CN107177693A - A kind of many times of PCR amplification methods - Google Patents
A kind of many times of PCR amplification methods Download PDFInfo
- Publication number
- CN107177693A CN107177693A CN201710591161.6A CN201710591161A CN107177693A CN 107177693 A CN107177693 A CN 107177693A CN 201710591161 A CN201710591161 A CN 201710591161A CN 107177693 A CN107177693 A CN 107177693A
- Authority
- CN
- China
- Prior art keywords
- sequence
- many times
- universal primer
- primer
- primers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000012408 PCR amplification Methods 0.000 title claims abstract description 20
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 27
- 238000004519 manufacturing process Methods 0.000 claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 22
- 230000003321 amplification Effects 0.000 claims abstract description 17
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 10
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 10
- 230000000295 complement effect Effects 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 230000004087 circulation Effects 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 4
- 238000002844 melting Methods 0.000 claims description 4
- 230000008018 melting Effects 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 101150030367 cls gene Proteins 0.000 claims description 2
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 239000000376 reactant Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000006073 displacement reaction Methods 0.000 abstract description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 3
- 102000039446 nucleic acids Human genes 0.000 abstract description 3
- 150000007523 nucleic acids Chemical class 0.000 abstract description 3
- 238000011005 laboratory method Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 16
- 239000002585 base Substances 0.000 description 11
- 241000607142 Salmonella Species 0.000 description 7
- 238000000137 annealing Methods 0.000 description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 241000186779 Listeria monocytogenes Species 0.000 description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- XMTQQYYKAHVGBJ-UHFFFAOYSA-N 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA Chemical compound CN(C)C(=O)NC1=CC=C(Cl)C(Cl)=C1 XMTQQYYKAHVGBJ-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000191070 Escherichia coli ATCC 8739 Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kind of many times of PCR amplification methods, expanded with many times of primers (low concentration) and a pair of universal primers (high concentration) of the multiple identical sequence compositions of a pair 5 ' ends and universal primer identical in same PCR reactions, archaeal dna polymerase used is the heat-resisting polymerase acted on strand displacement in amplification.Many times of primer sequences include two parts, i.e., 3 ' ends and target gene are complementary, and 5 ' ends carry at least one and universal primer identical sequence.The number that many times of the upstream and downstream end of primer 5 ' carries universal primer is identical or different, and the sequence of upstream and downstream universal primer can also be identical or different.This method one cycle produces multiple amplified productions simultaneously, and each amplified production also contains at least one upstream and a downstream universal primer sequence in addition to containing target amplification sequence.The present invention can produce the amplified production more than twice because of one cycle, therefore can significantly improve PCR amplification efficiency and detection sensitivity, be suitable as conventional molecular biological laboratory techniques and medical science detection of nucleic acids means be used for low template base because detection.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to a kind of many times of PCR amplification methods.
Background technology
Round pcr is as a kind of method of external rapid amplifying specific gene, the advantages of because of its high specific and sensitivity
It is widely used to each field.The key of PCR method is to design a pair of oligonucleotides with high preciseness according to target gene
Primer, rapid amplifying is realized by archaeal dna polymerase enzymatic reaction.
Since round pcr invention, although various improvement and derivative method emerge in an endless stream, but all fail to break through its base
The amplification of this pattern, i.e. each target gene is by being carried out with a pair of complementary upstream and downstream primers of template.Standard PCR
Expanded using pair of primers, circulation can only at most produce twice of amplified production every time, therefore its amplification efficiency is restricted.
The content of the invention
It is an object of the invention to provide a kind of many times of PCR amplification methods, to break through in the prior art, circulation is most every time
The limit of twice of amplified production can only be produced, so as to improve PCR amplification efficiency and shorten the PCR reaction time.
The many times of PCR amplification methods of the present invention, methods described includes carrying out the amplification in the reactive mixture, described anti-
Answer mixture to include one-to-many times of primer and a pair of universal primers, and replace conventional with the hot resistant DNA polymerase of chain substitution
Archaeal dna polymerase.Trigger PCR in initial several circulations with many times of primers of low concentration, produce and include universal primer sequence
First amplified production of row；Using the first amplified production as template, under with the effect of the hot resistant DNA polymerase of chain substitution,
PCR is expanded using the universal primer of higher concentration.For target gene, one cycle can produce the amplification production more than twice
Thing, so as to improve PCR amplification efficiency and shorten PCR proliferation times.
The first aspect of the invention is to provide a kind of many times of PCR amplification methods, using a pair 3 ' ends and target base
Because complementary specific sequence and 5 ' many times of primers being made up of multiple universal sequences complete first round amplification, produce comprising logical
With the first amplified production of primer sequence；Product using the first amplification is template, in the heat-resistant dna polymerization with chain substitution
Under enzyme effect, complete the second wheel using universal primer and expand, produce a plurality of amplified production for carrying target gene sequence；It is wherein many
Times primer and universal primer are added simultaneously, are reacted under same annealing temperature condition.
The second aspect of the invention is to provide the design that a kind of many times of PCR expand the primer, including many times of primers and
Universal primer.
The many times of primer concentrations are 5-100nM；Hold and target gene sequence complete complementary, its sequence the 3 ' of many times of primers
Primer Software for Design can be used, the high preciseness of usual outstanding primer should be met, sequence length is 10-30 bases；5 ' ends are comprising extremely
It can be connected between few one and the identical sequence of universal primer, its sequence with base；The alkali of many times of primer formation hairpin structures
Radix should be as far as possible low, and itself total complementary base radix should be as far as possible low.
The universal primer number that the upstream and downstream sequence of many times of primers is included is identical or different, but upstream and downstream are more in a system
Times primer includes universal primer sequence total number and is at least 3.
The dense universal primer is respectively l-10 μM；Universal primer sequence is unrelated with target gene sequence, and it designs completely only
Stand on by cls gene；Its thing sequence length is 10-30 bases；The annealing temperature of universal primer and many times of end sequences of primer 3 '
The annealing temperature difference of row is no more than 4 DEG C；The sequence of upstream universal primer and downstream universal primer can be with identical or different.
Many times of primers and universal primer are mixed together with other PCR reaction reagents, and two-wheeled PCR is expanded in identical annealing temperature
It is lower carry out, reaction first few circulation in have low concentration many times of primers complete, and universal primer due to its sequence with
Target gene is mismatched, and is not served completely.
The many times of PCR amplification methods of the present invention have many advantages than conventional PCR method：
First, the use of many times of primers of low concentration improves the preciseness of reaction, advantageously reduces or eliminate primer
Dimer and non-specific amplification product；
Second, close, therefore many times of primers and logical of the melting temperature of the specific sequence of many times of primers and universal primer
Reacted under same annealing temperature with primer, it is to avoid the expansion caused by melting temperature and joint efficiency of primer etc. are different
The heterogeneity of Increasing Efficiency；
3rd, the sequence of universal primer is independently of target template sequence and designed, with high preciseness.High concentration
Universal primer extends the straight line accumulated phase of many times of PCR amplified production, is conducive to improving the sensitivity detected and shortens amplification
Time.
Brief description of the drawings
The present invention is further described below in conjunction with the accompanying drawings.
Fig. 1 is many times of PCR amplification method schematic diagrames of the invention.Fm and Rm are respectively that many times of upstream and downstream draws in figure
Thing, FUP and RUP are respectively upstream and downstream universal primer.
Fig. 2A and Fig. 2 B are many times of primer schematic diagrames of the invention.101. upstream specific primer in figure, 102. connection alkali
Base, 103. upstream universal primers, 104. downstream specific primers, 105. connection bases, 106. downstream universal primers.
Fig. 3 A and 3B are respectively using the amplification electrophoretogram of 2 times of primers and 3 times of primers in embodiment 1.M is 50bp DNA
Molecular criteria.
The specificity experiments that Fig. 4 is many times of PCR of real-time fluorescence in the embodiment of the present invention 2.
Embodiment
As shown in figure 1, the invention provides a kind of many times of PCR amplification methods, i.e., with one-to-many times of primer (low concentration) and
A pair of universal primers (high concentration) are expanded in same PCR reactions, and archaeal dna polymerase used is made with strand displacement in amplification
Heat-resisting polymerase.Several circulations before PCR reactions, 3 ' ends of many times of primers are specifically bound with target gene, with
The extension of polymerase, produces the complete double-stranded DNA with 5 ' terminal sequences.Expansion is completed by universal primer in subsequent PCR cycle
Increase, because the strand displacement of polymerase is acted on, the chain that external primers are formed can displace the chain of inside with extension, so each
Multiple amplified productions can be produced after circulation, each of which amplified production also contains at least one in addition to containing target amplification sequence
Upstream and a downstream universal primer sequence.Fm and Rm are respectively many times of primers of upstream and downstream, and FUP and RUP are respectively upstream
With downstream universal primer.
As shown in Figure 2 A and 2 B, the two parts that are designed with of many times of upstream primer are constituted, i.e., the target genes at 3 ' ends are special
Property primer sequence 101 and 5 ' hold upstream universal primer sequence 103, between the two with connect base 102 be connected；Many times of downstream primer
Design be equally made up of two parts, i.e., 3 ' end target gene-specific primers sequences 104 and 5 ' hold upstream universal primer
Sequence 106, is connected with connecting base 105 between the two.The universal primer sequence at 5 ' ends of many times of upstream and downstream primer can with identical or
Difference, the number of its universal primer can also be identical or different.Between the universal primer sequence at 5 ' ends and the specific sequence at 3 ' ends
It can also be designed to that one section of specific probe sequence is used to detect.
The method can significantly improve PCR amplification efficiency and detection sensitivity, conventional molecular biological experiment is suitable as
Room means and medical science detection of nucleic acids means be used for low template base because detection.
The present invention is expanded on further with reference to specific embodiment.These embodiments be merely to illustrate the present invention and without limit
The scope of the present invention processed.In addition, after present disclosure has been read, those skilled in the art can make various change to the present invention
Dynamic or modification, these equivalent form of values equally fall into the application appended claims limited range.
SD archaeal dna polymerases and SD reaction buffers are purchased from BIORON GmbH (Germany) company, primer in the following example
And other related reagents are provided by Shanghai Sheng Gong bioengineering limited company.
Embodiment 1
Many times of PCR expand template DNA：
Using 69bp artificial synthesized template as amplification target, designed according to many times of primers in technical scheme and universal primer
Principle designs following primer (table 1).
Reaction condition is as follows：
PCR reaction systems cumulative volume be 25 μ L, the SD archaeal dna polymerases comprising 10U, 1 × SD reaction buffers, 3.5mM
MgCl2, 0.4mM dNTPs, each 30nM many times of upstream and downstream primers F m and Rm, each 1 μM of upstream and downstream universal primer FUP and
RUP, 0.5 μ L SYBR Geen I (20x), 2 μ L template.Negative control is made with sterilized water.
PCR amplification programs are：92 DEG C of denaturation 2min, subsequent 92 DEG C of denaturation 20s, 56 DEG C of renaturation 30s, 68 DEG C extend 20s, 25
Individual circulation.Take the above-mentioned amplification liquid of 5 μ L to add 1 μ L 6x sample loading buffers, take 5 μ L to carry out electrophoresis experiment, deposition condition after mixing
For：Voltage 100V, time 60min, 2% Ago-Gel, 4S red nucleic acid dye colour developings.
Fig. 3 A and 3B are respectively the amplification electrophoretogram using 2 times of primers and 3 times of primers in embodiment 1.M is 50bp DNA
Molecular criteria.
Above-mentioned many times of PCR amplifications：
(1) three amplified bands are obtained using 2 times of primer pairs (F2/R2), 113bp, 135bp and 157bp is divided respectively, represented
Produce four amplified productions, i.e., two each one of 135bp and 113bp and 157bp.
(2) five amplified bands are obtained using 3 times of primer pairs (F3/R3), 113bp, 135bp, 157bp, 179bp is divided respectively
And 201bp, represent to produce nine amplified productions, i.e., two 135bp, three 157bp, two 179bp and 113bp and 201bp are each
One.
The artificial synthesized template of table 1. and many times of primers and universal primer sequence
Embodiment 2
Many times of PCR detections of real-time quantitative of salmonella：
Using the ttr genes of salmonella as detection many times of primers of target design and universal primer (table 2).
Reaction condition is as follows：
PCR reaction systems cumulative volume be 25 μ L, the SD archaeal dna polymerases comprising 10U, 1 × SD reaction buffers, 4mM's
MgCl2, 0.4mM dNTPs, each 30nM 2 times of primers Fs 2 of upstream and downstream and R2, each 1 μM of upstream and downstream universal primer FUP and RUP,
0.5 μ L SYBR Geen I (20x), 2 μ L template.Negative control is made with sterilized water.
PCR amplification programs are：92 DEG C of denaturation 2min, subsequent 92 DEG C of denaturation 30s, 55 DEG C of renaturation 30s, 68 DEG C extend 1min,
Fluoroscopic examination, 35 circulations are carried out in extension.
Table 2 detects the primer sequence table of salmonella ttr genes
As shown in figure 4, the specificity experiments that Fig. 4 is many times of PCR of real-time fluorescence in the embodiment of the present invention 2.Salmonella in figure
Bacteria strain is mouse salmonella CMCC50071, and nonsalmonella bacterial strain is Escherichia coli ATCC 8739, vibrio parahemolyticus
CMCC 17802, Listeria monocytogenes CMCC 54003.
As a result interpretation：Primer according to designed by salmonella ttr genes is used for many times of PCR analyses, can from Fig. 4
Go out, only salmonella detects, and other nonsalmonella such as Escherichia coli, vibrio parahemolyticus, and Listeria monocytogenes are all
Fail detection, illustrate that the specificity of primer is very good.
The above described is only a preferred embodiment of the present invention, any formal limitation not is made to the present invention, therefore
It is every without departing from technical solution of the present invention content, what the technical spirit according to the present invention was made to above example any simply repaiies
Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.
Sequence table
<110>Zhongshan University
<120>A kind of many times of PCR amplification methods
<160> 11
<210> 1
<211> 69
<212> DNA
<213>Artificial sequence
<221>misc_feature
<400> 1
tgagcagcagacagcagacacaacgaacttatgatcggtagagtacacaagtgtcgtcgctgtgtctga 69
<210> 2
<211> 63
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400> 2
ggacagaggacagcagtgacttggacagaggacagcagtgactttgagcagcagacagcagac 63
<210> 3
<211> 63
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400> 3
ccacacagacgacacagaccttccacacagacgacacagacctttcagacacagcgacgacac 63
<210> 4
<211> 85
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400> 4
ggacagaggacagcagtgacttggacagaggacagcagtgacttggacagaggacagcagtgactttgagcag
cagacagcagac 85
<210> 5
<211> 85
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400> 5
ccacacagacgacacagaccttccacacagacgacacagaccttccacacagacgacacagacctttcagaca
cagcgacgacac 85
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400> 6
ggacagaggacagcagtgac 20
<210>7
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400> 7
ccacacagacgacacagacc 20
<210>8
<211> 66
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400>8
cagagatggtggcaggaagtaacagagatggtggcaggaagtaaggtagtcaggaagtcacgaatg 66
<210>9
<211> 65
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400>9
caggaggcagagacaagaggaacaggaggcagagacaagaggaagcacaggtaatggcaatcagt 65
<210>10
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400>10
cagagatggtggcaggaagt 20
<210>11
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<400>11
caggaggcagagacaagagg 20
Claims (8)
1. a kind of many times of PCR amplification methods, it is characterised in that：Methods described includes carrying out the amplification in the reactive mixture,
The reactant mixture includes one-to-many times of primer and a pair of universal primers, and the polymerization of the heat-resistant dna with chain substitution
Enzyme；Trigger PCR in first few circulation with many times of primers of low concentration, the first amplified production of generation general draws comprising multiple
Thing sequence；Using the first amplified production as template, PCR is expanded using the universal primer of higher concentration, is produced comprising target gene
Multiple second amplified productions.
2. many times of PCR amplification methods according to claim 1, it is characterised in that：Methods described further comprises step：
1) the first amplified reaction, many times of primers hybridize with template, produce and include at least two upstream universal primers and a downstream
First amplified production of universal primer or at least one upstream universal primer and two downstream universal primer sequences.
2) the second amplified reaction, using the first amplified production as template, universal primer is combined with the first amplified production, is replaced with chain
Under the hot resistant DNA polymerase effect for changing effect, produce it is multiple comprising target gene sequence and at least one upstream universal primer and
Second amplified production of one downstream universal primer.
3. method according to claim 2, it is characterised in that：Step 1) in, many times of primer concentrations are 5-100nM；It is described
The 3 ' of many times of primers are held includes at least one and universal primer sequence identical bound site with target gene complementary specificity, 5 ' ends
Point.
4. method according to claim 2, it is characterised in that：Step 1) in, what the upstream and downstream sequences of many times of primers was included
Universal primer number is identical or different, but the universal primer sequence total number that many times of primers are included in a system is at least 3.
5. method according to claim 2, it is characterised in that：Step 2) in, the dense universal primer is respectively l-10 μM；Institute
Universal primer is stated with target gene sequence without homology；The melting temperature of the universal primer and target gene-specific primers
Melting temperature difference is no more than 4 DEG C.
6. method according to claim 2, it is characterised in that：Step 2) in, the sequence of the upstream and downstream of the universal primer
It is identical or different.
7. method according to claim 2, it is characterised in that：Step 2) in, the 3 ' of many times of primers holds and target gene sequence
Row complete complementary, its sequence can use primer Software for Design, should meet the high preciseness of usual outstanding primer, sequence length is 10-
30 bases, 5 ' ends include and can connected between at least one and the identical sequence of universal primer, its sequence with base.
8. method according to claim 2, it is characterised in that：Universal primer sequence is unrelated with target gene sequence, and it sets
Meter is totally independent of by cls gene；Its thing sequence length is 10-30 bases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710591161.6A CN107177693B (en) | 2017-07-19 | 2017-07-19 | Multiple PCR amplification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710591161.6A CN107177693B (en) | 2017-07-19 | 2017-07-19 | Multiple PCR amplification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107177693A true CN107177693A (en) | 2017-09-19 |
| CN107177693B CN107177693B (en) | 2020-11-06 |
Family
ID=59838034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710591161.6A Active CN107177693B (en) | 2017-07-19 | 2017-07-19 | Multiple PCR amplification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107177693B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352350A (en) * | 2011-09-30 | 2012-02-15 | 北京万达因生物医学技术有限责任公司 | Same sequence primer transpositional nucleic acid amplification technology |
| CN103797366A (en) * | 2011-01-14 | 2014-05-14 | Cb生物技术公司 | Immunodiversity assessment method and its use |
| CA2894381A1 (en) * | 2012-12-07 | 2014-06-12 | Invitae Corporation | Multiplex nucleic acid detection methods |
| CN104293962A (en) * | 2014-10-20 | 2015-01-21 | 苏州大学 | Method for screening general primers |
| CN105705659A (en) * | 2013-08-28 | 2016-06-22 | 赛卢拉研究公司 | Massively parallel single cell analysis |
| CA2999902A1 (en) * | 2015-10-09 | 2017-04-13 | Accuragen Holdings Limited | Methods and compositions for enrichment of amplification products |
-
2017
- 2017-07-19 CN CN201710591161.6A patent/CN107177693B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103797366A (en) * | 2011-01-14 | 2014-05-14 | Cb生物技术公司 | Immunodiversity assessment method and its use |
| CN102352350A (en) * | 2011-09-30 | 2012-02-15 | 北京万达因生物医学技术有限责任公司 | Same sequence primer transpositional nucleic acid amplification technology |
| CA2894381A1 (en) * | 2012-12-07 | 2014-06-12 | Invitae Corporation | Multiplex nucleic acid detection methods |
| CN105705659A (en) * | 2013-08-28 | 2016-06-22 | 赛卢拉研究公司 | Massively parallel single cell analysis |
| CN104293962A (en) * | 2014-10-20 | 2015-01-21 | 苏州大学 | Method for screening general primers |
| CA2999902A1 (en) * | 2015-10-09 | 2017-04-13 | Accuragen Holdings Limited | Methods and compositions for enrichment of amplification products |
Non-Patent Citations (3)
| Title |
|---|
| Y. M. LONG ET AL.: ""An innovative SNP genotyping method adapting to multiple platforms and throughputs"", 《THEORETICAL AND APPLIED GENETICS》 * |
| 汪维鹏等: ""一种基于适配器连接介导的等位基因特异性扩增法测定多重SNP"", 《遗传》 * |
| 陈英等: ""一种高效筛选公共引物的方法"", 《中国血液流变学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107177693B (en) | 2020-11-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gill et al. | Nucleic acid isothermal amplification technologies—a review | |
| JP2021094038A (en) | Isothermal methods for preparing nucleic acids and related compositions | |
| CN103255227A (en) | Primer-mediated cyclized constant-temperature nucleic acid rolling circle amplification method and kit | |
| EP2171099A1 (en) | Nested multiplex amplification method for identification of multiple biological entities | |
| CN106884037B (en) | Gene chip kit for detecting bacterial drug resistance gene | |
| CN107893109A (en) | A kind of low abundance gene mutation enrichment method based on removal wild-type sequence | |
| CN106399577B (en) | A kind of real-time fluorescence PCR detection method for the double target nucleic acids of interest present detections of single channel | |
| CN102936593B (en) | Nucleic acid detection method applying multiplex PCR array technology | |
| JP2015023874A (en) | Rna detection method | |
| CN102286473A (en) | Reagent kit for directly carrying out nucleic acid amplification from whole blood sample and method | |
| KR20160088316A (en) | Nucleic Acid Amplification | |
| CN103146684A (en) | Method for isothermal amplification of nucleic acid segments | |
| EP2013366B1 (en) | Sequencing of the L10 codon of the HIV gag gene | |
| JP6940412B2 (en) | PCR method | |
| CN101329250B (en) | Method for detecting sequence of PCR-LCR gene polymorphism of fluorescence labeling probe | |
| Marciniak et al. | Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences | |
| CN107177693A (en) | A kind of many times of PCR amplification methods | |
| Qin et al. | Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR | |
| CN102424833A (en) | Chip and method for real-time PCR (polymerase chain reaction) gene detection at polygenic mutation site | |
| KR101785687B1 (en) | Method for detection of target nucleic acid sequence by multiple amplification nested signal amplification | |
| CN113789368B (en) | Nucleic acid detection kit, reaction system and method | |
| CN102559856A (en) | Method for deleting vector segments in sequencing library | |
| CN101845519A (en) | Loop-mediated lsothermal amplification (LAMP) kit for quick detection of prawn taura syndrome viruses (TSV) and detection method thereof | |
| CN110468179A (en) | The method of selective amplification nucleic acid sequence | |
| CN101445828B (en) | Method for nucleic acid quantitation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |