CN105718759B - A kind of bPrimer batch PCR primer design methods based on Primer3 - Google Patents
A kind of bPrimer batch PCR primer design methods based on Primer3 Download PDFInfo
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Abstract
The bPrimer batch PCR primer design methods based on Primer3 that the present invention provides a kind of, the method includes：Obtain the original series of target dna sequence；Extract the high frequency polymorphic site in DNA polymorphism data；The high frequency polymorphic site is marked；The annotated sequence of output token high frequency polymorphic site, the annotated sequence are identical with the bases longs of the original series；The annotated sequence is read, and generates candidate drugs；Screen candidate drugs etc..BPrimer batch PCR primer design methods provided by the invention based on Primer3 can avoid high frequency polymorphic site, reduce and expand failure caused by target group's genetic diversity；It is capable of the specificity of batch detection primer, reduces amplification failure caused by the reasons such as non-specific amplification, primer dimer；It can be used in assessing the specificity of existing primer；Long target fragment can be divided automatically.
Description
Technical field
The present invention relates to PCR primer design fields, more specifically, are related to a kind of based on Primer3's
BPrimer batch PCR primer design methods.
Background technology
PCR (Polymerase Chain Reaction, PCR) is real as most basic molecular biology
Test one of means, can in vitro millions of times ground amplification target dna copy number, thus be widely used in genetic engineering,
The fields such as diagnostic reagent.
One key factor of PCR experiment success or failure is the quality of PCR primer design, therefore, to design better primer,
The PCR primer design software of huge number is invented.Earliest and most basic PCR primer design software is Primer0.5, then
Again the Primer3 being widely used now is devised on the basis of Primer0.5 to increase income PCR primer design software.Primer3
Most basic function include design PCR primer and design hybridization probe, and have many advantages, such as freely, increase income, be cross-platform.Then
Researcher carries out secondary development on the basis of Primer3, forms such as NCBI Primer BLAST, BatchPrimer3
Etc. derived from primer-design software, further expanded the influence power of Primer3.
Although Primer3 and its derivative software are widely applied, cannot still fully meet in actual items
The individual demand of user, there are prodigious rooms for improvement.Primer3 and its derivative primer-design software exist such as 1 institute of table
The problem of showing：
Table 1：Primer3 and its derivative primer-design software there are the problem of
It, can not batch operation by upper table, it can be seen that Primer3 can only once input a segment；BatchPrimer3
Batch operation is improved emphatically, but it cannot prevent amplification failure caused by genetic diversity or homologous gene, it can not be straight
Connect the longer target sequence of processing；NCBI Primer BLAST mainly pass through BLAST technologies (Basic Local Alignment
Search Tool, BLAST) prediction non-specific amplification, but it does not support batch operation；MPRIMER improves emphatically non-specific
Amplification prediction and secondary structure detection, but equally cannot directly handle longer target sequence.
Meanwhile design of primers field is there is also numerous business softwares, such as Primer Premier6.O, LIGO 7,
Primer Analysis Software.Although business software has a clear superiority in terms of hommization and easy expenditure, it is suitble to not yet done
The experimenter for knowing bioinformatics uses；But they as business software, source code is opaque, it can not self-developing expansion
Function is opened up, it also can not voluntarily debugging, it is difficult to and the interaction of other open source softwares, it is cross-platform in poor shape, and also need
Payment authorization is wanted to take.
Invention content
The bPrimer batch PCR primer design methods based on Primer3 that the object of the present invention is to provide a kind of, to solve
Existing PCR primer design method described in background technology prevents genetic diversity because not having when designing PCR primer due to causes to draw
The problem of object design failure.
In order to solve the above technical problem, the present invention provides following technical solutions：
A kind of bPrimer batch PCR primer design methods based on Primer3, the method includes：
S01：Obtain the original series of target dna sequence；
S02：According to the high frequency polymorphic site in the original series extraction DNA polymorphism data of the target dna sequence；
S03：The high frequency polymorphic site is marked；
S04：The annotated sequence of output token high frequency polymorphic site, the base of the annotated sequence and the original series
Length is identical；
S05：The annotated sequence is read, and calculates melting temperature and generates candidate drugs；
S06：Candidate drugs are screened, primer is obtained.
Preferably, the method further includes：The automatic segmentation of long target fragment.
Preferably, the long target fragment it is automatic segmentation include：If the original series of the target dna sequence do not obtain
The original series of the target dna sequence are then equally divided into two subsequences, and separately design primer by candidate drugs；If institute
It states two subsequences and does not obtain candidate drugs, then continue to divide equally by described two subsequences, until the original of the target dna sequence
Beginning sequence has the length of candidate drugs or got subsequence to be less than the length for presetting minimum product.
Preferably, the original series of acquisition target dna sequence described in step S01 include：Build the seat of target dna sequence
File is marked, and every a line of the coordinate file is made to include a genomic coordinates.
Preferably, the form of the genomic coordinates is chrA:B+C forms.
Preferably, the high frequency polymorphic site in extraction DNA polymorphism data described in step S02 includes：From the DNA
High frequency polymorphic site corresponding with the coordinate file of the target dna sequence is extracted in polymorphism data.
Preferably, described in step S03 to the high frequency polymorphic site be marked including：With IUPAC standard degeneracys
Code, "<>" and " [] " respectively in the high frequency polymorphic site mononucleotide high frequency polymorphic site, insertion and deletion mark
Site and target dna sequence are labeled.
Preferably, candidate drugs are screened described in step S06 includes：
Screen out candidate drugs of the melting temperature not in setting range；
Screen out the candidate drugs that mononucleotide high frequency polymorphic site is more than given threshold；
Screen out 3 ' candidate drugs of the end containing mononucleotide high frequency polymorphic site；
Screen out 3 ' candidate drugs of the end containing insertion and deletion marker site；
Screen out the candidate drugs containing degeneracy base within the scope of 3 ' 5 bases in end；
Screen out the candidate drugs of non-specific amplification；
Screen out the candidate drugs there are risk.
Preferably, the candidate drugs for screening out non-specific amplification are to be predicted using iPCRess and In-silicoPCR
Candidate drugs whether non-specific amplification, and the candidate drugs of non-specific amplification are screened out.
The bPrimer batch PCR primer design methods based on Primer3 that the present invention provides a kind of, the method includes：
S01：Obtain the original series of target dna sequence；S02：Original series according to the target dna sequence extract DNA polymorphism
High frequency polymorphic site in data；S03：The high frequency polymorphic site is marked；S04：Output token high frequency is polymorphic
The annotated sequence in property site, the annotated sequence are identical with the bases longs of the original series；S05：Read the annotation sequence
Row, and calculate melting temperature and generate candidate drugs；S06：Candidate drugs are screened, primer is obtained.It is provided by the invention to be based on
The bPrimer batch PCR primer design methods of Primer3 can avoid high frequency polymorphic site, and then reduce because of target group
Failure is expanded caused by genetic diversity.Meanwhile primer design method provided by the invention is capable of the spy of batch detection primer
The opposite sex, and then amplification failure caused by the reasons such as non-specific amplification, primer dimer is reduced, and can be used in assessing existing draw
The specificity of object.
Description of the drawings
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, for those of ordinary skills, do not making the creative labor
Under the premise of, it can also be obtained according to these attached drawings other attached drawings.
Fig. 1 is the flow of the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3
Figure；
Fig. 2 is long mesh in the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3
The schematic diagram that standard film section is divided automatically；
Fig. 3 is that the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 design
ATMe59_F7, KLKB1e6_F12 and L_301_F7 carry out the electrophoretogram of PCR experiment；
Fig. 4 is that the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 design
L_301_F7 carries out the peak value figure of PCR experiment；
Fig. 5 is that the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 design
Chr1-51-F0, Chr1-69-F0 carry out the electrophoretogram of PCR experiment；
Fig. 6 is that the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 design
Chr1-51-F0 carries out the peak value figure of PCR experiment；
Fig. 7 is that the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 design
Chr1-69-F0 carries out the peak value figure of PCR experiment；
Fig. 8 is that the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 design
Depth map is sequenced in mammary cancer risk gene BRCA1, BRCA2 whole exon.
Specific implementation mode
The method of Batch Design targeting PCR primer provided in an embodiment of the present invention, solves existing PCR primer design method
The problem of preventing genetic diversity because not having when designing PCR primer due to design of primers caused to fail.
In order to make those skilled in the art more fully understand the technical solution in the embodiment of the present invention, and make of the invention real
The above objects, features, and advantages for applying example can be more obvious and easy to understand, below in conjunction with the accompanying drawings to the technology in the embodiment of the present invention
Scheme is described in further detail.
Attached drawing 1 is please referred to, attached drawing 1 shows the bPrimer batches PCR provided in an embodiment of the present invention based on Primer3
The flow chart of primer design method.It can be seen that the bPrimer provided in an embodiment of the present invention based on Primer3 from attached drawing 1
Batch PCR primer design method includes：
S01：The original series of the target dna sequence of FASTA formats are obtained from genome sequence file；
S02：The high frequency that original series according to the target dna sequence extract in the DNA polymorphism data of VCF formats is more
State property site；
S03：The high frequency polymorphic site is marked；
S04：The annotated sequence of output token high frequency polymorphic site, the base of the annotated sequence and the original series
Length is identical, and the sequence in sequential file is also consistent；
S05：The annotated sequence is read, and calculates melting temperature (melting temperature, Tm) and generates candidate
Primer；
S06：Candidate drugs are screened, primer is obtained.
BPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 can avoid high frequency
Polymorphic site, and then reduce and expand failure caused by target group's genetic diversity, reach designed primer amplification
Successful probability.
Further, the bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 are also wrapped
Include the automatic segmentation of long target fragment.If the original series of target dna sequence do not obtain candidate drugs, need to longer mesh
The original sequence-stretches of mark DNA sequence dna are split, to realize the design of primer.To the original of longer target dna sequence
When sequence fragment is split, the original sequence-stretches of longer target dna sequence are first equally divided into two subsequences, then
Design of primers is carried out respectively to two subsequences.If still not obtaining candidate drugs in above-mentioned two subsequence, by two
Subsequence carries out average segmentation again, and carries out design of primers respectively again.If still not waited in the subsequence being divided into
Primer is selected, then continues average segmentation, until the original series of target dna sequence have the length of candidate drugs or got subsequence
Less than the length for presetting minimum product.
Specifically, attached drawing 2 is please referred to, attached drawing 2 shows provided in an embodiment of the present invention based on Primer3's
The schematic diagram that long target fragment is divided automatically in bPrimer batch PCR primer design methods.It is learnt from attached drawing 2, if former target
The coordinate in region be (A, B), then two points arrive sub-goal region 1 (A, A+int ((B-A+S)/2)) and 2 (A+ of sub-goal region
Int ((B-A-S)/2), B)；Wherein, S is preset overlapping region length.Certainly, in practical applications, long target fragment
Automatic dividing function use Implementation of pseudocode.
In the step S01 of bPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3
The original series for obtaining target dna sequence include building the coordinate file of target dna sequence, and make each of the coordinate file
Row includes a genomic coordinates, and then forms the original series of target dna sequence.Form the genomic coordinates of coordinate file
Form is chrA:The form of B+C, such as MED12_exon1chrX:70360484+70360706.Wherein, A indicates that chromosome is compiled
Number, B and C indicate the starting and ending coordinate of target area respectively, and starting and ending coordinate is 1-based, i.e. chromosome
First base coordinate be 1；"+" number indicates that the normal chain of chromosome, "-" number indicate the minus strand of chromosome, such as " chrX:
70360484+70360706 " means that the region of the bit base from 70360484 to 70360706 in X chromosome normal chain.In institute's structure
In the coordinate file for the target dna sequence built, target designation is arranged in first row, since target designation can influence target area and draw
The name of object, therefore target designation is separated with genomic coordinates with tab, length of recommended be no more than 15 English, number or
Underscore character.If not providing target designation, acquiescence regard genomic coordinates itself as target designation.
The structure of coordinate file based on target dna sequence, it is provided in an embodiment of the present invention based on Primer3's
BPrimer batch PCR primer design methods need more from DNA in the high frequency polymorphic site in extracting DNA polymorphism data
State property extracting data high frequency polymorphic site corresponding with the coordinate file of constructed target dna sequence.It is extracting
It after high frequency polymorphic site, needs to be labeled extracted high frequency polymorphic site, with IUPAC standard degeneracys when mark
Code, "<>" and " [] " respectively to the mononucleotide high frequency polymorphic site (Single in the high frequency polymorphic site
Nucleotide Polymorphisms, SNP), insertion and deletion marker site (insertion-deletion, INDEL) and mesh
Mark DNA sequence dna is labeled, and wherein IUPAC standards degeneracy code is as shown in table 2.
Table 2：IUPAC standard degeneracy codes
The high frequency polymorphic site being marked is exported, and forms annotated sequence, is formed by annotated sequence and original
The bases longs of beginning sequence are identical, and annotated sequence must be consistent with sequence of the original series in sequential file, to avoid rear
Continuous result of calculation error.Further, annotated sequence is read, and candidate drugs are generated according to annotated sequence by Primer3 softwares
With calculating Tm.Due to when calculating Tm using annotated sequence, the Tm values due to annotated sequence contains degeneracy code and there are error, because
This then needs to recalculate Tm values, Tm values at this time are counted using original series when annotated sequence and original series all exist
It calculates.If designed candidate drugs include IUPAC degeneracy codes, and do not provide original series, then can be calculated by Primer3 softwares
The Tm of be possible to primer, and export the value range of Tm.
BPrimer batch PCR primer design methods provided in an embodiment of the present invention based on Primer3 are to candidate drugs
It is main to consider the following content when being screened：Screen out candidate drugs of the Tm not in setting range；It is more than to set to screen out SNP
Determine the candidate drugs of threshold value；Screen out 3 ' candidate drugs of the end containing SNP；Screen out 3 ' candidate drugs of the end containing INDEL；Screen out 3 '
Hold the candidate drugs containing degeneracy base within the scope of 5 bases；Screen out the candidate drugs of non-specific amplification；Screen out that there are risks
Candidate drugs.
Wherein, temperature when half, Tm values are lost when heated due to double-spiral structure that melting temperature Tm is DNA
The important reference of annealing temperature is reacted for PCR, and then when carrying out design of primers, need to screen out Tm values not in user's setting
Candidate drugs in range.
If it is designed go out candidate drugs there are SNP polymorphic sites and INDEL polymorphic sites, can cause actually to answer
Being reduced in the amplification efficiency of certain samples in can not even expand to obtain product, therefore for there are polymorphic positions on primer
Point, the amplification efficiency in order to improve primer need to screen out candidate drugs of the SNP more than given threshold, 3 ' candidates of the end containing SNP
Primer and 3 ' candidate drugs of the end containing INDEL.
When containing degeneracy base within the scope of 5 bases at 3 ' ends, need to screen out the candidate drugs, specifically, degeneracy
Then 5 base ranges that Primer3 softwares filter out 3 ' ends are arranged in base lowercase, standard base capitalization
Include the candidate drugs of lowercase.
For the candidate drugs of non-specific amplification, the embodiment of the present invention is by using iPCRess softwares and In-
SilicoPCR softwares come predicting candidate primer whether non-specific amplification, so that the candidate drugs of non-specific amplification be screened out.
Predicting candidate primer whether non-specific amplification when, recursive call iPCRess, only intercept a 3 ' end sub-sequences come build configuration
The range of file and specified maximum mispairing number, to complete the prediction of candidate drugs non-specific amplification, wherein predicting candidate
The standard of primer non-specific amplification refers to table 3.
Table 3：Predicting candidate primer non-specific amplification standard scale
To having carried out the above-mentioned candidate drugs screened out, it is also necessary to be screened out to the candidate drugs there are high risk.
It is screened out according to preset filter criteria when screening out the candidate drugs of high risk, sequence is preferentially chosen in principle in screening
Number is low, primer positioned at table forefront, which refers to table 4.In practical applications, by standard can according to
The experience at family is set.
Table 4：Filter criteria
| Project | Pass through standard |
| GC% | 40%≤GC%≤60% |
| Any_compl(MPprimer) | ≥-6 |
| 3'_compl | ≤6 |
| Pair_3'_compl | ≤6 |
| Pair_Any_compl | ≤9 |
| In-silico PCR iPCRess | Referring to table 3 |
BPrimer batch PCR primer design methods provided by the invention based on Primer3 can avoid high frequency polymorphism
Site, and then reduce and expand failure caused by target group's genetic diversity.Meanwhile design of primers side provided by the invention
Method is capable of the specificity of batch detection primer, and then reduces amplification failure caused by the reasons such as non-specific amplification, primer dimer,
And it can be used in assessing the specificity of existing primer.
The embodiment of the present invention additionally provides the verification to primer design method of the present invention, to the guiding price of confirmatory experiment
Value.In the design of primers stage, the primer of 3 pairs of typical prediction non-specific amplifications is picked, and picks 2 couples of prediction spies at random
The primer of different amplification, wherein the primer of 3 pairs of typical prediction non-specific amplifications is ATMe59_F7, KLKB1e6_F12 and L_
The primer of 301_F7,2 pairs of prediction specific amplifieds are Chr1-51-F0, Chr1-69-F0, and above-mentioned primer is provided according to the present invention
Predicting candidate primer whether the method for non-specific amplification predicts PCR's as a result, Caliper LabChip GX instrument is used in combination
Device verifies the product of practical PCR experiment.In this experiment, except next using iPCRess softwares and In-silicoPCR softwares
Whether predicting candidate primer also uses two web page tools of NCBI Primer Blast and MFEPrimer3 outside non-specific amplification
As a comparison, the result of prediction refers to table 5.
Table 5：The prediction result of non-specific amplification
, it can be seen that the electrophoresis result of the PCR experiment of ATMe59_F7 has multi-ribbon or without amplification from table 5；KLKB1e6_
The electrophoresis result of the PCR experiment of F12 has multi-ribbon；The electrophoresis result of the PCR experiment of L_301_F7 has multi-ribbon or without amplification；
The electrophoresis result of the PCR experiment of Chr1-51-F0, Chr1-69-F0 only has single band, that is, specific amplified occurs.
The result such as attached drawing 3-7 of the product of practical PCR experiment, attached drawing are verified using Caliper LabChip GX instruments
3-7 respectively illustrates the electrophoretogram and peak value figure of PCR experiment, wherein 17,18 and 19 in attached drawing 3 indicate ATMe59_ respectively
The electrophoresis result figure of F7, KLKB1e6_F12 and L_301_F7, G13 the and G14 items in attached drawing 5 indicate respectively Chr1-51-F0 and
The electrophoresis result figure of Chr1-69-F0., it can be seen that the PCR product of ATMe59_F7 is without apparent band from attached drawing 3-7, and
The PCR product of KLKB1e6_F12 and L_301_F7 has multiple main bands, and the size of primary product band in 1000bp or less
It is close with prediction result；The PCR product of Chr1-51-F0 only has a faint 662bp non-specificity band, there is a small amount of sheet
The PCR product specific amplification in the areas Duan Hu, Chr1-69-F0 is good, and there are a small amount of dimers, and the specificity of two primers is all very
It is good, meet expection.Illustrate that the consistency of non-specific prediction technique and experimental result provided in an embodiment of the present invention is preferable as a result,
And then can show that the specificity of PCR experiment can be effectively predicted in non-specific prediction technique provided in an embodiment of the present invention, and can
Effectively primer to be instructed to select.
The bPrimer batch PCR primer design methods based on Primer3 that embodiment provides to illustrate the invention can be criticized
Amount design PCR primer, the embodiment of the present invention also set the PCR primer of 6 breast cancer related genes full exon sequencing
It counts, and verifies the effect of design of primers with the result of practical PCR experiment, the essential information of target gene refers to table 6.
Table 6：The essential information of target gene
As seen from Table 6, it includes 123 exons that 6 genes, which have altogether, devises 133 pairs of candidate drugs altogether, and protect
Each exon is demonstrate,proved completely to be covered by one or more pairs of candidate drugs.When carrying out design of primers, 133 pairs of candidate drugs pass through respectively
The design for crossing 4 batches obtains, and is screened according to filter criteria provided in an embodiment of the present invention.Divide from PCR experiment result
It analyses, there is electrophoresis experiment of 130 pairs of primers after carrying out PCR amplification to have the correct band of size in 133 pairs of candidate drugs, and meet
PCR results are qualified；In 3 pairs of failed candidate drugs, there are 2 pairs of primers to cause to predict since Tm values are relatively low in design
The result is that non-specific amplification；Only a unsuccessful primer of PCR can not be predicted with design data, and the success rate of this experiment is high
In 97.7%.
The bPrimer batch PCR primer design methods based on Primer3 that embodiment provides to illustrate the invention can transport
For targeting in high-flux sequence, the embodiment of the present invention devises the full exon sequencing of mammary cancer risk gene BRCA1, BRCA2
Required targeting primer, and corresponding to 48 encoded exons of the two genes.By Fluidigm Access Array
Target fragment is expanded, is then sequenced on Illumina MiSeq high-flux sequence platforms, what final information analysis obtained
Depth profile, the distribution map please refer to attached drawing 8., it can be seen that 123 amplicons all Successful amplifications from attached drawing 8, wherein most
Low depth is 67, and highest depth is 516, and difference is less than 10 times.Amplicon mean depth is 155, and all amplicons are all put down at 5 times
In equal depth.It is considered that the sequencing depth of each amplicon is uniform, sequencing quality meets expection.
BPrimer batch PCR primer design methods provided by the invention based on Primer3 can avoid high frequency polymorphism
Site, and then reduce and expand failure caused by target group's genetic diversity.Meanwhile design of primers side provided by the invention
Method is capable of the specificity of batch detection primer, and then reduces amplification failure caused by the reasons such as non-specific amplification, primer dimer,
And it can be used in assessing the specificity of existing primer.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within made by modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of bPrimer batch PCR primer design methods based on Primer3, which is characterized in that the Primer3 is
Primer3 increases income PCR primer design software；The method includes：
S01：Obtain the original series of target dna sequence；It is described obtain target dna sequence original series include：Build target
The coordinate file of DNA sequence dna, and every a line of the coordinate file is made to include a genomic coordinates；The coordinate file is formed
The original series of the target dna sequence；
S02：According to the high frequency polymorphic site in the original series extraction DNA polymorphism data of the target dna sequence；It is described
High frequency polymorphic site includes mononucleotide high frequency polymorphic site and insertion and deletion marker site；
S03：The high frequency polymorphic site is marked；
S04：The annotated sequence of output token high frequency polymorphic site, the bases longs of the annotated sequence and the original series
It is identical；
S05：The annotated sequence is read, and calculates melting temperature and Mass production candidate drugs；
S06：Candidate drugs are screened, primer is obtained.
2. the bPrimer batch PCR primer design methods according to claim 1 based on Primer3, which is characterized in that
The method further includes：The automatic segmentation of long target fragment.
3. the bPrimer batch PCR primer design methods according to claim 2 based on Primer3, which is characterized in that
The long target fragment it is automatic segmentation include：
If the original series of the target dna sequence do not obtain candidate drugs, the original series of the target dna sequence are put down
Two subsequences are divided into, and separately design primer；
If described two subsequences do not obtain candidate drugs, described two subsequences are continued to divide equally, until the target dna
The original series of sequence have the length of candidate drugs or got subsequence to be less than the length for presetting minimum product.
4. the bPrimer batch PCR primer design methods according to claim 2 based on Primer3, which is characterized in that
The form of the genomic coordinates is chrA:B+C forms, wherein chr is chromosome name prefix；A is chromosome numbers；B and
C indicates origin coordinates and end coordinate of the target dna sequence on homologue respectively.
5. the bPrimer batch PCR primer design methods according to claim 1 based on Primer3, which is characterized in that
Described in step S02 extract DNA polymorphism data in high frequency polymorphic site include：It is carried from the DNA polymorphism data
Take high frequency polymorphic site corresponding with the coordinate file of the target dna sequence.
6. the bPrimer batch PCR primer design methods according to claim 1 based on Primer3, which is characterized in that
Described in step S03 to the high frequency polymorphic site be marked including：
With IUPAC standard degeneracys code, "<>" and " [] " it is polymorphic to the mononucleotide high frequency in the high frequency polymorphic site respectively
Property site, insertion and deletion marker site and target dna sequence are labeled.
7. the bPrimer batch PCR primer design methods according to claim 6 based on Primer3, which is characterized in that
Candidate drugs are screened described in step S06 includes：
Screen out candidate drugs of the melting temperature not in setting range；
Screen out the candidate drugs that mononucleotide high frequency polymorphic site is more than given threshold；
Screen out 3 ' candidate drugs of the end containing mononucleotide high frequency polymorphic site；
Screen out 3 ' candidate drugs of the end containing insertion and deletion marker site；
Screen out the candidate drugs containing degeneracy base within the scope of 3 ' 5 bases in end；
Screen out the candidate drugs of non-specific amplification；
Screen out the candidate drugs there are risk.
8. the bPrimer batch PCR primer design methods according to claim 7 based on Primer3, which is characterized in that
The candidate drugs for screening out non-specific amplification are to be using iPCRess softwares and In-si licoPCR software prediction candidate drugs
No non-specific amplification, and the candidate drugs of non-specific amplification are screened out.
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| CN106367481B (en) * | 2016-08-26 | 2019-11-08 | 广州永诺健康科技有限公司 | A kind of multiple PCR primer expanding BRCA1/2 gene and a kind of design method of multiple PCR primer |
| CN107937497A (en) * | 2017-11-29 | 2018-04-20 | 拓普基因科技（广州）有限责任公司 | A kind of multiple PCR primer design method based on Primer3 |
| CN110491448B (en) * | 2019-07-15 | 2023-02-07 | 广州奇辉生物科技有限公司 | Method, system, platform and storage medium for processing PCR primers |
| CN110957005B (en) * | 2019-08-19 | 2023-03-24 | 北京康普森生物技术有限公司 | Design of primer for amplicon sequencing and construction method of amplicon sequencing library |
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| CN103571833A (en) * | 2013-11-18 | 2014-02-12 | 四川农业大学 | Design method of SSR label primer and wheat SSR label primers |
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| CN101475988A (en) * | 2009-02-03 | 2009-07-08 | 戴立忠 | Design method for realtime fluorescent quantitative PCR experiment interior label |
| CN103571833A (en) * | 2013-11-18 | 2014-02-12 | 四川农业大学 | Design method of SSR label primer and wheat SSR label primers |
| CN104673884A (en) * | 2014-05-24 | 2015-06-03 | 四川农业大学 | Method of developing polymorphic EST-SSR marker by utilizing complete genome and EST data |
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