELEVENFVKALN
SLKVKPEVIYIDSADVKAERFAENIRSRLAYE AKVVAEHKADAKYEIVSAASILAKVIRDREIE
KLKAEYGDFGSGYPSDPRTKKWLEEWYSKH GNFPPIVRRTWDTAKKIEEKFKRAQLTLDNFL
KRFRN 2 199 P13T MKVAGADEAGRGTVIGPLVIVAAVVEEDKIR
SLTKLGVKDSKQLTPAQREKLFDEIVKVLDD YSVVIVSPQDIDGRKGSMNELEVENFVKALN
SLKVKPEVIYIDSADVKAERFAENIRSRLAYE AKVVAEHKADAKYEIVSAASILAKVIRDREIE
KLKAEYGDFGSGYPSDPRTKKWLEEWYSKH GNFPPIVRRTWDTAKKIEEKFKRAQLTLDNFL
KRFRN Location of mutations are shown in bold and underlined
typeface.
[0155] To determine whether these mutant RNase H2 enzymes increase
mismatch discrimination when the mismatch is opposite the RNA,
1.sup.st generation quantitative rhPCR assays were utilized against
the rs4939827 SNP present in SMAD7. This SNP has been utilized in
the past (Dobosy et al, 2011, and U.S. Pat. No. 8,911,948 B2) to
characterize rhPCR efficiency and specificity, and its response
under differing conditions is well understood. The primers utilized
in these assays are shown in Table 23, SEQ ID NOs: 200-203. Assays
were done in 10 .mu.L reaction volumes. Thermal cycling and data
collection was performed with a CFX384.RTM. Real Time System
(Bio-Rad.RTM., Hercules, Calif.). Briefly, 1.times. of the iQ.TM.
SYBR.RTM. Green Supermix.RTM. was utilized in combination with 200
nM (2 pmol) of either the blocked forward primer (SEQ ID NOs: 202
or 203) and 200 nM (2 pmol) of the unblocked reverse primer (SEQ ID
NO: 201), or 200 nM (2 pmol) of unblocked control forward primer
(SEQ ID NO: 200) and 200 nM (2 pmol) of unblocked reverse primer
(SEQ ID NO: 201). 5 mU of either WT (SEQ ID NO: 197) or each mutant
RNase H2 enzyme (SEQ ID NOs: 198 or 199) was added to each
reaction. 10 ng of genomic cell line DNA (cell lines NA1852 and
NA18537, Coriell Institute for Medical Research, Camden, N.J.),
representing the two homozygous genotypes at the rs4939827 SNP, was
included in each reaction. Reactions were cycled under the
following conditions 95.degree. C..sup.3:00.fwdarw.(95.degree.
C..sup.0:10.fwdarw.60.degree. C..sup.0:30).times.85. Fluorescence
data was collected after each extension time point. After the
reaction was completed, data was analyzed, and the average Cq
values for each point was calculated. The results are presented in
Table 24.
TABLE-US-00023 TABLE 23 Sequences and SEQ IDs for the primers used
in the experiment described in Example 11. SEQ ID NO: Name Sequence
200 rs4939827 CAGCCTCATCCAAAAGAGGAAA unblocked 201 rs4939827 Rev
CTCACTCTAAACCCCAGCATT unblocked 202 rs4939827 rC
CAGCCTCATCCAAAAGAGGAAAcAGGA-x 4dmx 203 rs4939827 rU
CAGCCTCATCCAAAAGAGGAAAuAGGA-x 4dmx DNA is uppercase, RNA is
lowercase. x = C3 spacer (propanediol) blocker group.
TABLE-US-00024 TABLE 24 Cq and .DELTA.Cq values for the experiment
described in Example 11. RNase H2 Primer Forward primer CC TT
enzyme name SEQ ID NO template template .DELTA.Cq WT Unblocked 200
23.2 23.1 rC 4dmx 202 24.6 34.6 10.0 rU 4dmx 203 36.3 23.5 12.8
P13T Unblocked 200 23.8 23.0 (Mut ID 1) rC 4dmx 202 25.2 35.2 10.0
rU 4dmx 203 39.0 26.0 13.0 G12A Unblocked 200 21.6 21.7 (Mut ID 2)
rC 4dmx 202 64.8 81.6 16.9 rU 4dmx 203 54.6 41.5 13.1
[0156] These data show that these mutations do not significantly
increase mismatch discrimination when the SNP is located
immediately opposite the RNA residue in the rhPCR primer. The
possible exception of the G12A mutation is noted, but insufficient
amount of RNase H2 was utilized in this experiment resulting in
inefficient cleavage of primers and a severely delayed
amplification compared to the match template.
Example 12
[0157] This example demonstrates the effect of certain mutations on
the activity of P.a. RNase H2 enzyme. In particular, mutations G12A
and P13T increase mismatch discrimination when the mismatch is
placed 5' of the RNA nucleotide.
[0158] To determine whether mismatch discrimination can be
increased with the G12A and P13T mutant RNase H2 enzymes when the
mismatch is located 5' of the RNA nucleotide, assays targeting
rs113488022, the V600E SNP in the human BRAF gene were designed
with the mismatch located 5' of the RNA. The primers utilized in
these assays are shown in Table 25, SEQ ID NOs: 204-207. Assays
were performed in 10 .mu.L reaction volumes. Thermal cycling and
data collection were carried out with a CFX384.RTM. Real Time
System (Bio-Rad.RTM., Hercules, Calif.). Briefly, 1.times. of the
iQ.TM. SYBR.RTM. Green Supermix.RTM. was utilized in combination
with 200 nM (2 pmol) of either the blocked forward primer (SEQ ID
NOs: 206 or 207) and 200 nM (2 pmol) of the unblocked reverse
primer (SEQ ID NO: 205), or 200 nM (2 pmol) of unblocked control
forward primer (SEQ ID NO: 204) and 200 nM (2 pmol) of unblocked
reverse primer (SEQ ID NO: 205). 10 mU or 25 mU of either WT (SEQ
ID NO: 197) or each mutant RNase H2 enzyme (SEQ ID NOs: 198 or 199)
was added to each reaction. The 2,000 copies of synthetic
double-stranded gBlock.RTM. (IDT, Coralville, Iowa) template,
representing the two homozygous genotypes at the rs113488022 SNP
(SEQ ID NOs: 208 or 209), were also added to each reaction. Samples
were cycled under the following conditions 95.degree.
C..sup.3:00.fwdarw.(95.degree. C..sup.0:10.fwdarw.60.degree.
C..sup.0:30).times.85. Fluorescence intensity was collected after
each 60.degree. C. extension step. The Cq values were averaged from
3 replicates. The results are shown in Table 26.
TABLE-US-00025 TABLE 25 Sequences and SEQ ID NOs for the primers
and templates used in the experiment described in Example 12. SEQ
ID NO: Name Sequence 204 rs113488022 GTGATTTTGGTCTAGCTACAGT
unblocked 205 rs113488022 CCTCAATTCTTACCATCCACAAA Rev unblocked 206
rs113488022 GTGATTTTGGTCTAGCTACAGTgAAATG-x TrG 4dmx 207 rs113488022
GTGATTTTGGTCTAGCTACAGAgAAATG-x ArG 4dmx 208 gBlock .RTM. T
TAAGAGGAAAGATGAAGTACTATGTTTTAAA template
GAATATTATATTACAGAATTATAGAAATTAG ATCTCTTACCTAAACTCTTCATAATGCTTGCT
CTGATAGGAAAATGAGATCTACTGTTTTCCTT TACTTACTACACCTCAGATATATTTCTTCATG
AAGACCTCACAGTAAAAATAGGTGATTTTGG TCTAGCTACAGTGAAATCTCGATGGAGTGGG
TCCCATCAGTTTGAACAGTTGTCTGGATCCAT TTTGTGGATGGTAAGAATTGAGGCTATTTTTC
CACTGATTAAATTTTTGGCCCTGAGATGCTGC TGAGTTACTAGAAAGTCATTGAAGGTCTCAA
CTATAGTATTTTCATAGTTCCCAGTATTCACA AAAATCAGTGTTCTTATTTTTT 209 gBlock
.RTM. A TAAGAGGAAAGATGAAGTACTATGTTTTAAA template
GAATATTATATTACAGAATTATAGAAATTAG ATCTCTTACCTAAACTCTTCATAATGCTTGCT
CTGATAGGAAAATGAGATCTACTGTTTTCCTT TACTTACTACACCTCAGATATATTTCTTCATG
AAGACCTCACAGTAAAAATAGGTGATTTTGG TCTAGCTACAGAGAAATCTCGATGGAGTGGG
TCCCATCAGTTTGAACAGTTGTCTGGATCCAT TTTGTGGATGGTAAGAATTGAGGCTATTTTTC
CACTGATTAAATTTTTGGCCCTGAGATGCTGC TGAGTTACTAGAAAGTCATTGAAGGTCTCAA
CTATAGTATTTTCATAGTTCCCAGTATTCACA AAAATCAGTGTTCTTATTTTTT DNA is
uppercase, RNA is lowercase. X = C3 spacer (propanediol) blocker
group. Location of the mismatch is shown in bold and underlined in
the gBlocks .RTM..
TABLE-US-00026 TABLE 26 Cq and .DELTA.Cq values for the experiments
in Example 12. 10 mU RNase H2 25 mU RNase H2 RNase Forward T A T A
H2 Primer template template .DELTA.Cq template template .DELTA.Cq
WT Unblocked 26.2 25.6 26.0 26.0 TrG 26.8 29.1 2.3 26.9 29.0 2.1
ArG 37.2 26.8 10.4 36.1 26.8 9.3 P13T Unblocked 26.2 25.8 25.8 25.8
TrG 27.2 31.4 4.2 26.5 30.0 3.4 ArG 39.0 27.0 12.0 38.8 26.9 11.9
G12A Unblocked 26.1 26.0 26.0 26.0 TrG 28.6 36.5 7.9 27.1 33.2 6.2
ArG 42.1 30.7 11.4 38.8 27.7 11.1
[0159] These data show that the P13T RNase H2 mutant improved the
.DELTA.Cq quantification of the mismatch discrimination with the
TrG primer from 2.3 cycles with WT to 4.3 cycles (with 10 mU RNase
H2). The P13T RNase H2 also improved mismatch discrimination with
the ArG primer, increasing the .DELTA.Cq from 10.4 to 12.0 cycles.
While modest, these changes can be significant in sensitive assays.
The data also shows that the G12A RNase H2 mutant improved the
.DELTA.Cq quantification of the mismatch discrimination with the
TrG primer from 2.3 cycles with WT (with 10 mU RNase H2) to 6.2
cycles (with 25 mU RNase H2). The G12A RNase H2 also improved
mismatch discrimination with the ArG primer, increasing the
.DELTA.Cq from 10.4 to 11.4 cycles. The change with the ArG primer
is less important here, as it already was effective. Both of these
mutations therefore improve mismatch discrimination when the
mismatch is located 5' of the RNA nucleotide.
Example 13
[0160] This example demonstrates the effect of certain mutations on
the activity of P.a. RNase H2 enzyme. In particular, mutations G12A
and P13T increase mismatch discrimination when the mismatch is
placed 3' of the RNA.
[0161] To determine whether mismatch discrimination can be
increased with the G12A and P13T mutant RNase H2 enzymes when the
mismatch is located 3' of the RNA, assays targeting rs113488022,
the V600E SNP in the human BRAF gene were designed with the
mismatch located 3' of the RNA. The primers utilized in these
assays are shown in Table 27, SEQ ID NOs: 204, 205, 210, and 211.
Assays were conducted in 10 .mu.L reaction volumes. Thermal cycling
and data collection was performed with a CFX384.RTM. Real Time
System (Bio-Rad.RTM., Hercules, Calif.). Briefly, 1.times. of the
iQ.TM. SYBR.RTM. Green Supermix.RTM. was utilized in combination
with 200 nM (2 pmol) of either the blocked forward primer (SEQ ID
NOs: 210 or 211) and 200 nM (2 pmol) of the unblocked reverse
primer (SEQ ID NO: 205), or 200 nM (2 pmol) of unblocked control
forward primer (SEQ ID NO: 204) and 200 nM (2 pmol) of unblocked
reverse primer (SEQ ID NO: 205). 5 mU of either WT (SEQ ID NO: 197)
or each mutant RNase H2 enzyme (SEQ ID NOs: 198 or 199) was added
to each reaction. The 2,000 copies of synthetic double-stranded
gBlock.RTM. (IDT, Coralville, Iowa) template, representing the two
homozygous genotypes at the rs113488022 SNP (SEQ ID NOs: 208 or
209), were also added to each reaction. Reactions were cycled under
the following conditions 95.degree. C..sup.3:00.fwdarw.(95.degree.
C..sup.0:10.fwdarw.60.degree. C..sup.0:30).times.85. Fluorescence
intensity was collected after each extension step. The average Cq
values were averaged from 3 replicates. The results are shown in
Table 28.
TABLE-US-00027 TABLE 27 Sequences and SEQ IDs for the primers and
templates used in the experiment described in Example 13. SEQ ID
NO: Name Sequence 204 rs113488022 GTGATTTTGGTCTAGCTACAGT unblocked
205 rs113488022 CCTCAATTCTTACCATCCACAAA Rev unblocked 210
rs113488022 GTGATTTTGGTCTAGCTACArGTAAAA-x rGT 4dmx 211 rs113488022
GTGATTTTGGTCTAGCTACArGTAAAA-x rGA 4dmx DNA is uppercase, RNA is
lowercase. X = C3 spacer (propanediol) blocker group.
TABLE-US-00028 TABLE 28 Cq and .DELTA.Cq values for the experiments
in Example 13. 10 mU RNase H2 25 mU RNase H2 RNase Forward T A T A
H2 Primer template template .DELTA.Cq template template .DELTA.Cq
WT Unblocked 26.4 26.0 25.9 26.0 rGT 4dmx 26.4 26.5 0.2 25.7 26.3
0.6 rGA 4dmx 33.3 26.0 7.3 31.3 25.9 5.3 P13T Unblocked 25.8 25.7
25.5 25.8 rGT 4dmx 31.7 37.3 5.6 26.1 30.6 4.5 rGA 4dmx 42.0 27.0
15.0 41.4 26.4 15.0 G12A Unblocked 25.8 25.8 25.7 25.9 rGT 4dmx
55.7 66.8 11.1 41.7 50.3 8.6 rGA 4dmx 75.6 48.2 27.4 58.0 35.4
22.5
[0162] These data show that the P13T RNase H2 mutant improved the
.DELTA.Cq discrimination with the rGT primer from 0.2 cycles (seen
for WT) to 4.5 cycles (seen for mutant at 10 mU RNase H2). The P13T
RNase H2 also improved mismatch discrimination with the rGA primer,
increasing the .DELTA.Cq from 7.3 to 15.0 cycles. These
improvements turned a failed assay into successful one, in
particular when the rGT primer was employed. Furthermore, the G12A
RNase H2 mutant improved the .DELTA.Cq of the mismatch
discrimination with the rGT primer from 0.2 cycles to 8.6 cycles.
The G12A RNase H2 also improved mismatch discrimination with the
rGA primer, increasing the .DELTA.Cq from 7.3 to 22.5 cycles. This
change came at the cost of some amplification efficiency, but this
would likely be overcome with the addition of more G12A mutant
RNase H2 enzyme. In conclusion, both of these mutations improve
mismatch discrimination when the mismatch is located 3' of the RNA
nucleotide.
Example 14
[0163] This example demonstrates the effect of certain mutations on
the activity of P.a. RNase H2 enzyme. In particular, mutation G169A
of P.a. RNase H2 has no effect on mismatch discrimination when the
mismatch is located opposite the RNA.
[0164] Towards identifying mutant RNase H2 enzymes with the desired
properties, an additional P.a. RNase H2 mutant was developed with
by substituting glycine with alanine at position 169 (G169A).
Similar mutations in RNase H2 enzymes from other species that
support rhPCR may likewise show similar improved properties
relative to their WT forms.
[0165] 6.times.-His-tagged-mutant and WT P.a. RNase H2 proteins
were generated by standard site-directed mutagenesis (SDM)
techniques (see, e.g., Weiner M. et al., Gene, 151:119-123 (1994)).
The primers used for SDM of the P.a. RNase H2 enzyme are shown in
Table 29. The mutagenized proteins were sequence verified and
expressed in E. coli using standard methods. Purification was done
by affinity purification over a charged Ni.sup.2+ column, as
described in Dobosy et al. (2011) and U.S. Pat. No. 8,911,948 B2).
Final amino acid sequences for the mutagenized proteins are shown
in Table 30. After expression and purification, specific activity
and unit definitions were determined using previously described
techniques (U.S. Pat. No. 8,911,948 B2).
TABLE-US-00029 TABLE 29 SDM primers for mutagenesis of the RNase H2
enzymes used in Example 14. SEQ Specific Primer ID AA name NO:
changes Sequence G169A 212 G169A AGCCGAATACGGTGATTTTGGTTCCGCATA
Forward CCCGTCTGATCCGCGTACTAAGA Oligo G169A 213 G169A
TCTTAGTACGCGGATCAGACGGGTATGCG Reverse GAACCAAAATCACCGTATTCGGCT
Oligo All bases are DNA. Characters shown in bold are the mutagenic
nucleotides.
TABLE-US-00030 TABLE 30 Amino acid sequences for the RNase H2
proteins in Example 14. Mut SEQ AA ID ID Specific # NO: changes
Sequence N/A 197 WT RNase H2 MKVAGADEAGRGPVIGPLVIVAAVVEEDKIR
SLTKLGVKDSKQLTPAQREKLFDEIVKVLDD YSVVIVSPQDIDGRKGSMNELEVENFVKALN
SLKVKPEVIYIDSADVKAERFAENIRSRLAYE AKVVAEHKADAKYEIVSAASILAKVIRDREIE
KLKAEYGDFGSGYPSDPRTKKWLEEWYSKH GNFPPIVRRTWDTAKKIEEKFKRAQLTLDNFL
KRFRN 3 214 G169A MKVAGADEAGRGPVIGPLVIVAAVVEEDKIR
SLTKLGVKDSKQLTPAQREKLFDEIVKVLDD YSVVIVSPQDIDGRKGSMNELEVENFVKALN
SLKVKPEVIYIDSADVKAERFAENIRSRLAYE AKVVAEHKADAKYEIVSAASILAKVIRDREIE
KLKAEYGDFGSAYPSDPRTKKWLEEWYSKH GNFPPIVRRTWDTAKKIEEKFKRAQLTLDNFL
KRFRN Location of mutations are shown in bold and underlined
typeface.
[0166] To determine whether this mutant RNase H2 enzyme increases
mismatch discrimination when the mismatch is opposite the RNA,
quantitative rhPCR assays were utilized against the rs4939827 SNP
present in SMAD7. This SNP has been utilized in the past (Dobosy et
al, 2011, and U.S. Pat. No. 8,911,948 B2) to characterize rhPCR
efficiency and specificity, and its response under differing
conditions is well understood. The primers utilized in these assays
are shown in Table 31, SEQ ID NOs: 215-218. Assays were done in 10
reaction volumes. Thermal cycling and data collection was performed
with a CFX384.RTM. Real Time System (Bio-Rad.RTM., Hercules,
Calif.). Briefly, 1.times. of the iQ.TM. SYBR.RTM. Green
Supermix.RTM. was utilized in combination with 200 nM (2 pmol) of
either the blocked forward primer (SEQ ID NOs: 217 or 218) and 200
nM (2 pmol) of the unblocked reverse primer (SEQ ID NO: 216), or
200 nM (2 pmol) of unblocked control forward primer (SEQ ID NO:
215) and 200 nM (2 pmol) of unblocked reverse primer (SEQ ID NO:
216). 5 mU of either WT (SEQ ID NO: 197) or G169 mutant RNase H2
enzyme (SEQ ID NO: 214) was added to each reaction. 10 ng of
genomic cell line DNA (cell lines NA1852 and NA18537, Coriell
Institute for Medical Research, Camden, N.J.), representing the two
homozygous genotypes at the rs4939827 SNP, was included in each
reaction. Reactions were cycled under the following conditions
95.degree. C..sup.3:00.fwdarw.(95.degree.
C..sup.0:10.fwdarw.60.degree. C..sup.0:30).times.85. Fluorescence
data was collected after each extension time point. After the
reaction was completed, data was analyzed, and the average Cq
values for each point was calculated. The results are presented in
Table 24.
TABLE-US-00031 TABLE 31 Sequences and SEQ IDs for the primers used
in the experiment described in Example 14. SEQ ID NO: Name Sequence
215 rs4939827 CAGCCTCATCCAAAAGAGGAAA unblocked 216 rs4939827 Rev
CTCACTCTAAACCCCAGCATT unblocked 217 rs4939827 rC
CAGCCTCATCCAAAAGAGGAAAcAGGA-x 4dmx 218 rs4939827 rU
CAGCCTCATCCAAAAGAGGAAAuAGGA-x 4dmx DNA is uppercase, RNA is
lowercase. x = C3 spacer (propanediol) blocker group.
TABLE-US-00032 TABLE 32 Cq and .DELTA.Cq values for the experiment
described in Example 14. RNase H2 Primer Forward primer CC TT
enzyme name SEQ ID NO template template .DELTA.Cq WT Unblocked 7
22.4 23.0 rC 4dmx 9 22.9 34.3 11.5 rU 4dmx 10 32.6 23.3 9.3 G169A
Unblocked 7 22.2 22.0 (Mut ID 3) rC 4dmx 9 23.2 31.1 8.0 rU 4dmx 10
39.2 27.5 11.8
[0167] These data show that these mutations do not significantly
increase mismatch discrimination when the SNP is located
immediately opposite the RNA residue in the rhPCR primer.
Example 15
[0168] This example demonstrates the effect of certain mutations on
the activity of P.a. RNase H2 enzyme. In particular, mutation of
P.a. RNase H2 at G169A increases mismatch discrimination when the
mismatch is placed 5' of the RNA nucleotide.
[0169] To determine whether mismatch discrimination can be
increased with the G169A mutant RNase H2 enzyme when the mismatch
is located 5' of the RNA nucleotide, assays targeting rs113488022,
the V600E SNP in the human BRAF gene were designed with the
mismatch located 5' of the RNA. The primers utilized in these
assays are shown in Table 27, SEQ ID NOs: 204-207. Assays were
performed in 10 .mu.L reaction volumes. Thermal cycling and data
collection were carried out with a CFX384.RTM. Real Time System
(Bio-Rad.RTM., Hercules, Calif.). Briefly, 1.times. of the iQ.TM.
SYBR.RTM. Green Supermix.RTM. was utilized in combination with 200
nM (2 pmol) of either the blocked forward primer (SEQ ID NOs: 206
or 207) and 200 nM (2 pmol) of the unblocked reverse primer (SEQ ID
NO: 205), or 200 nM (2 pmol) of unblocked control forward primer
(SEQ ID NO: 204) and 200 nM (2 pmol) of unblocked reverse primer
(SEQ ID NO: 205). 25 mU of either WT (SEQ ID NO: 197) or G169
mutant RNase H2 enzyme (SEQ ID NO: 214) was added to each reaction.
The 2,000 copies of synthetic double-stranded gBlock.RTM. (IDT,
Coralville, Iowa) template, representing the two homozygous
genotypes at the rs113488022 SNP (SEQ ID NOs: 208 or 209), were
also added to each reaction. Reactions were cycled under the
following conditions 95.degree. C..sup.3:00.fwdarw.(95.degree.
C..sup.0:10.fwdarw.60.degree. C..sup.0:30).times.85. Fluorescence
intensity was collected after each extension step. The average Cq
values were averaged from 3 replicates. The results are shown in
Table 33.
TABLE-US-00033 TABLE 33 Cq and .DELTA.Cq values for the experiments
in Example 15. 10 mU RNase H2 25 mU RNase H2 RNase Forward T A T A
H2 Primer template template .DELTA.Cq template template .DELTA.Cq
WT Unblocked 26.8 25.9 26.1 25.8 rGT 4dmx 28.1 28.1 0.0 25.6 25.9
0.3 rGA 4dmx 40.5 26.3 14.2 28.1 25.6 2.5 G169A Unblocked 26.2 26.0
26.9 26.7 rGT 4dmx 29.2 39.3 10.1 26.1 27.3 1.2 rGA 4dmx 43.2 26.4
16.8 31.7 25.9 5.9
[0170] These data show that the G169A RNase H2 mutant improved the
.DELTA.Cq discrimination with the rGT primer from 0.0 cycles (seen
for WT) to 10.1 cycles (seen for mutant at 5 mU RNase H2). The
G169A RNase H2 also slightly improved mismatch discrimination with
the rGA primer, increasing the .DELTA.Cq from 14.2 to 16.8 cycles.
These improvements turned failed assay into successful one, in
particular when the rGT primer was employed. In conclusion, this
mutation improves mismatch discrimination when the mismatch is
located 3' of the RNA nucleotide.
[0171] The RNase H2 mutations examined in Examples 11-15 enhanced
mismatch discrimination more when the mutation was located 5' or 3'
of the RNA than when located opposite of the RNA nucleotide.
Together, these results suggest that limitations of mutation
discrimination of rhPCR are based not only on the ability of the
RNase H2 to recognize the mismatch and the ability of a polymerase
to extend the cleaved primer, but also from the conversion of the
mismatched template to the primer sequence. This conversion masks
the ability of the RNase H2 enzyme to recognize the mismatch when
the mismatch is located at the RNA nucleotide. This could happen in
several ways. Most (if not all) conversions spring from a cleavage
occurring on the 3' side of the mismatch. Once this type of
cleavage occurs, it can be extended by the DNA polymerase, after
which the newly generated template fully matches the primers. The
converted template then contaminates the sample because it is
expected to be amplified in subsequent cycles.
[0172] RNase H2 usually cleaves strand at 5' side of RNA
nucleotide. Experiments (not shown) have indicated that the rare
cleavage on the 3' side of RNA nucleotide occurs at a rate of
approximately 1 per 1,000 cleavage reactions. If the mismatch is
located at the RNA site, such rare cleavage will cause template
conversion. Introducing the mismatch 5' of the RNA nucleotide makes
conversion of the template to the primer sequence occur in the
first cycle of replication. In this case, mismatch discrimination
depends solely on the ability of the RNase H2 to recognize the
mismatch and the ability of polymerase to extend the mismatched
primer, since the polymerase is expected to be inhibited by a
mismatch at the 3' end of a primer.
[0173] On the other hand, placing the mismatch 3' of the RNA
nucleotide reduces (or completely eliminates) the template
conversion. Again, in this position, mismatch discrimination
depends solely on the ability of RNase H2 to recognize the mismatch
and the ability of the polymerase to extend the mismatched primer.
However, the polymerase is not expected to be inhibited in this
case since the mismatch site is cleaved out of the primer. The
results may also have arisen because the mutations revealed here
are more susceptible to mutations present 5' and 3' of the RNA than
they are opposite the RNA. These hypotheses are not intended to
limit the scope of the invention.
Example 16
[0174] This example demonstrates the effect of certain mutations on
the activity of P.a. RNase H2 enzyme. In particular, mutations G12A
and P13T increase mismatch discrimination when the mismatch is
placed 3' of the RNA with multiple SNPs.
[0175] To determine whether mismatch discrimination can be
increased with the G12A and P13T mutant RNase H2 enzymes when the
mismatch is located 3' of the RNA, assays targeting rs12744607,
rs60118785, rs60607502, rs7992897, rs3117947, rs7583169, rs4853850,
rs28520334, rs13316719, rs61514082, rs12908737, rs6572257,
rs2242527, rs1076939, rs35593667, and rs6046375 were designed with
the mismatch located 3' of the RNA. Assays were conducted in 10
.mu.L reaction volumes. Thermal cycling and data collection was
performed with a CFX384.TM. Real-Time System (Bio-Rad.RTM.,
Hercules, Calif.). Briefly, 1.times. of the iQ.TM. SYBR.RTM. Green
Supermix.RTM. was utilized in combination with 200 nM (2 pmol) of
either the blocked forward primer and 200 nM (2 pmol) of the
unblocked reverse primer. 10 mU of either WT (SEQ ID NO: 197) or 20
mU P13T mutant RNase H2 enzyme (SEQ ID NOs: 198 or 199) or 50 mU
G12A mutant RNase H2 enzyme (SEQ ID NO: 199) was added to each
reaction. The 10 ng of NA12878 or NA24385 genomic cell line DNA,
representing the two homozygous genotypes at each of these SNPs,
were also added to each reaction. Reactions were cycled under the
following conditions 95.degree. C..sup.3:00.fwdarw.(95.degree.
C..sup.0:10.fwdarw.60.degree. C..sup.0:30).times.85 cycles.
Fluorescence intensity was collected after each extension step. The
average Cq values were averaged from 3 replicates. The results are
shown in Table 34.
TABLE-US-00034 TABLE 34 Cq and .DELTA.Cq values for the experiments
in Example 16. WT RN2 P13T G12A NA12878 NA24385 .DELTA.Cq NA12878
NA24385 .DELTA.Cq NA12878 NA24385 .DELTA.Cq rs12744607 >85
>85 N/A 73.0 >85 12.0 38.2 61.5 23.3 rs13316719 27.5 25.3 2.2
26.4 25.5 1.0 26.4 27.0 -0.7 rs60118785 25.6 37.8 12.3 40.8 63.7
22.9 26.7 37.6 11.0 rs61514082 24.7 27.2 2.5 25.8 52.5 26.7 26.0
36.5 10.5 rs60607502 25.4 25.5 0.0 25.9 34.8 8.9 29.8 44.0 14.1
rs12908737 34.7 70.6 35.9 75.3 >85 9.7 35.7 52.9 17.2 rs7992897
25.1 27.9 2.8 24.5 24.4 0.1 25.9 30.7 4.8 rs6572257 >85 71.5
13.5 71.3 40.9 30.4 49.2 27.7 21.5 rs3117947 47.3 62.4 15.1 33.3
43.5 10.2 25.6 26.3 0.6 rs2242527 29.4 47.9 18.5 39.2 67.7 28.5
32.6 50.5 17.9 rs7583169 27.6 26.0 1.6 31.6 35.7 4.0 29.1 41.8 12.7
rs1076939 33.7 40.2 6.5 34.1 59.6 25.5 36.3 49.9 13.6 rs4853850
30.2 31.9 1.7 36.6 56.1 19.5 29.3 34.0 4.7 rs35593667 31.7 34.5 2.7
42.3 64.9 22.7 38.3 73.2 34.9 rs28520334 26.1 25.1 1.0 24.9 24.3
0.6 24.7 25.0 0.3 rs6046375 >85 61.1 24.0 80.4 46.1 34.3 53.1
28.2 24.9
[0176] These data show that the P13T RNase H2 mutant improved the
.DELTA.Cq discrimination at multiple SNPs. An excellent example is
rs61514082, where the .DELTA.Cq increased from 2.5 cycles (seen for
WT) to 26.7 cycles (seen for mutant at 20 mU RNase H2). The P13T
RNase H2 also improved mismatch discrimination at the rs60607502
SNP, increasing the .DELTA.Cq from 0.0 to 8.9 cycles. Other SNPs
also improved their specificity. The two SNPs that did not improve
.DELTA.Cq (r513316719 and r528520334) show that this mutation can
be further improved, and does not solve all issues with all assays,
despite a wide-ranging improvement being observed. These
improvements turned many failed assays into successful ones,
showing the broad improvements in .DELTA.Cq that this mutation
confers.
[0177] The G12A RNase H2 mutant also improved the .DELTA.Cq of the
mismatch discrimination at multiple SNPs. The rs61514082 SNP
increased in .DELTA.Cq from 2.5 cycles to 10.5 cycles. The G12A
RNase H2 also improved mismatch discrimination with the rs60607502
SNP, increasing the .DELTA.Cq from 0.0 to 14.1 cycles. In
conclusion, both mutations improve mismatch discrimination when the
mismatch is located 3' of the RNA nucleotide.
[0178] 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.
[0179] 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.
[0180] 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
218124DNAArtificial SequenceSynthetic 1gctgtgattt tggtctagct acag
24225DNAArtificial SequenceSynthetic 2gctgtgattt tggtctagct acagt
25325DNAArtificial SequenceSynthetic 3gctgtgattt tggtctagct acaga
25427DNAArtificial SequenceSyntheticmisc_feature(1)..(1)5'
6-carboxyfluoresceinmisc_feature(9)..(10)internal ZEN
quenchermisc_feature(27)..(27)3' Iowa Black fluorescence quencher
4tcccatcagt ttgaacagtt gtctgga 27531DNAArtificial
SequenceSyntheticmisc_feature(26)..(26)RNAmisc_feature(31)..(31)3'
C3 propanediol spacer block 5gctgtgattt tggtctagct acagtgaaat g
31631DNAArtificial
SequenceSyntheticmisc_feature(26)..(26)RNAmisc_feature(31)..(31)3'
C3 propanediol spacer block 6gctgtgattt tggtctagct acagagaaat g
31731DNAArtificial
SequenceSyntheticmisc_feature(26)..(26)RNAmisc_feature(31)..(31)3'
C3 propanediol spacer block 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
SequenceSyntheticmisc_feature(26)..(26)RNAmisc_feature(27)..(28)two
C3 propanediol spacers 10gctgtgattt tggtctagct acagtgatg
291129DNAArtificial
SequenceSyntheticmisc_feature(26)..(26)RNAmisc_feature(27)..(28)two
C3 propanediol spacers 11gctgtgattt tggtctagct acagagatg
291231DNAArtificial
SequenceSyntheticmisc_feature(26)..(26)RNAmisc_feature(31)..(31)3'
C3 propanediol spacer 12gccctcaatt cttaccatcc acaaaatgga a
311323DNAArtificial SequenceSynthetic 13cgccgcgtat agtcccgcgt aaa
231413DNAArtificial SequenceSyntheticmisc_feature(1)..(1)5'
6-carboxyfluoresceinmisc_feature(1)..(1)LNA
residuemisc_feature(5)..(7)LNA residuesmisc_feature(11)..(11)LNA
residuemisc_feature(13)..(13)3' Iowa Black fluorescence quencher
14ccatcaccgt gct 131513DNAArtificial
SequenceSyntheticmisc_feature(1)..(1)5' 6-carboxy-2',4,4',5',7,7'-
hexachlorofluoresceinmisc_feature(5)..(7)LNA
residuesmisc_feature(11)..(11)LNA residuemisc_feature(13)..(13)3'
Iowa Black fluorescence quencher 15caatccccga gct
131653DNAArtificial
SequenceSyntheticmisc_feature(48)..(48)RNAmisc_feature(53)..(53)3'
C3 propanediol spacer block 16gcccatgtcc cagcgaacca tcaccgtgct
agccctcgat acagcccggc cac 531752DNAArtificial
SequenceSyntheticmisc_feature(46)..(46)RNAmisc_feature(52)..(52)3'
C3 propanediol spacer block 17gcccatgtcc cagcgaacaa tccccgagct
gccctcgata cagcctggcc ac 521826DNAArtificial
SequenceSyntheticmisc_feature(21)..(21)RNAmisc_feature(26)..(26)3'
C3 propanediol spacer block 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
SequenceSyntheticmisc_feature(1)..(1)5' Yakima
yellowmisc_feature(1)..(1)LNA residuemisc_feature(5)..(7)LNA
residuesmisc_feature(11)..(11)LNA residuemisc_feature(13)..(13)3'
Iowa Black fluorescence quencher 22caatccccga gct
132356DNAArtificial
SequenceSyntheticmisc_feature(51)..(51)RNAmisc_feature(56)..(56)3'
C3 propanediol spacer block 23gcccatgtcc cagcgaacca tcaccgtgct
acttcccaca ccctcatatc utgtta 562459DNAArtificial
SequenceSyntheticmisc_feature(54)..(54)RNAmisc_feature(59)..(59)3'
C3 propanediol spacer block 24gcccatgtcc cagcgaacaa tccccgagct
cttacttccc acaccctcat atautgtta 592532DNAArtificial
SequenceSyntheticmisc_feature(27)..(27)RNAmisc_feature(32)..(32)3'
C3 propanediol spacer block 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
SequenceSyntheticmisc_feature(54)..(54)RNAmisc_feature(59)..(59)3'
C3 propanediol spacer block 28gcccatgtcc cagcgaacca tcaccgtgct
ttctcttctg gactccctat aatattgtg 592959DNAArtificial
SequenceSyntheticmisc_feature(54)..(54)RNAmisc_feature(59)..(59)3'
C3 propanediol spacer block 29gcccatgtcc cagcgaacaa tccccgagct
ttctcttctg gactccctat aacattgtg 593026DNAArtificial
SequenceSyntheticmisc_feature(21)..(21)RNAmisc_feature(26)..(26)3'
C3 propanediol spacer block 30gcggattgat gcagcagtga gtcatg
263156DNAArtificial
SequenceSyntheticmisc_feature(51)..(51)RNAmisc_feature(56)..(56)3'
C3 propanediol spacer block 31gcccatgtcc cagcgaacca tcaccgtgct
ctccgttgtt ttccagaaac gatttc 563256DNAArtificial
SequenceSyntheticmisc_feature(51)..(51)RNAmisc_feature(56)..(56)3'
C3 propanediol spacer block 32gcccatgtcc cagcgaacaa tccccgagct
ctccgttgtt ttccagaaat gatttc 563356DNAArtificial
SequenceSyntheticmisc_feature(51)..(51)RNAmisc_feature(56)..(56)3'
C3 propanediol spacer block 33gcccatgtcc cagcgaacaa tccccgagct
ctccgttgtt ttccagaaag gatttc 563428DNAArtificial
SequenceSyntheticmisc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
C3 propanediol spacer block 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
SequenceSyntheticmisc_feature(51)..(51)RNAmisc_feature(56)..(56)3'
C3 propanediol spacer block 38gcccatgtcc cagcgaacca tcaccgtgct
gtctttgctt tcctggtgag cccatg 563955DNAArtificial
SequenceSyntheticmisc_feature(49)..(49)RNAmisc_feature(55)..(55)3'
C3 propanediol spacer block 39gcccatgtcc cagcgaacaa tccccgagct
gtctttgctt tcctggtgac ccatg 554032DNAArtificial
SequenceSyntheticmisc_feature(27)..(27)RNAmisc_feature(32)..(32)3'
C3 propanediol spacer block 40gcgttggaac taccacattg ctttatugta ct
324119DNAArtificial SequenceSynthetic 41gcggagggaa gctcatcag
194222DNAArtificial SequenceSynthetic 42ccctagtctc agaccttccc aa
224324DNAArtificial SequenceSyntheticmisc_feature(1)..(1)5' Yakima
yellowmisc_feature(24)..(24)3' Iowa Black fluorescence quencher
43ccacgagctg agtgcgtcct gtca 244450DNAArtificial SequenceSynthetic
44aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct
504555DNAArtificial SequenceSynthetic 45caagcagaag acggcatacg
agatggacct atgtgactgg agttcagacg tgtgc 554621DNAArtificial
SequenceSyntheticmisc_feature(1)..(1)5 universal domain
(UniFor-UniPro-)misc_feature(16)..(16)RNAmisc_feature(21)..(21)3'
propanediol (C3) spacer blocking group 46ccctgggtcc ctgggagaat c
214726DNAArtificial SequenceGCK-356-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(21)..(21)RNAmisc_feature(26)..(26)3'
propanediol (C3) spacer blocking group 47cgaggagaac cacattctcc
cagggt 264818DNAArtificial SequenceERBB3-33-1misc_feature(1)..(1)5
universal domain
(UniFor-UniPro-)misc_feature(14)..(14)RNAmisc_feature(18)..(18)3'
propanediol (C3) spacer blocking group 48gggcggccgt gacucacc
184924DNAArtificial SequenceERBB3-33-2misc_feature(1)..(1)5
universal domain
(UniFor-UniPro-)misc_feature(18)..(18)RNAmisc_feature(24)..(24)3'
propanediol (C3) spacer blocking group 49gagggaaggg ggtgagtcac ggcg
245025DNAArtificial SequenceTTR-1257-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(20)..(20)RNAmisc_feature(25)..(25)3'
propanediol (C3) spacer blocking group 50cctgggagcc atttgcctcu
gggtt 255127DNAArtificial SequenceTTR-1257-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 51ctttggcaac ttacccagag
gcaaatc 275222DNAArtificial
SequenceHAMP-253-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(17)..(17)RNAmisc_feature(22)..(22)3'
propanediol (C3) spacer blocking group 52gcactgagct cccagauctg gc
225322DNAArtificial SequenceHAMP-253-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(17)..(17)RNAmisc_feature(22)..(22)3'
propanediol (C3) spacer blocking group 53gcaagcggcc cagatcuggg ac
225427DNAArtificial SequenceBIRC5-606-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 54gacgacccca tgtaagtctt
cuctggg 275523DNAArtificial
SequenceBIRC5-606-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(17)..(17)RNAmisc_feature(23)..(23)3'
propanediol (C3) spacer blocking group 55cgaggctggc cagagaagac ttt
235636DNAArtificial SequenceSAA 146-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(31)..(31)RNAmisc_feature(36)..(36)3'
propanediol (C3) spacer blocking group 56ctttcccaac aagattatca
tttcctttaa aaaaat 365730DNAArtificial SequenceSAA
146-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(25)..(25)RNAmisc_feature(30)..(30)3'
propanediol (C3) spacer blocking group 57cgccccagga taactatttt
ttttaaaggt 305823DNAArtificial
SequenceIDO1-97-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(18)..(18)RNAmisc_feature(23)..(23)3'
propanediol (C3) spacer blocking group 58agacactgag gggcaccaga ggt
235926DNAArtificial SequenceIDO1-97-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(21)..(21)RNAmisc_feature(26)..(26)3'
propanediol (C3) spacer blocking group 59cttgtagtct gctcctctgg
ugcccg 266033DNAArtificial SequenceIDO1-176-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(28)..(28)RNAmisc_feature(33)..(33)3'
propanediol (C3) spacer blocking group 60agtaaagagt accatattga
tgaagaagtg gga 336124DNAArtificial
SequenceIDO1-176-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(19)..(19)RNAmisc_feature(24)..(24)3'
propanediol (C3) spacer blocking group 61gcagagcaaa gcccacttct tcaa
246225DNAArtificial SequenceCYP27A-31016-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(20)..(20)RNAmisc_feature(25)..(25)3'
propanediol (C3) spacer blocking group 62cctttggtga ggactcccag
atggc 256321DNAArtificial
SequenceCYP27A-31016-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(16)..(16)RNAmisc_feature(21)..(21)3'
propanediol (C3) spacer blocking group 63cctgggcccc atctgggagt g
216430DNAArtificial SequenceSAA 226-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(25)..(25)RNAmisc_feature(30)..(30)3'
propanediol (C3) spacer blocking group 64tctcctctga tctagagagg
taagcaggga 306524DNAArtificial SequenceSAA
226-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(19)..(19)RNAmisc_feature(24)..(24)3'
propanediol (C3) spacer blocking group 65accaggcccg accctgctua cctg
246626DNAArtificial SequenceKIF11-369-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(21)..(21)RNAmisc_feature(26)..(26)3'
propanediol (C3) spacer blocking group 66gagaagggga agaacatcca
ggtgga 266724DNAArtificial
SequenceKIF11-369-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(19)..(19)RNAmisc_feature(24)..(24)3'
propanediol (C3) spacer blocking group 67gcatctcacc accacctgga tgta
246828DNAArtificial SequenceC3-1394-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
propanediol (C3) spacer blocking group 68ctggacagca ctagtttttt
gcctgggt 286922DNAArtificial
SequenceC3-1394-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(17)..(17)RNAmisc_feature(22)..(22)3'
propanediol (C3) spacer blocking group 69ccacgacttc ccaggcaaaa at
227023DNAArtificial SequenceHOGA-505-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(18)..(18)RNAmisc_feature(23)..(23)3'
propanediol (C3) spacer blocking group 70cactgcagag gtggactatg ggt
237129DNAArtificial SequenceHOGA-505-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(24)..(24)RNAmisc_feature(29)..(29)3'
propanediol (C3) spacer blocking group 71gattctcctc cagtttccca
taguccacg 297230DNAArtificial
SequenceEGFR-123344-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(25)..(25)RNAmisc_feature(30)..(30)3'
propanediol (C3) spacer blocking group 72ccagaggatg ttcaataact
gtgaggtgga 307325DNAArtificial
SequenceEGFR-123344-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(20)..(20)RNAmisc_feature(25)..(25)3'
propanediol (C3) spacer blocking group 73caaattccca aggaccaccu
cacac 257425DNAArtificial
SequenceALDH2-15144-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(20)..(20)RNAmisc_feature(25)..(25)3'
propanediol (C3) spacer blocking group 74tgaaggggac aaggtgagaa
ctgga 257527DNAArtificial
SequenceALDH2-15144-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 75cccaaggtaa gtcaccagtt
cucacca 277625DNAArtificial
SequenceAGXT-140-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(20)..(20)RNAmisc_feature(25)..(25)3'
propanediol (C3) spacer blocking group 76ccatggcctc tcacaagctg
ctgga 257721DNAArtificial SequenceAGXT-140-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(16)..(16)RNAmisc_feature(21)..(21)3'
propanediol (C3) spacer blocking group 77gggggtcacc agcagcttgt c
217830DNAArtificial SequenceAPOC-2929-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(25)..(25)RNAmisc_feature(30)..(30)3'
propanediol (C3) spacer blocking group 78ccgttaagga caagttctct
gagtuctggc 307927DNAArtificial
SequenceAPOC-2929-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 79tcagggtcca aatcccagaa
cucagac 278036DNAArtificial SequenceMet
27554-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(31)..(31)RNAmisc_feature(36)..(36)3'
propanediol (C3) spacer blocking group 80aattttattt acttcttgac
ggtccaaagg gaaaca 368128DNAArtificial SequenceMet
27554-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
propanediol (C3) spacer blocking group 81gtctgagcat ctagagtttc
ccuttggt 288227DNAArtificial SequenceSAA 88-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 82aggtgaggag cacaccaagg
agtgata 278335DNAArtificial SequenceSAA 88-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(30)..(30)RNAmisc_feature(35)..(35)3'
propanediol (C3) spacer blocking group 83gaaaacagag taagttttaa
aaatcactcc ttgga 358420DNAArtificial
SequenceHIF1A-293-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(15)..(15)RNAmisc_feature(20)..(20)3'
propanediol (C3) spacer blocking group 84tcgcaccccc acctctggag
208527DNAArtificial SequenceHIF1A-293-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 85gaaggaaagg caagtccaga
ggtgggc 278636DNAArtificial SequenceMet
27475-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(31)..(31)RNAmisc_feature(36)..(36)3'
propanediol (C3) spacer blocking group 86aattttattt acttcttgac
ggtccaaagg gaaaca 368728DNAArtificial SequenceMet
27475-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
propanediol (C3) spacer blocking group 87gtctgagcat ctagagtttc
ccuttggt 288821DNAArtificial
SequenceHAMP-295-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(16)..(16)RNAmisc_feature(21)..(21)3'
propanediol (C3) spacer blocking group 88ctcgccagcc tgaccagtgg g
218925DNAArtificial SequenceHAMP-295-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(20)..(20)RNAmisc_feature(25)..(25)3'
propanediol (C3) spacer blocking group 89gggaaaacag agccactggu
caggg 259022DNAArtificial
SequenceGRHPR-2234-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(17)..(17)RNAmisc_feature(22)..(22)3'
propanediol (C3) spacer blocking group 90gcctcctctc cgaccacgtg gt
229126DNAArtificial SequenceGRHPR-2234-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(20)..(20)RNAmisc_feature(26)..(26)3'
propanediol (C3) spacer blocking group 91ggatcctctt gtccacgtgg
tcggac 269220DNAArtificial SequenceHAMP-88-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(15)..(15)RNAmisc_feature(20)..(20)3'
propanediol (C3) spacer blocking group 92ggcgccacca ccttcttggt
209327DNAArtificial SequenceHAMP-88-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 93gctctgtctc atttccaaga
aggtgga 279428DNAArtificial SequenceMet
27254-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
propanediol (C3) spacer blocking group 94gagccaaagt cctttcatct
gtaaaggt 289530DNAArtificial SequenceMet
27254-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(25)..(25)RNAmisc_feature(30)..(30)3'
propanediol (C3) spacer blocking group 95gaagttgatg aaccggtcct
ttacagatgt 309626DNAArtificial
SequenceGRHPR-2264-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(21)..(21)RNAmisc_feature(26)..(26)3'
propanediol (C3) spacer blocking group 96acaagaggat cctggatgct
gcagga 269723DNAArtificial
SequenceGRHPR-2264-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(18)..(18)RNAmisc_feature(23)..(23)3'
propanediol (C3) spacer blocking group 97cgctctagct ccttggcagg gaa
239827DNAArtificial SequenceSerpina 279-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 98actcagttcc acaggtggga
gggaggc 279927DNAArtificial SequenceSerpina
279-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 99cactctaagc cctgctgtcc
cacctga 2710027DNAArtificial SequenceMyc
459-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 100cgggaggcta ttctgcccat
tugggat 2710124DNAArtificial SequenceMyc
459-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(19)..(19)RNAmisc_feature(24)..(24)3'
propanediol (C3) spacer blocking group 101cggggaagtg tccccaaaug
ggct 2410223DNAArtificial SequenceSerpina
130-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(18)..(18)RNAmisc_feature(23)..(23)3'
propanediol (C3) spacer blocking group 102gctgctgctg ccaggaautc cac
2310324DNAArtificial SequenceSerpina 130-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(19)..(19)RNAmisc_feature(24)..(24)3'
propanediol (C3) spacer blocking group 103cccctccaac ctggaattcc
tggg 2410423DNAArtificial SequenceMyc 490-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(18)..(18)RNAmisc_feature(23)..(23)3'
propanediol (C3) spacer blocking group 104ctgccaggac ccgcttcuct gat
2310526DNAArtificial SequenceMyc 490-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(21)..(21)RNAmisc_feature(26)..(26)3'
propanediol (C3) spacer blocking group 105caaggagagc ctttcagaga
agcggc 2610623DNAArtificial
SequenceGRHPR-2179-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(18)..(18)RNAmisc_feature(23)..(23)3'
propanediol (C3) spacer blocking group 106gatgagccca tccctgccaa ggt
2310723DNAArtificial SequenceGRHPR-2179-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(18)..(18)RNAmisc_feature(23)..(23)3'
propanediol (C3) spacer blocking group 107cgctctagct ccttggcagg gaa
2310821DNAArtificial SequenceGYG-2851-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(16)..(16)RNAmisc_feature(21)..(21)3'
propanediol (C3) spacer blocking group 108tcgccacccc tcagguctca c
2110926DNAArtificial SequenceGYG-2851-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(21)..(21)RNAmisc_feature(26)..(26)3'
propanediol (C3) spacer blocking group 109acctcatgga gtctgagacc
ugaggc 2611021DNAArtificial
SequenceGYG-2793-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(16)..(16)RNAmisc_feature(21)..(21)3'
propanediol (C3) spacer blocking group 110gccctggtcc tgggaucatc a
2111128DNAArtificial SequenceGYG-2793-2misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
propanediol (C3) spacer blocking group 111ctgtgctgtt tcagagatga
tcccaggt 2811228DNAArtificial SequenceSerpina
79-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
propanediol (C3) spacer blocking group 112caagagtcct gagctgaacc
aagaaggt 2811325DNAArtificial SequenceSerpina
79-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(20)..(20)RNAmisc_feature(25)..(25)3'
propanediol (C3) spacer blocking group 113cgaccccctc ctccttcttg
gttct 2511428DNAArtificial SequenceMyc 538-1misc_feature(1)..(1)5'
universal domain
(UniFor-UniPro-)misc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
propanediol (C3) spacer blocking group 114ctgcttagac gctggatttt
ttucggga 2811527DNAArtificial SequenceMyc
538-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(22)..(22)RNAmisc_feature(27)..(27)3'
propanediol (C3) spacer blocking group 115ctggttttcc actacccgaa
araaaaa 2711631DNAArtificial
SequenceGYG-2744-1misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(26)..(26)RNAmisc_feature(31)..(31)3'
propanediol (C3) spacer blocking group 116ctttgtatta agatcaggcc
tttgtgacac a 3111728DNAArtificial
SequenceGYG-2744-2misc_feature(1)..(1)5' universal domain
(UniFor-UniPro-)misc_feature(23)..(23)RNAmisc_feature(28)..(28)3'
propanediol (C3) spacer blocking group 117catcgtttgt ggttagtgtc
acaaaggg 2811819DNAArtificial SequenceRNase P For 118gcggagggaa
gctcatcag 1911922DNAArtificial SequenceRNase P Rev 119ccctagtctc
agaccttccc aa 2212024DNAArtificial SequenceRNase P
probemisc_feature(1)..(1)5' 6-Fluorescein fluorescent dye
(FAM)misc_feature(24)..(24)3' Iowa Black fluorescent quencher
(IBFQ) 120ccacgagctg agtgcgtcct gtca 2412122DNAArtificial
SequenceGCK-356-1 121gaggaaactg tgactgaacc tc 2212216DNAArtificial
SequenceGCK-356-2 122ccaaggcttc tccgcc 1612317DNAArtificial
SequenceERBB3-33-1 123gagtccgggg agggatg 1712419DNAArtificial
SequenceERBB3-33-2 124caatccctac tccagcctc 1912521DNAArtificial
SequenceTTR-1257-1 125atgtgagcct ctctctacca a 2112623DNAArtificial
SequenceTTR-1257-2 126gtcctctgat ggtcaaagtt cta
2312716DNAArtificial SequenceHAMP-253-1 127cactggtcag gctggc
1612818DNAArtificial SequenceHAMP-253-2 128caagctcaag acccagca
1812925DNAArtificial SequenceBIRC5-606-1 129caactcaaat cttttgacaa
ctcag 2513016DNAArtificial SequenceBIRC5-606-2 130ggagctggaa ggctgg
1613123DNAArtificial SequenceSAA 146-1 131ttcagaatgg tatggctgta tgc
2313220DNAArtificial SequenceSAA 146-2 132cacagatcag gtgaggagca
2013319DNAArtificial SequenceIDO1-97-1 133gttttccata gcgtgtgcc
1913417DNAArtificial SequenceIDO1-97-2 134gtggtcactg gctgtgg
1713523DNAArtificial SequenceIDO1-176-1 135ttcccacatt ttactgcctt
ctc 2313620DNAArtificial SequenceIDO1-176-2 136cgctatggaa
aactcctgga 2013718DNAArtificial SequenceCYP27A-31016-1
137caggtctgtg catcagcg 1813820DNAArtificial SequenceCYP27A-31016-2
138ctttctggaa gcgatacctg 2013920DNAArtificial SequenceSAA 226-1
139cgcacagaac tcaacatggg
2014024DNAArtificial SequenceSAA 226-2 140aatagttatc ctggggcata
cagc 2414118DNAArtificial SequenceKIF11-369-1 141gctcggaatc
ctgtcagc 1814218DNAArtificial SequenceKIF11-369-2 142cagccaaatt
cgtctgcg 1814322DNAArtificial SequenceC3-1394-1 143gggatgttcc
agtcactgtt ac 2214415DNAArtificial SequenceC3-1394-2 144ggttggtggc
agggg 1514516DNAArtificial SequenceHOGA-505-1 145aggggaaggt gcccag
1614617DNAArtificial SequenceHOGA-505-2 146aaggtggaca ttgcggg
1714723DNAArtificial SequenceEGFR-123344-1 147tcataattcc tctgcacata
ggt 2314818DNAArtificial SequenceEGFR-123344-2 148gccaaggcac
gagtaaca 1814928DNAArtificial SequenceALDH2-15144-1 149cgtataaaat
agaagacgaa tccatccc 2815015DNAArtificial SequenceALDH2-15144-2
150atggcacgat gccgt 1515115DNAArtificial SequenceAGXT-140-1
151ggcttgagca gggcc 1515215DNAArtificial SequenceAGXT-140-2
152tggccaaggc cagtg 1515315DNAArtificial SequenceAPOC-2929-1
153tcaggcagcc acggc 1515418DNAArtificial SequenceAPOC-2929-2
154gtgaccgatg gcttcagt 1815527DNAArtificial SequenceMet 27554-1
155catacgcagc ctgaagtata ttaaaca 2715626DNAArtificial SequenceMet
27554-2 156tagatgctca gacttttcac acaaga 2615723DNAArtificial
SequenceSAA 88-1 157ttcagaatgg tatggctgta tgc 2315825DNAArtificial
SequenceSAA 88-2 158agcagggaag gctcagtata aatag
2515916DNAArtificial SequenceHIF1A-293-1 159taagcgctgg ctccct
1616020DNAArtificial SequenceHIF1A-293-2 160ctctagtctc acgaggggtt
2016126DNAArtificial SequenceMet 27475-1 161ctcttttctg tgagaataca
ctccag 2616224DNAArtificial SequenceMet 27475-2 162taccccatta
agtatgtcca tgcc 2416317DNAArtificial SequenceHAMP-295-1
163tctcccatcc ctgctgc 1716415DNAArtificial SequenceHAMP-295-2
164ccgcttgcct cctgc 1516516DNAArtificial SequenceGRHPR-2234-1
165cacccagtgt gcacct 1616618DNAArtificial SequenceGRHPR-2234-2
166gccaaggagc tagagcga 1816716DNAArtificial SequenceHAMP-88-1
167gaggcggtgg tctgag 1616817DNAArtificial SequenceHAMP-88-2
168tgttccctgt cgctctg 1716927DNAArtificial SequenceMet 27254-1
169ctttagcctt ctcactgata tcgaatg 2717025DNAArtificial SequenceMet
27254-2 170gcatattctc cccacagata gaaga 2517115DNAArtificial
SequenceGRHPR-2264-1 171cctgcccacc cagtg 1517218DNAArtificial
SequenceGRHPR-2264-2 172ctgtgaggtg gagcagtg 1817321DNAArtificial
SequenceSerpina 279-1 173tagctcctgg gcatttcttc c
2117421DNAArtificial SequenceSerpina 279-2 174agcttgagga gagcaggaaa
g 2117520DNAArtificial SequenceMyc 459-1 175cctggttttc cactacccga
2017621DNAArtificial SequenceMyc 459-2 176cactggaact tacaacaccc g
2117721DNAArtificial SequenceSerpina 130-1 177ttcctgctct cctcaagctc
t 2117821DNAArtificial SequenceSerpina 130-2 178gagctgaacc
aagaaggagg a 2117921DNAArtificial SequenceMyc 490-1 179aggcattcga
ctcatctcag c 2118021DNAArtificial SequenceMyc 490-2 180tgcactggaa
cttacaacac c 2118117DNAArtificial SequenceGRHPR-2179-1
181tcggagagga ggcagag 1718217DNAArtificial SequenceGRHPR-2179-2
182ttctcctgag ggcctcc 1718321DNAArtificial SequenceGYG-2851-1
183acagggagaa ggatgtcaga g 2118422DNAArtificial SequenceGYG-2851-2
184gtcctgggat catctctgaa ac 2218521DNAArtificial SequenceGYG-2793-1
185acagggagaa ggatgtcaga g 2118621DNAArtificial SequenceGYG-2793-2
186cactaaccac aaacgatgcc t 2118718DNAArtificial SequenceSerpina
79-1 187gaattcctgg cagcagca 1818818DNAArtificial SequenceSerpina
79-2 188ctactgcctc cacccgaa 1818922DNAArtificial SequenceMyc 538-1
189taggcattcg actcatctca gc 2219021DNAArtificial SequenceMyc 538-2
190tgcactggaa cttacaacac c 2119117DNAArtificial SequenceGYG-2744-1
191ggaccagggc acctttg 1719223DNAArtificial SequenceGYG-2744-2
192ggctttctcc agataagata ctg 2319353DNAArtificial SequenceSynthetic
193tgcaggtgca gatgaagctg gtcgtgcccc agttattggt ccgctggtta ttg
5319453DNAArtificial SequenceSynthetic 194caataaccag cggaccaata
actggggcac gaccagcttc atctgcacct gca 5319553DNAArtificial
SequenceSynthetic 195aggtgcagat gaagctggtc gtggtacggt tattggtccg
ctggttattg ttg 5319653DNAArtificial SequenceSynthetic 196caacaataac
cagcggacca ataaccgtac cacgaccagc ttcatctgca cct
53197224PRTPyrococcus abyssi 197Met Lys Val Ala Gly Ala Asp Glu Ala
Gly Arg Gly Pro Val Ile Gly1 5 10 15Pro Leu Val Ile Val Ala Ala Val
Val Glu Glu Asp Lys Ile Arg Ser 20 25 30Leu Thr Lys Leu Gly Val Lys
Asp Ser Lys Gln Leu Thr Pro Ala Gln 35 40 45Arg Glu Lys Leu Phe Asp
Glu Ile Val Lys Val Leu Asp Asp Tyr Ser 50 55 60Val Val Ile Val Ser
Pro Gln Asp Ile Asp Gly Arg Lys Gly Ser Met65 70 75 80Asn Glu Leu
Glu Val Glu Asn Phe Val Lys Ala Leu Asn Ser Leu Lys 85 90 95Val Lys
Pro Glu Val Ile Tyr Ile Asp Ser Ala Asp Val Lys Ala Glu 100 105
110Arg Phe Ala Glu Asn Ile Arg Ser Arg Leu Ala Tyr Glu Ala Lys Val
115 120 125Val Ala Glu His Lys Ala Asp Ala Lys Tyr Glu Ile Val Ser
Ala Ala 130 135 140Ser Ile Leu Ala Lys Val Ile Arg Asp Arg Glu Ile
Glu Lys Leu Lys145 150 155 160Ala Glu Tyr Gly Asp Phe Gly Ser Gly
Tyr Pro Ser Asp Pro Arg Thr 165 170 175Lys Lys Trp Leu Glu Glu Trp
Tyr Ser Lys His Gly Asn Phe Pro Pro 180 185 190Ile Val Arg Arg Thr
Trp Asp Thr Ala Lys Lys Ile Glu Glu Lys Phe 195 200 205Lys Arg Ala
Gln Leu Thr Leu Asp Asn Phe Leu Lys Arg Phe Arg Asn 210 215
220198224PRTArtificial SequenceSynthetic 198Met Lys Val Ala Gly Ala
Asp Glu Ala Gly Arg Ala Pro Val Ile Gly1 5 10 15Pro Leu Val Ile Val
Ala Ala Val Val Glu Glu Asp Lys Ile Arg Ser 20 25 30Leu Thr Lys Leu
Gly Val Lys Asp Ser Lys Gln Leu Thr Pro Ala Gln 35 40 45Arg Glu Lys
Leu Phe Asp Glu Ile Val Lys Val Leu Asp Asp Tyr Ser 50 55 60Val Val
Ile Val Ser Pro Gln Asp Ile Asp Gly Arg Lys Gly Ser Met65 70 75
80Asn Glu Leu Glu Val Glu Asn Phe Val Lys Ala Leu Asn Ser Leu Lys
85 90 95Val Lys Pro Glu Val Ile Tyr Ile Asp Ser Ala Asp Val Lys Ala
Glu 100 105 110Arg Phe Ala Glu Asn Ile Arg Ser Arg Leu Ala Tyr Glu
Ala Lys Val 115 120 125Val Ala Glu His Lys Ala Asp Ala Lys Tyr Glu
Ile Val Ser Ala Ala 130 135 140Ser Ile Leu Ala Lys Val Ile Arg Asp
Arg Glu Ile Glu Lys Leu Lys145 150 155 160Ala Glu Tyr Gly Asp Phe
Gly Ser Gly Tyr Pro Ser Asp Pro Arg Thr 165 170 175Lys Lys Trp Leu
Glu Glu Trp Tyr Ser Lys His Gly Asn Phe Pro Pro 180 185 190Ile Val
Arg Arg Thr Trp Asp Thr Ala Lys Lys Ile Glu Glu Lys Phe 195 200
205Lys Arg Ala Gln Leu Thr Leu Asp Asn Phe Leu Lys Arg Phe Arg Asn
210 215 220199224PRTArtificial SequenceSynthetic 199Met Lys Val Ala
Gly Ala Asp Glu Ala Gly Arg Gly Thr Val Ile Gly1 5 10 15Pro Leu Val
Ile Val Ala Ala Val Val Glu Glu Asp Lys Ile Arg Ser 20 25 30Leu Thr
Lys Leu Gly Val Lys Asp Ser Lys Gln Leu Thr Pro Ala Gln 35 40 45Arg
Glu Lys Leu Phe Asp Glu Ile Val Lys Val Leu Asp Asp Tyr Ser 50 55
60Val Val Ile Val Ser Pro Gln Asp Ile Asp Gly Arg Lys Gly Ser Met65
70 75 80Asn Glu Leu Glu Val Glu Asn Phe Val Lys Ala Leu Asn Ser Leu
Lys 85 90 95Val Lys Pro Glu Val Ile Tyr Ile Asp Ser Ala Asp Val Lys
Ala Glu 100 105 110Arg Phe Ala Glu Asn Ile Arg Ser Arg Leu Ala Tyr
Glu Ala Lys Val 115 120 125Val Ala Glu His Lys Ala Asp Ala Lys Tyr
Glu Ile Val Ser Ala Ala 130 135 140Ser Ile Leu Ala Lys Val Ile Arg
Asp Arg Glu Ile Glu Lys Leu Lys145 150 155 160Ala Glu Tyr Gly Asp
Phe Gly Ser Gly Tyr Pro Ser Asp Pro Arg Thr 165 170 175Lys Lys Trp
Leu Glu Glu Trp Tyr Ser Lys His Gly Asn Phe Pro Pro 180 185 190Ile
Val Arg Arg Thr Trp Asp Thr Ala Lys Lys Ile Glu Glu Lys Phe 195 200
205Lys Arg Ala Gln Leu Thr Leu Asp Asn Phe Leu Lys Arg Phe Arg Asn
210 215 22020022DNAArtificial SequenceSynthetic 200cagcctcatc
caaaagagga aa 2220121DNAArtificial SequenceSynthetic 201ctcactctaa
accccagcat t 2120227DNAArtificial
SequenceSyntheticmisc_feature(23)..(23)RNA
basemisc_feature(27)..(27)3' C3 spacer (propanediol) blocker group
202cagcctcatc caaaagagga aacagga 2720327DNAArtificial
SequenceSyntheticmisc_feature(23)..(23)RNA
basemisc_feature(27)..(27)3' C3 spacer (propanediol) blocker group
203cagcctcatc caaaagagga aauagga 2720422DNAArtificial
SequenceSynthetic 204gtgattttgg tctagctaca gt 2220523DNAArtificial
SequenceSynthetic 205cctcaattct taccatccac aaa 2320628DNAArtificial
SequenceSyntheticmisc_feature(23)..(23)RNA
basemisc_feature(28)..(28)3' C3 spacer (propanediol) blocker group
206gtgattttgg tctagctaca gtgaaatg 2820728DNAArtificial
SequenceSyntheticmisc_feature(23)..(23)RNA
basemisc_feature(28)..(28)3' C3 spacer (propanediol) blocker group
207gtgattttgg tctagctaca gagaaatg 28208401DNAArtificial
SequenceSynthetic 208taagaggaaa gatgaagtac tatgttttaa agaatattat
attacagaat tatagaaatt 60agatctctta cctaaactct tcataatgct tgctctgata
ggaaaatgag atctactgtt 120ttcctttact tactacacct cagatatatt
tcttcatgaa gacctcacag taaaaatagg 180tgattttggt ctagctacag
tgaaatctcg atggagtggg tcccatcagt ttgaacagtt 240gtctggatcc
attttgtgga tggtaagaat tgaggctatt tttccactga ttaaattttt
300ggccctgaga tgctgctgag ttactagaaa gtcattgaag gtctcaacta
tagtattttc 360atagttccca gtattcacaa aaatcagtgt tcttattttt t
401209401DNAArtificial SequenceSynthetic 209taagaggaaa gatgaagtac
tatgttttaa agaatattat attacagaat tatagaaatt 60agatctctta cctaaactct
tcataatgct tgctctgata ggaaaatgag atctactgtt 120ttcctttact
tactacacct cagatatatt tcttcatgaa gacctcacag taaaaatagg
180tgattttggt ctagctacag agaaatctcg atggagtggg tcccatcagt
ttgaacagtt 240gtctggatcc attttgtgga tggtaagaat tgaggctatt
tttccactga ttaaattttt 300ggccctgaga tgctgctgag ttactagaaa
gtcattgaag gtctcaacta tagtattttc 360atagttccca gtattcacaa
aaatcagtgt tcttattttt t 40121026DNAArtificial
SequenceSyntheticmisc_feature(21)..(21)RNA
basemisc_feature(26)..(26)3' C3 spacer (propanediol) blocker group
210gtgattttgg tctagctaca gtaaaa 2621126DNAArtificial
SequenceSyntheticmisc_feature(21)..(21)RNA
basemisc_feature(26)..(26)3' C3 spacer (propanediol) blocker group
211gtgattttgg tctagctaca gaaaaa 2621253DNAArtificial
SequenceSynthetic 212agccgaatac ggtgattttg gttccgcata cccgtctgat
ccgcgtacta aga 5321353DNAArtificial SequenceSynthetic 213tcttagtacg
cggatcagac gggtatgcgg aaccaaaatc accgtattcg gct
53214224PRTArtificial SequenceSynthetic 214Met Lys Val Ala Gly Ala
Asp Glu Ala Gly Arg Gly Pro Val Ile Gly1 5 10 15Pro Leu Val Ile Val
Ala Ala Val Val Glu Glu Asp Lys Ile Arg Ser 20 25 30Leu Thr Lys Leu
Gly Val Lys Asp Ser Lys Gln Leu Thr Pro Ala Gln 35 40 45Arg Glu Lys
Leu Phe Asp Glu Ile Val Lys Val Leu Asp Asp Tyr Ser 50 55 60Val Val
Ile Val Ser Pro Gln Asp Ile Asp Gly Arg Lys Gly Ser Met65 70 75
80Asn Glu Leu Glu Val Glu Asn Phe Val Lys Ala Leu Asn Ser Leu Lys
85 90 95Val Lys Pro Glu Val Ile Tyr Ile Asp Ser Ala Asp Val Lys Ala
Glu 100 105 110Arg Phe Ala Glu Asn Ile Arg Ser Arg Leu Ala Tyr Glu
Ala Lys Val 115 120 125Val Ala Glu His Lys Ala Asp Ala Lys Tyr Glu
Ile Val Ser Ala Ala 130 135 140Ser Ile Leu Ala Lys Val Ile Arg Asp
Arg Glu Ile Glu Lys Leu Lys145 150 155 160Ala Glu Tyr Gly Asp Phe
Gly Ser Ala Tyr Pro Ser Asp Pro Arg Thr 165 170 175Lys Lys Trp Leu
Glu Glu Trp Tyr Ser Lys His Gly Asn Phe Pro Pro 180 185 190Ile Val
Arg Arg Thr Trp Asp Thr Ala Lys Lys Ile Glu Glu Lys Phe 195 200
205Lys Arg Ala Gln Leu Thr Leu Asp Asn Phe Leu Lys Arg Phe Arg Asn
210 215 22021522DNAArtificial SequenceSynthetic 215cagcctcatc
caaaagagga aa 2221621DNAArtificial SequenceSynthetic 216ctcactctaa
accccagcat t 2121727DNAArtificial
SequenceSyntheticmisc_feature(23)..(23)RNA
basemisc_feature(27)..(27)3' C3 spacer (propanediol) blocker group
217cagcctcatc caaaagagga aacagga 2721827DNAArtificial
SequenceSyntheticmisc_feature(23)..(23)RNA
basemisc_feature(27)..(27)3' C3 spacer (propanediol) blocker group
218cagcctcatc caaaagagga aauagga 27
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