CN108753935A - A kind of method that primer dimer expands in reduction multiplex polymerase chain re-action - Google Patents
A kind of method that primer dimer expands in reduction multiplex polymerase chain re-action Download PDFInfo
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Abstract
The invention discloses the method that primer dimer in a kind of reduction multiplex polymerase chain re-action expands, method includes：Forward primer and reverse primer are obtained, the forward primer and reverse primer are both designed as template hybridization region and template extension area；Multiple dSpacer and/or C3Spacer modifications are separated between on the template hybridization region, and there are one dSpacer or C3Spacer to modify between the template hybridization region and template extension area.The present invention's reduces the method that primer dimer expands in multiplex polymerase chain re-action, greatly reduces the generation probability of primer dimer, improves the spreadability and homogeneity of structure gene library.
Description
Technical field
The present invention relates to gene amplification fields in molecular biology, and in particular to a kind of reduction Multiplex polymerase chain formula
The method that primer dimer expands in reaction.
Background technology
PCR is always the most effective DNA sequence dna acquisition methods of biotechnology, and with advances in technology, PCR has very
More updates and application.High throughput sequencing technologies are the most widely used technologies of biotechnology during the nearly last ten years, from
Since 2005 are born, single nucleotide polymorphism, genome structure variation, group's polymorphism can see by the way that scientist is sequenced
Equal important informations.
Multiplex PCR refers in a tube reaction system, and the parallel thousands of above PCR of 2- that carry out react, its effective solution
Multidigit point, polygenic target capture demand.But PCR tuples are more, then the primer dimer of PCR is more serious, such as Fig. 1 institutes
Show, primer dimer is exactly 3 ' the end part base complementrities combination of pair of primers or primer itself in fact, in the effect of Taq enzyme
Under extend from 3 ' ends and be formed by the double chain DNA fragment of small-molecular-weight.
It is often inevitable in the number of base complementary theory at 3 ends when multiplex PCR tuple height is thousands of to hundreds of.Primer two
The generation of aggressiveness, can make target product yield reduce even without, while also influence multiplex PCR tuple.Have at present very much
DMSO (dimethyl sulfoxide) is such as added in the method for solving primer dimer, using thermal starting polymerase, improves annealing temperature etc., but
All from the root cause solve primer between Complementarity Problem.
Patent No. CN201510761091.5 " a kind of multiple PCR method of three-wheel amplification " is provided in careful Chinese patent
A kind of method of multiplex PCR, but there is no solve the problem of that primer dimer generates when multiplex PCR.The patent No.：
A kind of patent of CN201210193352.4 patents entitled " universal multiple PCR method " describe it is a kind of add at 5 end of primer it is general
Sequence obtains universal amplification primer, method that parallel multiplex amplification is realized by different PCR cycles, and this method due to
Primer length is increased, the generation probability of primer dimer may be caused to become larger.The patent No.：ZL 2,015 1 0555967.0,
CN201410586158.1, CN201410236110, CN 201010262894 etc. is all based on the multiple of a small amount of amplicon
Gene tester.In foreign patent, the patent of PCT/US2014/067666 propose it is a kind of realized using OMGA rings it is more
The method of re-spread increasing can also realize the parallel amplification of a large amount of PCR.
In above-mentioned patent, CN201510761091.5 needs 3 wheel PCR of expansion that library construction could be realized for being sequenced.And
The generation of primer dimer is not avoided from background.PCT/US2014/067666 is easy production as a result of longer primer
Raw primer dimer.
Invention content
The method that primer dimer expands in multiplex polymerase chain re-action is reduced the purpose of the present invention is to provide a kind of,
Primer dimer is easy tod produce to solve existing multi-PRC reaction process, gene library spreadability is built and homogeneity is poor
Defect.
To achieve the above object, the present invention provide it is a kind of reduction multiplex polymerase chain re-action in primer dimer expand
Method, the method includes：
Forward primer and reverse primer are obtained, by the forward primer and reverse primer along 5, -3, is set on extending direction
It is calculated as template hybridization region and template extension area；
It is separated with multiple dSpacer and/or C3Spacer modifications between on the template hybridization region, and hybridizes in the template
There are one dSpacer or C3Spacer to modify between area and template extension area.
Preferably, the length of the template hybridization region is 15-30 bases, and TM values range is in 60-65 degree.
Preferably, the length of the template extension area is 10-20 base, and TM values range is in 45-55 degree.
Preferably, the dSpacer between the template hybridization region and template extension area or the subsequent bases of C3Spacer
For T, base T is replaced with into base U.
Preferably, the final concentration of 100-300nmol of the use of the primer.
Preferably, the condition of the multiplex polymerase chain re-action is high annealing temperature after first low temperature thermal oxidation.
Preferably, the multiplex polymerase chain re-action using Taq birdss of the same feather flock together synthase or high-fidelity is birdsed of the same feather flock together synthase.
Preferably, in the template hybridization region, at interval of 10 bases, there are one dSpacer or C3Spacer to modify.
The invention has the advantages that：
The present invention's reduces the method that primer dimer expands in multiplex polymerase chain re-action, greatly reduces primer
The generation probability of dimer improves the spreadability and homogeneity of structure gene library.
Description of the drawings
Fig. 1 is that the Modify to primer A of the present invention and the unmodified BPCR primer dimers of primer generate comparison schematic diagram.
Fig. 2 is the structural schematic diagram of the Mdification primer of the present invention.
Fig. 3 is to reduce the method Library development flow that primer dimer expands in multiplex polymerase chain re-action using the present invention
Schematic diagram.
Fig. 4 is the Mdification primer of the present invention and unmodified primer extension product electrophoretogram, and A1, A2 are that unmodified primer expands
Increase production object, B1, B2 are the primer extension products after modification, and M is 50bp MARKER.
Fig. 5 be unmodified primer gene amplification product electrophoretogram, wherein 0 be non-gene mentation group negative control, A1, A2,
A3 is the amplified production of 3 genomes of unmodified MIX, and M is 50bp MARKER.
Fig. 6 is the electrophoretogram of Mdification primer gene amplification product, wherein 0 is the negative control of non-gene mentation group, B1, B2, B3
To modify the amplified production of 3 genomes of MIX, M is 50bp MARKER.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
In the present invention, " primer dimer " is the potential by-product of PCR, primer dimer due to complementary base in primer and
The primer molecule composition hybridized each other.
In the present invention, dSpacer or C3Spacer modification be primer synthesis in a kind of modification, be a kind of carbon bone
Frame acts on, similar with the single base length that DNA pentoses and its phosphodiester bond provide, in this way can with one base position of filling-in,
Allow DNA base that pulling force or pressure caused by lacking nucleotide are not present in no Mismatching.
When forward primer and reverse primer are when its 3 ' end has complementary region, it may occur however that the formation of primer dimer.
Due to the high concentration of primer, which can be as short as 2-3 nucleotide to cause primer dimer to expand.When the complementation
When region is at least 4 or 5 nucleotide, unwanted primer dimer amplification almost surely occurs.The reduction of the present invention is multiple
The method that primer dimer expands in PCR, the problem of amplification of primer dimer can be well solved.
As depicted in figs. 1 and 2, the method for reducing primer dimer amplification in multiplex polymerase chain re-action of the invention,
It includes：Forward primer and reverse primer are obtained, forward primer and reverse primer are divided into template hybridization region and template is prolonged
Stretch area；It is separated with multiple dSpacer and/or C3Spacer modifications between on template hybridization region, and prolongs in template hybridization region and template
Stretch between area that there are one dSpacer or C3Spacer to modify.The present invention by reduce template extension area primer length, and
There are upper multiple dSpacer and/or C3Spacer modifications on forward primer and reverse primer, since polymerase cannot identify this kind of repair
Base is adornd, causes primer effectively complementary can not to extend, while not reducing template binding ability, effectively reduces and draw
The probability that object dimerization zoarium generates.
Specifically, in template hybridization region, at interval of 10 bases, there are one dSpacer or C3Spacer to modify.Mould
There are one dSpacer or C3Spacer to modify for every 10 bases in plate hybridization region, between template hybridization region and template extension area
There are one dSpacer or C3Spacer to modify, for blocking primer dimer caused by non-specific binding between primer.It is logical
It crosses after addition dSpacer or C3Spacer modification and carries out high temperature conjunction after complementary series annealing, formed and new can be used for high pass
The library construction of sequence is measured, while reducing the amplification of primer dimer in multiplex polymerase chain re-action, realizes dual area annealing,
Specificity is good.
In the primer of design, template hybridization region and template extension area, template hybridization are followed successively by from 5, -3, extending direction
The length in area is 15-30 bases, and TM values range is in 60-65 degree.The length of template extension area is 10-20 base, TM value ranges
In 45-55 degree, it is used for the extension of template area, the condition of multiplex polymerase chain re-action to be high annealing temperature after first low temperature thermal oxidation
Degree, multiplex polymerase chain re-action using Taq birdss of the same feather flock together synthase or high-fidelity is birdsed of the same feather flock together synthase.
Preferably, template hybridization region and template extension area junction are equipped with dSpacer or C3Spacer, at this
When the subsequent bases of dSpacer or C3Spacer are T, base T is replaced with into base U.As shown in figure 3, in order to coordinate high pass
Measure sequencing library structure, between template hybridization region and template extension area there are one the subsequent bases of dSpacer be located next to T,
When synthesis, T is replaced with into U in favor of being directly connected to after subsequent digestion.Can directly USER digestions and connection, eliminate conventional build
The end in library is repaired, phosphorylation and plus A processes.
In the present invention, the final concentration of 100-300nmol of use of primer, multiple primers are mixed to form multiplex PCR mixture,
In the case where polymerizeing enzyme effect, PCR amplification is carried out, available polymerase is that common Taq birdss of the same feather flock together synthase or high-fidelity is birdsed of the same feather flock together synthase,
Such as：Platinum Taq enzyme winged Sai Mo, (ThermoFisher, PlatinumTMTaq DNA Polymerase Cat:
10966018), the OneTaq thermal starting archaeal dna polymerases (cat of NEB:#M0481), the Q5 polymerases of NEB, (cat:#M0492).
As shown in figure 3, PCR product USER digestions, 3 ' end A PCR structures outstanding are formed after removing U.Meanwhile in conjunction with ligase with connect
Head connection directly forms the library with connector, and connection product carries out magnetic beads for purifying, and PCR amplifies, and forms final library production
Object.
Embodiment 1：The method that primer dimer expands in multiplex polymerase chain re-action is reduced using the present invention
1, the designed PCR primers as follows：
A：Unmodified Primers complementary pair, wherein label underscore is template extension area, non-dashed part is template hybridization region
Unmodified forward primer：
F:GGCAGGAGACTGGCGGTGGACCAGGTGGAGCCGAA
GCCTGTAAACAGGC
Unmodified reverse primer
R:AAAAGTTGCCAAGGTAGCTCAGTCTTGTGCTGCT
GCCTGTTTACAGGCC
B：Primers complementary pair after modification, wherein label underscore is template extension area, non-dashed part is template hybridization
Area
Forward primer after modification
Modify_F：GGCAGGAGACTGGCGG(dSpacer)GGACCAGGTGGAGCCGAA(dSpacer)GGCCTGTAAACAGGC
Reverse primer after modification
Modify_R：AAAAGTTGCCAAGGTAGC(dSpacer)CAGTCTTGTGCTGCT(dSpacer)GCCTGTTTACAGGCC
2, following PCR reactions are carried out using above-mentioned primer：
Using 0.2ml PCR pipes, each pair of primer reacts in super-clean bench according to following system configurations in A/B groups：It is super
20 μ l of fidelity 2X premixed liquids；Primer F(300nM)8μl；Primer R(300nM)8μl；H2O, in total 40 μ l.
Amplification program is arranged：95℃3min；95 DEG C of 15s, 60 DEG C of 20s, 72 DEG C of 4min, 2-4 steps 20 cycle.72℃
4min, 10 DEG C of preservations.
Each pair of primer does 2 parallel laboratory tests, and after PCR, all products carry out electrophoresis detections detection figure, as shown in figure 4,
With unmodified A primer ratios, the B primer pairs modified by dSpacer do not generate primer dimer N between 50-100bp.
Embodiment 2 reduces the method that primer dimer expands in multiplex polymerase chain re-action using the present invention
In the present embodiment, using 120 multipair Mdification primers and unmodified primer, Genomic PCR is carried out.The primer of modification
As described before, it is modified using dSpacer, and in the template hybridization region and the design of template extension area of forward primer and reverse primer
DSpacer, since the present embodiment is without building library subsequent operation, so dU modifications closely are not essential.
1, prepared by primer MIX, and every primer is diluted to 100uM, then non-modified and modification forward primer and reversed
Primer, by Mdification primer and unmodified primers respectively according to 1：1 is mixed into MIX A and B, and every primer takes 4ul to mix to totality
Product 4ml, final concentration 100nM.
2, to each primer mixing MIX, following PCR reactions are carried out；
Using 0.2ml PCR pipes, each pair of primers of A/B react in super-clean bench according to following system configurations：Super fidelity
5 μ l of 2X premixed liquids；8 μ l of PrimerMixA or B；Human gene group DNA 20ng, H2O, in total 30 μ l.
Amplification program is arranged：95℃3min；95 DEG C of 15s, 60 DEG C of 20s, 72 DEG C of 4min, 2-4 steps 2 cycle.95℃
15s, 50 DEG C of 20s, 60 DEG C of 20s, 72 DEG C of 4min, 5-8 steps 18 cycle, 72 DEG C of 4min, 10 DEG C of preservations.
Each pair of primer does 3 parallel laboratory tests, and one negative control for being not added with genome of band.After PCR, all products into
Row electrophoresis detection detection figure, as shown in figure 5, unmodified primer sets MIX PCR, 200 or so purposes band unobvious L, 50-100
Between primer dimer it is very more.As Fig. 6 by the B primer sets MIX that modifies in the apparent L of 200 or so purposes, and in 50-100bp
Between do not generate primer dimer N or very weak.
Embodiment 3 is built library using the method that primer dimer in the reduction multiplex polymerase chain re-action of the present invention expands and is flowed
Journey
The present embodiment carries out Genomic PCR using 440 multipair Mdification primers, and carries out USER digestions and Jian Ku, has completed
Whole Library development flow, and inspection is sequenced.Mdification primer is as described before, is modified using dSpacer, and in template hybridization region and template
Extension area have dSpacer and closely dU modification.
Table 1：The unmodified primer in part of Genomic PCR：
| Segment | F | R |
| Y300 | TCCCTCAGTGGATACTTGCAGGCAGGCTTT | GAGCTGTCAGCCTGCCTAAGCAGAGGAAAA |
| Y369 | TCTGCCTCTGCCATTTTCCTCTGCTTAGGCAGGC | ACAATCAGGCCTGATGTCCTGAGACTAGGGTA |
| Y293 | CATGGATGTCTGGTGTGGCAATTTCTCAACATCATCTGC | AGGCAAGTCCTTGCACGGGCTATCTGGGAAAGGCAG |
| Y332 | TGTTCTTGACTTCCCTGTCTCACTTCATCCTGC | TGCTCAACTTGGACTTGCCTTTGTCATGGT |
| Y305 | ATTTACCTCTAATTCCGTTCCCAGTTGAGCAG | ACTCTATGACCCAGAATGAAAACCCAAGGGGA |
| Y308 | TGTTTCATTGTCTGCAGTGAACCCAGGAAGAGA | CATGACATGAAGCACCTGCTCAACTGGGAACTG |
Table 2：The part Mdification primer of Genomic PCR：
1, prepared by primer MIX, and every primer is diluted to 100Um, then according to 1：1 is mixed into PrimerMIX, every primer
4ul is taken to mix to total volume 2ml, final concentration 200nM.
2, to each primer mixing MIX, PCR reactions are carried out；
Using 0.2ml PCR pipes, by each pair of primer in Mdification primer and unmodified primers according to as follows in super-clean bench
System configurations are reacted：Super 15 μ l of fidelity 2X premixed liquids；PrimerMix 8μl；Human gene group DNA 20ng；H2O, in total
30μl。
Amplification program is arranged：95℃3min；95 DEG C of 15s, 60 DEG C of 20s, 72 DEG C of 4min, 2-4 steps 2 cycle.50℃
20s, 60 DEG C of 20s, 72 DEG C of 4min, 5-8 steps 18 cycle, 72 DEG C of 4min, 10 DEG C of preservations.
3, USER digestions
Prepare reaction mixture, the 2nd step PCR product 30ul according to following proportioning；USER enzymes (NEB, CAT：#M5505)
1ul；PCR instrument is adjusted to 37 degree, heat lid setting 42-45 degree reacts 15min.
4, library connector connects
Prepare reaction mixture according to following proportioning：3rd step USER digestion products 31ul；USER enzymes 1ul；T4DNA
ligase buffer 10ul；Adapter oligo mix(15uM)3ul；DNA ligase 1ul；Add water to total volume
100ul。
PCR instrument is adjusted to 16 degree, reacts 120min, after reaction, reaction product adds 150ul Ampure XP
Beads Beads carry out product purification according to purification process, finally use 20ul system eluted dnas.
5, library PCR reacts：The DNA product 20ul that step 4 purifies；Super 25 μ l of fidelity 2X premixed liquids；Library
Primer F(10uM)2.5μl；Library Primer R (10uM) 2.5 μ l；H2O, in total 30 μ l.
PCR reaction conditions：
In the present invention, PCR reaction products add 150ul Ampure XP Beads Beads, are produced according to purification process
Object purifies, and finally uses 20ul system eluted dnas.Purification step is carried out according to raw work biological PCR purification kit operating procedure.
Library can carry out high-flux sequence, and sequencing result statistics is as shown in Table 3 and Table 4, shows the amplification method of the present invention
For the genome coverage of multiplex PCR capture close to 100%, homogeneity is more than 98%, and in the present invention, homogeneity refers to sequencing fragment
Depth is more than the segment number of average sequencing depth 20% and total segments purpose percentage.
Table 3：Statistical form is sequenced
Table 4：Sequencing data original table (part)
The present invention's reduces the method that primer dimer expands in multiplex polymerase chain re-action using the present invention, carries out
In Genomic PCR, the coverage of genome is high, and close to 100%, homogeneity is more than 98%.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention belong to the scope of protection of present invention.
Claims (8)
1. a kind of reducing the method that primer dimer expands in multiplex polymerase chain re-action, which is characterized in that the method packet
It includes：
Forward primer and reverse primer are obtained, by the forward primer and reverse primer along 5, -3, is designed as on extending direction
Template hybridization region and template extension area；
Be separated with multiple dSpacer and/or C3Spacer modifications between on the template hybridization region, and in the template hybridization region and
There are one dSpacer or C3Spacer to modify between template extension area.
2. the method as described in claim 1, which is characterized in that
The length of the template hybridization region is 15-30 bases, and TM values range is in 60-65 degree.
3. the method as described in claim 1, which is characterized in that
The length of the template extension area is 10-20 base, and TM values range is in 45-55 degree.
4. the method as described in claim 1, which is characterized in that
DSpacer or the subsequent bases of C3Spacer between the template hybridization region and template extension area are T, by base T
Replace with base U.
5. the method as described in claim 1, which is characterized in that
The final concentration of 100-300nmol of use of the primer.
6. the method as described in claim 1, which is characterized in that
The condition of the multiplex polymerase chain re-action is high annealing temperature after first low temperature thermal oxidation.
7. the method as described in claim 1, which is characterized in that
The multiplex polymerase chain re-action using Taq birdss of the same feather flock together synthase or high-fidelity is birdsed of the same feather flock together synthase.
8. the method as described in claim 1, which is characterized in that
In the template hybridization region, at interval of 10 bases, there are one dSpacer or C3Spacer to modify.
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| CN114369649A (en) * | 2022-02-08 | 2022-04-19 | 山东见微生物科技有限公司 | Specific selective amplification and multiplex PCR method and application |
| WO2022148388A1 (en) * | 2021-01-06 | 2022-07-14 | 上海慧众同康生物科技有限公司 | Multi-amplification method |
| CN117568450A (en) * | 2023-11-17 | 2024-02-20 | 厦门飞朔生物技术有限公司 | Improved construction method and application of amplicon library carrying specificity molecular tag |
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Application publication date: 20181106 |