U.S. patent application number 15/053170 was filed with the patent office on 2016-06-16 for endonuclease-enhanced helicase-dependent amplification.
The applicant listed for this patent is BioHelix Corporation. Invention is credited to Huimin Kong, Yanhong Tong.
Application Number | 20160168630 15/053170 |
Document ID | / |
Family ID | 42542368 |
Filed Date | 2016-06-16 |
United States Patent
Application |
20160168630 |
Kind Code |
A1 |
Kong; Huimin ; et
al. |
June 16, 2016 |
ENDONUCLEASE-ENHANCED HELICASE-DEPENDENT AMPLIFICATION
Abstract
The invention provides methods and compositions for enhancing
the speed and sensitivity of helicase-dependent amplification
through the use of an endonuclease.
Inventors: |
Kong; Huimin; (Wenham,
MA) ; Tong; Yanhong; (Boxford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioHelix Corporation |
Beverly |
MA |
US |
|
|
Family ID: |
42542368 |
Appl. No.: |
15/053170 |
Filed: |
February 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14197130 |
Mar 4, 2014 |
9303288 |
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15053170 |
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13146602 |
Oct 7, 2011 |
8685648 |
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PCT/US2010/023089 |
Feb 3, 2010 |
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14197130 |
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61149390 |
Feb 3, 2009 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 1/6844 20130101;
C12P 19/34 20130101; C12Q 1/6844 20130101; C12Q 2521/531 20130101;
C12Q 2521/301 20130101; C12Q 1/686 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A kit for amplifying a target nucleic acid sequence in a sample,
comprising a type II restriction endonuclease, a helicase, a DNA
polymerase, and at least a first primer and a second primer,
wherein the sample may or may not contain a DNA molecule which
comprises the target nucleic acid; wherein the target nucleic acid
is one which can hybridize to at least the first and second primer
and is amplified in the presence of the composition to generate an
amplification product wherein the 5' end of the amplification
product is defined by the 5' end of the first primer and the 3' end
of the amplification product is defined by the 5' end of the second
primer; and wherein the type II restriction endonuclease is one
which cannot recognize or cleave the amplification product.
2. The kit according to claim 1, wherein the type II restriction
endonuclease is one which cleaves the target DNA within 5000
nucleotides of the target nucleic acid.
3. The kit according to claim 1, wherein the type II restriction
endonuclease is one which can recognize and cleave the DNA within
500 nucleotides of the target nucleic acid.
4. The kit according to claim 1, wherein the helicase is a
thermostable helicase.
5. The kit according to claim 1, further comprising a buffer.
6. The kit according to claim 1, further comprising
deoxynucleotides.
7. The kit according to claim 1, further comprising magnesium.
8. A method for detecting a DNA molecule comprising a target
nucleic acid in a sample, the method comprising (a) treating the
DNA molecule with a restriction endonuclease; (b) incubating the
DNA molecule with at least a first primer and a second primer and
amplifying the nucleic acid in a helicase-dependent amplification
reaction to generate an amplified product, wherein the amplified
product is not cleaved by the restriction endonuclease.
9. The method of claim 8, wherein step (a) is performed prior to
step (b).
10. The method of claim 8, wherein step (a) and step (b) are
performed simultaneously.
11. The method of claim 8, wherein the restriction endonuclease is
a type II restriction endonuclease.
12. The method of claim 8, wherein the restriction endonuclease is
a type IIs restriction endonuclease.
13. The method of claim 8, wherein the restriction endonuclease
cleaves the nucleic acid within 5000 nucleotides of a sequence that
hybridizes to the first and/or second primer.
14. The method of claim 8, wherein the restriction endonuclease
cleaves the nucleic acid within 500 nucleotides of a sequence that
hybridizes to the first and/or second primer.
15. The method of claim 8, wherein the helicase-dependent
amplification reaction comprises a helicase, a DNA polymerase, a
buffer, and the at least first and second primers.
16. The method of claim 15, wherein the helicase-dependent
amplification reaction further comprises magnesium.
17. The method of claim 15, wherein the helicase-dependent
amplification reaction further comprises deoxynucleotides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/197,130, filed Mar. 4, 2014, which is a continuation of U.S.
application Ser. No. 13/146,602, filed Oct. 7, 2011, now U.S. Pat.
No. 8,685,648, which is a National Stage filing under 35 U.S.C.
.sctn.371 of PCT/US2010/023089, filed Feb. 3, 2010, and Publication
No. WO/2010/091111, published Aug. 12, 2010, which claims the
benefit of U.S. Application No. 61/149,390, filed Feb. 3, 2009.
CROSS-REFERENCE TO A SEQUENCE LISTING
[0002] A Sequence Listing is being submitted electronically via EFS
in the form of a text file, created Feb. 25, 2016, and named
0418960903_SequenceListing.txt (750 bytes), the contents of which
are incorporated herein by reference in their entirety.
FIELD
[0003] The present invention relates to methods and compositions to
improve the speed and sensitivity of helicase-dependent
amplification.
BACKGROUND
[0004] Nucleic acid amplification has been widely used for research
and molecular diagnostics. PCR (polymerase chain reaction) is a
traditional method for nucleic acid amplification which requires
thermocycling. Heat denaturation during PCR thermocycling is a
powerful and important step to uniformly separate DNA duplexes,
generating single-stranded templates for subsequent primer
annealing. Several platforms exist for isothermal amplification,
including SDA (strand displacement amplification), TMA
(transcription-mediated amplification), RCA (rolling cycle
amplification), LAMP (loop-mediated amplification), NASBA (nucleic
acid sequence-based amplification), and HDA (helicase-dependent
amplification). HDA is different from other isothermal technologies
because the HDA technology employs a helicase to separate the
double-stranded nucleic acid. It is desirable to improve the speed
and sensitivity of nucleic acid amplification technologies with
high speed and high sensitivity, particularly for use in research
and diagnostic applications.
[0005] Several methods have been developed to improve speed and
sensitivity for PCR. Some of the methods that can improve PCR
performance have also been successful for improving HDA
performance. However, HDA is different from PCR, as HDA can be
performed isothermally and relies on a helicase to separate the
strands of double-stranded nucleic acids. Several specific methods
have been applied to improve HDA. For example, previous studies
have found that the protein concentrations of DNA polymerase and
helicase can be optimized to improve the performance of HDA.
Although increasing amounts of enzymes (helicases and DNA
polymerases) can increase detection speed, the Limit-of-Detection
(LoD), the minimum number of target nucleic acids required for
reliable detection, generally remains unchanged at, for example,
5-50 copies.
[0006] HDA uses helicase(s) to separate a DNA duplex. Helicase is
not a sequence-specific protein and therefore does not specifically
recognize a target region. The efficiency of the helicase
separating the double-stranded nucleic acid, especially in the
target region specified by forward and reverse primers, is
therefore a rate-limiting factor. Based on the unwinding direction,
helicases can be grouped into two major types: the 3'-5' helicases
and the 5'-3' helicases. The well-studied E. coli UvrD helicase
unwinds DNA in a 3' to 5' direction (Matson, S. W. J. Biol. Chem.,
261, 10169-10175. (1986)). Some helicases can unwind duplex nucleic
acid from blunt ends; advantages of using such helicases are
described in U.S. Pat. No. 7,282,328. Other helicases require a
single-stranded "tail" on the substrate duplex, generally a 3' tail
for 3'-5' helicases and a 5' tail for the 5-3' enzymes.
SUMMARY OF THE INVENTION
[0007] The present invention is based, in part, on the discovery
that the speed and sensitivity of helicase-dependent amplification
can be improved through the use of an endonuclease, such as a
restriction endonuclease. The extraction and purification of
nucleic acid substrates typically shears or nicks them, generating
randomly-distributed accessible ends for a helicase to load onto
the duplex. Perhaps as a result, the art has not previously
appreciated any need for or benefit from introducing additional
free ends in a target for helicase-dependent amplification.
Surprisingly, the inventors have now discovered that treating the
target nucleic acid with an endonuclease can significantly improve
both the sensitivity and speed of helicase-dependent
amplification.
[0008] Accordingly, in one aspect the invention provides an
improved composition for helicase-dependent amplification. The
composition includes a helicase, a DNA polymerase, at least one
primer, and an endonuclease. The endonuclease can be a restriction
endonuclease, such as a Type II or Type IIs restriction
endonuclease. Exemplary endonucleases include Avail, BanII, AseI,
SspI, SpeI, MboI, MboII, SwaI, BsrI, BtsCI, CviQI, CviAII, Tsp45I,
Fnu4HI, Ms1I, MspA1I, SacII, AgeI, DraI, and XmnI. The helicase is
preferably thermostable. The composition can include additional
components, such as deoxynucleotides, buffer, magnesium, sodium,
one or more additional primers, and/or single stranded binding
proteins, for example.
[0009] The invention also provides methods for assaying a sample by
combining a composition like those described above with a sample
containing or potentially containing a nucleic acid that hybridizes
to one or more primers in the composition. In this way, the
presence or absence of the target nucleic acid can be detected, the
amount of a target nucleic acid can be measured, or a target
nucleic acid known to be present can be amplified to facilitate its
subsequent characterization (e.g. determining the presence or
absence of a genetic variation) or other use. For example, the
sample can be a biological sample from a subject and can include
human tissue, cells or body fluid (e.g. blood, serum, plasma,
lymph, urine, sweat, tears, semen, vaginal fluid, nipple aspirate,
sputum, ejaculate, saliva, bronchial lavage, pleural effusion,
peritoneal fluid, amniotic fluid, glandular fluid, fine needle
aspirates, spinal fluid, conjunctival fluid, or cerebrospinal
fluid), and the target nucleic acid can be a human nucleic acid or
a foreign nucleic acid (e.g. from a virus, bacterium, fungus,
parasite, etc.). Alternatively, the sample could be an
environmental, animal or industrial sample. In one embodiment, the
sample contains no more than 1 ng of DNA (e.g. no more than 0.1 ng
or no more than 0.01 ng). In some embodiments, the sample contains
no more than about 300 copies of the target nucleic acid (e.g. no
more than about 30 copies or no more than about 3 copies.).
[0010] The invention also provides methods for amplifying a nucleic
acid by treating the nucleic acid with an endonuclease and
amplifying the treated nucleic acid in a helicase-dependent
amplification reaction. The amplification can follow the treatment
step, or the two processes can be concurrent. The endonuclease can
be a restriction endonuclease such as a Type II or a Type IIs
restriction endonuclease. Exemplary endonucleases include Avail,
BanII, AscII, SspII, SpeII, MboII, MboIIII, SwaII, BsrII, BtsCII,
CviQII, CviAIIII, Tsp451, Fnu4HI, Ms1I, MspA1I, SacII, AgeI, DraI,
and XmnI. In one embodiment, the helicase-dependent amplification
reaction includes a helicase (preferably thermostable), a DNA
polymerase, a buffer, deoxynucleotides, magnesium, and at least one
primer.
[0011] In each of the various methods of practicing the invention,
the endonuclease preferably, but not necessarily, cleaves the
nucleic acid within 5 kb (and perhaps within 500 bp) of the
nucleotide sequence in the nucleic acid that hybridizes to the
primer (i.e. the primer binding site).
[0012] Additional aspects and advantages of the invention will be
apparent from the following description of certain particular
embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 and FIG. 2 are graphs showing the effects of
restriction endonuclease SspI on helicase-dependent amplification
from 50,000 copies of plasmid DNA (FIG. 1) or from 20 ng human
genomic DNA (FIG. 2) for a one hour amplification at 63.5.degree.
C. FIG. 1 shows amplification from 50,000 copies plasmid DNA. Ct
(the threshold cycle, defined as the cycle number at which the
fluorescence level has exceeded the detection threshold) is 14 for
the assays without SspI, but is reduced to around 11.5 for the
assays with SspI. FIG. 2 shows amplification from 20 ng human
genomic DNA; Ct is around 14 for the assays with or without
SspI.
[0014] FIG. 3 and FIG. 4 are graphs showing the effects of
restriction endonucleases SpeI (FIG. 3) and BanII (FIG. 4)
(generating 5' or 3' ends after cleavage, respectively) on
helicase-dependent amplification from 50,000 copies of plasmid DNA
with a one hour amplification at 63.5.degree. C. FIG. 3 shows the
effects of SpeI. Ct is 14 for the assays without SspI, and is
reduces to around 11.5 for the assays with SspI. FIG. 4 shows the
effects of BanII: Ct is 13 for the assays without BanII, and is
reduced to about 11 with BanII.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0015] The invention improves the sensitivity of, and accelerates,
helicase-dependent amplification through the use of endonucleases.
Restriction endonucleases, nicking endonucleases, and homing
endonucleases recognize and cleave specific nucleic acid sequences,
generating 5'ss ends, 3'ss ends or blunt ends. More than 3,000
restriction endonucleases with over two hundred different
specificities have been isolated from bacteria (Roberts and
Macelis, Nucleic Acids Res. 26:338-350 (1998)). Type II restriction
endonucleases are the most commonly used restriction enzymes for
molecular biology applications: they cut DNA at defined positions
close to or within their recognition sequences. They produce
discrete restriction fragments and distinct gel banding patterns
and are the only class of restriction enzyme commonly used in the
laboratory for DNA analysis and gene cloning. Examples of type II
enzymes include EcoRI, HindIII, SspI, and BanII, each cleaving DNA
within its recognition sequence. The next most common Type II
enzymes, usually referred to as `type IIs," are those such as FokI
and A1wI that cleave outside of their recognition sequence to one
side. During an HDA reaction, the selected restriction
endonucleases generate break points (or ends) of the nucleic acid
substrates that are near a target region. These resulting ends
recruit helicases to the adjacent target region and thus increase
the chance or frequency of the helicase unwinding a target
region.
[0016] In an embodiment, an endonuclease is used prior to an HDA
reaction (separate from the HDA reaction, in a two-step format). In
such a two-step format, restriction endonucleases digest the DNA
targets first at the optimal working temperature, typically in an
HDA reaction buffer or in a buffer that does not inhibit the
following HDA reaction. The digested nucleic acid substrates can
then be used in the subsequent HDA reaction. In another embodiment,
the endonuclease is used together with an HDA enzyme mix (in a
one-step format, generally including a helicase, a DNA polymerase,
and optionally single-stranded DNA binding proteins). In such a
one-step format, the reaction speed of the endonucleases should be
fast enough to generate a certain number of specific ends during
the time of reaction set-up (including, if applicable, the time of
adding the master reaction mix to each sample in the individual
tube, mixing, and bringing the sample to an incubator which has
already been set to the HDA reaction temperature). In general, the
set-up time may be in the range of about 2 to about 15 minutes,
depending on how many samples are to be tested. As an illustrative
example, the "time-saver qualified enzymes" from New England
BioLabs Inc, (NEB, Ipswich, Mass.) provide restriction
endonucleases with fast reaction speeds. This group of fast
endonucleases digest about 1 .mu.g of DNA in about 5 minutes using
about 1 .mu.l of enzyme under recommended conditions. The one-step
format is easier to perform as there is no additional step required
to add the restriction endonucleases. On the other hand, fewer
restriction endonucleases are as suitable for the one-step format
because it requires fast speed and high digestion efficiency. In
general, an enzyme that is suitable for the one-step format can
always be used in a two-step reaction.
[0017] Several preferred restriction endonucleases for use in the
present invention arc selected on the basis of one or more the
following factor(s): [0018] Fast reaction speed. If the
endonucleases are used together with an HDA enzyme mix, reaction
speed is an important factor in the improvement in amplification.
The "time-saver qualified enzymes" from NEB are good choices, for
example. However, if the endonucleases are used separately in a
two-step reaction, reaction speed need not be an issue. [0019] The
buffer for endonuclease cleavage is preferably compatible with HDA
reaction buffer: the enzyme's activity is not inhibited by the
reaction buffer used for HDA. [0020] The endonuclease does not or
is not known to recognize, bind or cleave within the target
sequence to be amplified. If the target sequence is cleaved by a
restriction enzyme, the truncated target region can no longer be
used as a template for amplification. [0021] The endonuclease does
not recognize, bind or cut the corresponding internal control
sequence, if an internal control is included in the HDA reaction.
[0022] Proximity of the cleavage site to the target region.
Ideally, the cleavage site can be within 5,000 bp of the target
region. More preferably, the cleavage site is only 1-500 bp away
from the target region. [0023] To improve reaction performance
without cleaving the target sequence, the sequence in the region
for restriction enzyme digestion and HDA amplification should be
relatively conserved. Highly polymorphic regions of a target
nucleic acid are preferably avoided. [0024] The cleavage frequency
of an endonuclease is determined, in part, by the number of base
pairs it recognizes. Some restriction endonucleases recognize 6
base pairs. For example, the restriction endonuclease SspI
recognizes AATATT and cleaves in the middle. As another example,
restriction endonuclease SpeI recognizes ACTAGT and cleaves between
A and C. In general, restriction endonucleases that recognize
longer sequences (less frequent cutters such as SspI and SpeI) show
more enhancement in most of the HDA reactions. The high-frequency
cutters (for example, those that recognize 4 base pairs) will not
only generate restriction ends close to the targets, but also
generate more ends in the entire nucleic acid substrates. Since all
ends will recruit helicases, higher numbers of available ends in
the nucleic acid substrates may compete with ends that are adjacent
to the specific target sequence. This "dilution" effect can reduce
the effectiveness of the restriction enzyme in enhancing HDA.
[0025] In an embodiment, the restriction endonucleases are enzymes
which digest the DNA at a lower temperature during reaction setup
(for example, from about 0.degree. C. to about 37.degree. C.)
[0026] In an embodiment, the improvement effects of the
endonucleases depend on the amount of DNA. In a sample having very
low amounts of the initial targets, for example 1-50 copies of DNA
(for example, plasmid DNA, bacteria or virus genomic DNA, or human
genomic DNA), endonuclease cleavage can greatly improve the chance
of helicases binding to the limited number of targets, and make the
reaction more consistent at low target concentrations. When an HDA
reaction with low-initial-target is evaluated in real-time, the Ct
(the threshold cycle, defined as the cycle number at which the
fluorescence level has exceeded the detection threshold) is reduced
if an endonuclease is used. This indicates that the HDA speed is
enhanced by the restriction enzyme, evidenced by the earlier
appearance of the fluorescent signal across the Ct line. In
addition, the success rate of the HDA reaction with
low-initial-target is improved by using appropriate endonucleases:
the standard deviation of Ct is reduced. Restriction endonucleases
also reduce primer dimer formation (caused by non-specific primer
annealing and amplification) at low target concentrations. In a
sample having a medium amount of the input targets, for example,
more than 50 copies of DNA, endonuclease cleavage still improves
the detection speed. If the experiment is evaluated by real-time
assay, the time needed to see a positive fluorescent signal across
the Ct line is reduced by about 2 to about 6 minutes. On the other
hand, in HDA reactions with very high amounts of input substrate,
for example, about 20 ng of human genomic DNA, there will be an
overwhelming number of non-specific ends, exceeding the number of
helicases. In this case, no significant enhancement effects by the
restriction endonucleases are observed.
[0027] In an embodiment, a mixture of one or more compatible
endonucleases for use with the corresponding HDA can be selected.
For example, an endonuclease which can cleave the upstream sequence
of the target can be combined with an endonuclease which can cleave
the downstream sequence of the target.
[0028] In an embodiment, the use of restriction endonucleases in an
HDA reaction is compatible with other methods to improve HDA
performance. For example, the methods include, but are not limited
to, protein engineering or modification to improve the function of
the protein (helicases, polymerases, other accessory proteins).
[0029] The following experimental examples are provided to
illustrate certain embodiments of the invention, but are not
intended to limit the invention.
EXAMPLES
Example 1
Use of Endonucleases to Improve Reaction Speed of HDA
[0030] In this example, a method is disclosed to improve HDA
reaction speed by restriction endonuclease cleavage. The target of
amplification was within Homo sapiens breast cancer 1 (Genbank
accession No. NT 010755 REGION: 4967748 . . . 4971174, 3427 bp).
This region was cloned into pCR-Blunt II-TOPO (Invitrogen,
Carlsbad, Calif.), named as pBRCA-TOPO. Specific primers BRCA2F
(AGCTTAGCAGGAGTCCTAGCCCTTT) (SEQ ID NO: 1) and BRCA2R
(TCTGAGGACTCTAATTTCTTGGCCC) (SEQ ID NO: 2), were used to amplify an
85 bp fragment from this region. In order to compare the effects of
endonuclease cleavage from a medium amount of DNA and a high amount
of DNA (with overwhelming numbers of ends), the same endonuclease
was tested with different targets: plasmid DNA (50,000 copies of
pBRCA-TOPO, around 0.38 .mu.g) vs. human genomic DNA (20 ng, around
6000 copies). 20 ng of human genomic DNA contain many more ends
than 0.38 .mu.g of plasmid DNA.
[0031] The restriction endonuclease SspI (New England BioLabs, Inc,
Ipswich, Mass.) was chosen to show the effects of enzyme cleavage,
which can cleave the sequence around 187 bp downstream of the
specific amplification region. The target sequence of the enzyme is
AAT.sup..dwnarw.ATT. After cleavage, blunt ends are generated. The
HDA reaction was performed first by combining the following
components to produce a reaction master mix with a final volume of
45.mu./reaction (make 4.times. reaction master mix for 2 tested
samples):
[0032] 5 .mu.l 10.times. Annealing buffer II (see below*)
*10.times. Annealing buffer II contains 100 mM KCl and 200 mM
Tris-HCl (pH 8.8 at 25.degree. C.)
[0033] 0.75 .mu.l 5.0 .mu.M BRCA2F primer (SEQ ID NO: 1)
[0034] 0.75 .mu.l 5.0 .mu.M BRCA2R primer (SEQ ID NO: 2)
[0035] 2.0 .mu.l 100 mM MgSO.sub.4
[0036] 2.0 .mu.l 500 mM NaCl
[0037] 2.0 .mu.l 10 mM dNTP
[0038] 1.5 .mu.l 100 mM dATP
[0039] 0.5 .mu.l EvaGreen (20.times., Biotium, Hayward, Calif.)
[0040] 1 .mu.l ROX dye (50.times., Invitrogen, Carlsbad,
Calif.)
[0041] 3.5 .mu.l IsoAmpII enzyme mix (BioHelix, Beverly, Mass.)
[0042] 1 .mu.l SspI or H.sub.2O (for the control without
endonuclease)
add dH.sub.2O to total 45 .mu.l.
[0043] For the test with plasmid DNA, in 4 individual PCR reaction
tubes, 5 pi of 10,000 copies/.mu.1 pBRCA-TOPO was added to each
tube. 45 .mu.1 of reaction master mix with SspI was added to 2 of
the tubes, and 45 .mu.l of reaction master mix without SspI was
added to the other two tubes. The reactions were mixed by pipetting
up and down several times and then covered with mineral oil. The
reaction mixture was immediately incubated in an ABI7300 real-time
PCR machine with the well inspector setting: reporter dye: SYBR;
quencher: none; passive reference dye: ROX. An ABI7300 real-time
PCR instrument was used to monitor the HDA reaction in real-time
because no isothermal real-time fluorescent instrument was
commercially available. To perform an isothermal reaction on an
ABI7300 real-time PCR instrument, which requires a minimum of 1
degree of temperature variation between different cycles, the
following program was used:
Stage 1: (30.times.)
[0044] Step I: 64.5.degree. C. for 0:05
[0045] Step2: 63.5.degree. C. for 1:55
[0046] Data collection and real-time analysis enabled
Stage 2: (1.times.)
Dissociation Stage
[0047] Melt curve data collection and analysis enabled
[0048] For the test with human genomics DNA, a similar procedure
was performed. The only difference was that in 4 individual PCR
reaction tubes, 5 .mu.l of 4 ng/.mu.1 human genomic DNA (Promega,
Madison, Wis.) were added to each tube.
[0049] The graphs in FIG. 1 and FIG. 2 were generated by the
software of the ABI7300 realtime PCR System as log view of delta Rn
versus cycle. When 0.38-pg of plasmid DNA were used in the
reactions (FIG. 1), the Ct number was reduced by about 2.5 cycles
from around Ct 14 to 11.5 (corresponding to a reduction of
amplification time by 5 minutes) in the reaction with SspI compared
to the reaction without SspI. However, when 20 ng of human genomic
DNA were used in the tests (FIG. 2), there was no obvious
difference comparing the reactions with or without SspI. These
results demonstrate that in the presence of a high amount of DNA
(with an overwhelming number of ends for helicase loading), no
significant enhancement to the speed of the HDA reaction was
observed.
Example 2
Use of Endonucleases to Improve Detection Sensitivity of HDA when
the Target Copy Number is Low
[0050] In this example, endonuclease cleavage was used to improve
the sensitivity of HDA detection. The same target (human genomic
DNA) and primers were used as in Example 1 and the same components
of the reaction master mix were prepared.
[0051] For the assay with SspI endonuclease, 12.times. reaction
master mix with SspI was prepared for 10 samples (same components
as described in Example 1). For the assay without SspI, 12.times.
reaction master mix without SspI was prepared for 10 samples. In 20
individual PCR reaction tubes, 5 .mu.l of the following samples
were added to each tube:
1-2: 0.2 ng/.mu.l human genomic DNA (around 300 copies per
reaction); 3-8: 2 .mu.g/.mu.l human genomic DNA (around 3 copies
per reaction); 9-10: H.sub.2O (negative control); 11-12: 0.2
ng/.mu.l human genomic DNA (around 300 copies per reaction); 13-18:
2 .mu.g/.mu.l human genomic DNA (around 3 copies per reaction);
19-20: H.sub.2O (negative control).
[0052] Then, 45 .mu.l of reaction master mix with SspI were added
to tubes 1-10, and 45 .mu.l of reaction master mix without SspI
were added to tubes 11-20. The reactions were mixed by pipetting up
and down several times and then covered with mineral oil. The
reaction mixture was immediately incubated in an ABI7300 real-time
PCR machine with the same program as in Example 1. The results are
given in Table 1 below.
TABLE-US-00001 TABLE 1 Comparison of Sspl effects from the
amplification of low copy number of human genomic DNA. Ct from 1 ng
DNA Ct from 0.01 ng DNA Success rate from (300 copies) (3 copies)
0.01 ng DNA +Sspl 14.62 .+-. 0.08 18.36 .+-. 1.08 100% (6/6
positive) -Sspl 16.16 .+-. 0.04 22.87 16.67% (2/6 fail, 3/6
dimmer)
[0053] For a medium copy number of targets (300 copies of human
genomic DNA), the Ct was decreased by about 1.5 cycles (from Ct
16.1 to Ct 14.5) by the endonuclease. For a low copy number of
targets (3 copies of human genomic DNA), without the help of SspI,
only 1 out of 6 reactions was successful (success rate around 17%).
Two reactions failed and 3 reactions generated primer dimers.
However, with the help of SspI, the success rate was 100% (6 out of
6 worked), with a standard deviation of Ct of about 1 Ct. This
demonstrates that the robustness and sensitivity of the assay has
been significantly improved by endonuclease cleavage.
Example 3
Use of Endonucleases Generating a 5' End or a 3' End to Improve the
Reaction Speed of HDA
[0054] In this example, a method is disclosed to improve HDA
reaction speed by endonucleases digestion which can generate 5'
ends or 3' ends. The same plasmid target pBRCA-TOPO was used as in
Example 1 (50,000 copies for each reaction). The target sequence of
SpeI is A.sup..dwnarw.CTAGT; the closest site to the amplification
region is around 66 bp upstream of the primer binding site. Upon
cleavage, 5' ends are generated. The target sequence of BanII is
GRGCY.sup..dwnarw.C (R represents A or G, Y represents C or T), the
closest site to the amplification region is around 387 bp upstream
of the primer binding site. Upon cleavage, 3' ends are
generated.
[0055] The similar reaction master mix was prepared as Example 1.
4.times. reaction master mix were prepared for 2 tested samples
(45.mu./reaction):
[0056] 5 .mu.l 10.times. Annealing buffer II (see below*)
[0057] O.75 .mu.l 5.0 .mu.M BRCA2F primer (SEQ ID NO:1)
[0058] 0.75 .mu.l 5.0 .mu.M BRCA2R primer (SEQ ID NO:2)
[0059] 2.0 .mu.l 100 mM MgSO.sub.4
[0060] 2.0 .mu.l 500 mM NaCl
[0061] 2.0 .mu.l 10 mM dNTP
[0062] 1.5 .mu.l 100 mM dATP
[0063] 0.5 .mu.l EvaGreen (20.times., Biotium, Hayward, Calif.)
[0064] 1 .mu.l ROX dye (50.times., Invitrogen, Carlsbad,
Calif.)
[0065] 3.5 .mu.l IsoAmpII enzyme mix (BioHelix, Beverly, Mass.)
[0066] 1 .mu.l SpeI, or BanII or H.sub.2O (for the control without
endonuclease)
add dH.sub.2O to total 45 .mu.l.
[0067] For the test with SpeI, in 4 individual PCR reaction tubes,
5 .mu.l of 10,000 copies/.mu.l pBRCA-TOPO were added to each tube.
45 .mu.l of reaction master mix with SpeI were added to 2 of the
tubes, and 45 .mu.l of reaction master mix without SpeI were added
to 2 of the other tubes. The reactions were mixed well by pipetting
up and down several times and then covered with mineral oil. The
reaction mixture was immediately incubated in an ABI7300 real-time
PCR machine with the same program as Example 1.
[0068] A similar procedure was performed with BanII.
[0069] The graphs in FIG. 3 and FIG. 4 were generated by the
software of the ABI7300 realtime PCR System. The Ct was reduced by
about 2 cycles (corresponding to 4 minutes, from around Ct 14 to
12) in the reaction with SpeI compared to the reaction without
SpeI. The Ct was also reduced by about 2 cycles (corresponding to 4
minutes, from around Ct 13 to 11) in the reaction with BanII
compared to the reaction without BanII. (The corresponding melt
cure analysis is not shown here.) This demonstrates that the HDA
reaction speed can be improved by the endonucleases that can
generate either 5' ends or 3' ends, or blunt ends as Example 1.
[0070] It will be appreciated that several of the above-disclosed
and other features and functions, or alternatives thereof, may be
desirably combined into many other different systems or
applications.
Sequence CWU 1
1
2125DNAHomo sapiens 1agcttagcag gagtcctagc ccttt 25225DNAHomo
sapiens 2tctgaggact ctaatttctt ggccc 25
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