CN110491448A - A kind of method, system, platform and storage medium handling PCR primer - Google Patents
A kind of method, system, platform and storage medium handling PCR primer Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, and in particular to a kind of method, system, platform and storage medium for handling PCR primer.Obtain target primer data information to be processed；According to target primer data information to be processed, coordinate data of the target primer region on reference genome is obtained；Division is split to target primer region；The index object area completed is divided according to segmentation and refers to genome, obtains the DNA sequence dna of target primer region；According to the DNA sequence dna and custom parameter of target primer region, processing obtains candidate drugs；Candidate drugs are dispensed into different groupings, and generate PCR primer design report.The full-automatic design that the full exon of gene, the site snp, transcript and the multiple PCR primer of other hot spot regions may be implemented, can greatly shorten the design time of such primer, but also all standing of target area may be implemented.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of method, system, platform and storage for handling PCR primer
Medium.
Background technique
Polymerase chain reaction abbreviation PCR (Polymerase Chain Reaction) (also known as: polymerase chain reaction)
PCR is a kind of method of external enzyme' s catalysis specific DNA fragment, by high-temperature denaturation, low-temperature annealing (renaturation) and appropriate temperature extension etc.
Reaction composition a cycle, circulation carry out, and rapidly amplify target DNA, have high specificity, high sensitivity, operation letter
Just, the features such as time saving.It cannot be only used for the basic research such as Gene Isolation, clone and nucleic acid sequence analysis, can also be used in disease
Diagnosis or any have DNA, the place of RNA.And multiplex PCR (multiplex PCR), also known as Multiplex PCR or compound
PCR, it is the PCR reaction for adding two pairs or more primers in same PCR reaction system, while amplifying multiple nucleic acid fragments,
Its reaction principle, reaction reagent and operating process are identical as general PCR, and multiple PCR technique is in high-throughput gene sequencing technology
The important technical that the DNA fragmentation of capture particular region of interest is sequenced.
Most PCR application at present is all the object of research with certain genes or certain hot spot regions, and biological in gene
Learn the exon region that the mostly important part of meaning is gene.And in current PCR design method, manual queries are needed mostly
Public database website obtains the exon fragment sequence or hot spot target regional sequence of target gene, then utilizes design of primers
Software is manually designed, and finally also needs to be checked the specificity of primer with manually method and with the presence or absence of primer dimer
Situations such as.These steps are comparatively laborious, time-consuming and laborious.
Summary of the invention
For above rapid comparatively laborious, the time-consuming and laborious technical problem, the present invention provides a kind of side for handling PCR primer
The full exon of gene, the site snp, transcript and other hot spot regions may be implemented in method, system, platform and storage medium
The full-automatic design of multiple PCR primer, can greatly shorten the design time of such primer, but also target area may be implemented
All standing.
A method of processing PCR primer, the method specifically comprise the following steps:
Obtain target primer data information to be processed；
According to target primer data information to be processed, coordinate data of the target primer region on reference genome is obtained；
Division is split to target primer region；
The index object area completed is divided according to segmentation and refers to genome, obtains the DNA sequence dna of target primer region；
According to the DNA sequence dna and custom parameter of target primer region, processing obtains candidate drugs；
Candidate drugs are dispensed into different groupings, and generate PCR primer design report.
Further, the target primer data information specifically includes:
Target gene title, No. rs of the site ncbi database snp, transcript number and hot spot region.
It is further, described that division is split to target primer region, specifically:
According to customized expanding fragment length range, longer target area is divided into lesser target primer region.
Further, the custom parameter, specifically includes:
Expanding fragment length range, primer G/C content range, primer annealing temperature range, returns and draws primer length range
Object quantity and maximum annealing temperature difference.
Further, candidate drugs are dispensed into different groupings in step, and generate PCR primer design report it
In, it further comprises the steps of:
Obtain design of primers parameter information, primer grouping information, primer sequence and primer attribute.
To achieve the above object, the present invention also provides a kind of system for handling PCR primer, the system is specifically included:
First acquisition unit, for obtaining target primer data information to be processed；
Second acquisition unit, for obtaining target primer region in reference base according to target primer data information to be processed
Because of the coordinate data in group；
Divide division unit, for being split division to target primer region；
Third acquiring unit obtains target and draws for dividing the index object area completed according to segmentation and referring to genome
The DNA sequence dna of object area；
Processing unit, for the DNA sequence dna and custom parameter according to target primer region, processing obtains candidate drugs；
Generation unit for candidate drugs to be dispensed into different groupings, and generates PCR primer design report.
Further, the system further include:
First obtains module, for obtaining design of primers parameter information, primer grouping information, primer sequence and primer category
Property.
To achieve the above object, the present invention also provides a kind of platforms for handling PCR primer, comprising:
Processor, memory and processing PCR primer platform courses program；
Wherein the processing PCR primer platform courses program, the processing PCR primer platform are executed in the processor
Control program is stored in the memory, and the processing PCR primer platform courses program realizes the processing PCR
The method and step of primer.
To achieve the above object, the present invention also provides a kind of computer-readable storage mediums, described computer-readable
Storage medium be stored with processing PCR primer platform courses program, the processing PCR primer platform courses program, realize described in
Handle the method and step of PCR primer.
Compared with prior art, the invention has the following advantages:
The present invention by it is a kind of handle PCR primer method,
Obtain target primer data information to be processed；
According to target primer data information to be processed, coordinate data of the target primer region on reference genome is obtained；
Division is split to target primer region；
The index object area completed is divided according to segmentation and refers to genome, obtains the DNA sequence dna of target primer region；
According to the DNA sequence dna and custom parameter of target primer region, processing obtains candidate drugs；
Candidate drugs are dispensed into different groupings, and generate PCR primer design report.
And correspondingly system unit and module:
First acquisition unit, for obtaining target primer data information to be processed；
Second acquisition unit, for obtaining target primer region in reference base according to target primer data information to be processed
Because of the coordinate data in group；
Divide division unit, for being split division to target primer region；
Third acquiring unit obtains target and draws for dividing the index object area completed according to segmentation and referring to genome
The DNA sequence dna of object area；
Processing unit, for the DNA sequence dna and custom parameter according to target primer region, processing obtains candidate drugs；
Generation unit for candidate drugs to be dispensed into different groupings, and generates PCR primer design report.Into one
Step ground, the system further include:
First obtains module, for obtaining design of primers parameter information, primer grouping information, primer sequence and primer category
Property.
And correspondingly platform and storage medium；
The complete of the full exon of gene, the site snp, transcript and the multiple PCR primer of other hot spot regions may be implemented
Automated Design, can greatly shorten the design time of such primer, but also all standing of target area may be implemented.
Detailed description of the invention
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, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing.
Fig. 1 is a kind of method framework flow diagram for handling PCR primer of the present invention；
Fig. 2 is a kind of method framework preferred embodiment schematic diagram for handling PCR primer of the present invention；
Fig. 3 is a kind of system architecture schematic diagram for handling PCR primer of the present invention；；
Fig. 4 is a kind of platform architecture schematic diagram for handling PCR primer of the present invention；
Fig. 5 is computer-readable storage medium configuration diagram in an embodiment of the present invention；
The object of the invention is realized, the embodiments will be further described with reference to the accompanying drawings for functional characteristics and advantage.
Specific embodiment
Purposes, technical schemes and advantages to facilitate the understanding of the present invention are clearer, with reference to the accompanying drawing and have
The invention will be further described for the embodiment of body, and those skilled in the art can be by content disclosed in the present specification easily
Understand further advantage and effect of the invention.
The present invention also can be implemented or be applied by other different specific examples, and the various details in this specification is also
Various modifications and change can be carried out without departing from the spirit of the present invention based on different viewpoints and application.
It is to be appreciated that if relating to directionality instruction (such as up, down, left, right, before and after ...) in the embodiment of the present invention,
Then directionality instruction be only used for explain under a certain particular pose (as shown in the picture) between each component relative positional relationship,
Motion conditions etc., if the particular pose changes, directionality instruction is also correspondingly changed correspondingly.
In addition, being somebody's turn to do " first ", " second " etc. if relating to the description of " first ", " second " etc. in the embodiment of the present invention
Description be used for description purposes only, be not understood to indicate or imply its relative importance or implicitly indicate indicated skill
The quantity of art feature." first " is defined as a result, the feature of " second " can explicitly or implicitly include at least one spy
Sign.It secondly, the technical solution between each embodiment can be combined with each other, but must be with those of ordinary skill in the art's energy
Based on enough realizations, when the combination of technical solution appearance is conflicting or cannot achieve, it will be understood that this technical solution
In conjunction with being not present, also not the present invention claims protection scope within.
Preferably, a kind of method for handling PCR primer of the present invention is applied in one or more terminal or server.
The terminal be it is a kind of can be according to the instruction for being previously set or store, automatic progress numerical value calculating and/or information processing are set
Standby, hardware includes but is not limited to microprocessor, specific integrated circuit (Application Specific Integrated
Circuit, ASIC), programmable gate array (Field-Programmable Gate Array, FPGA), digital processing unit
(Digital Signal Processor, DSP), embedded device etc..
The terminal can be desktop PC, notebook, palm PC and cloud server etc. and calculate equipment.It is described
Terminal can carry out human-computer interaction by modes such as keyboard, mouse, remote controler, touch tablet or voice-operated devices with client.
The present invention is to realize a kind of method, system, platform and storage medium for handling PCR primer.
As shown in Figure 1, being the flow chart of the method for processing PCR primer provided in an embodiment of the present invention.
In the present embodiment, the method for the processing PCR primer, can be applied to the terminal for having display function or solid
Determine in terminal, the terminal be not limited to PC, smart phone, tablet computer, the desktop computer for being equipped with camera or
All-in-one machine etc..
What the method for the processing PCR primer also can be applied to be attached by terminal and by network and the terminal
In the hardware environment that server is constituted.Network includes but is not limited to: wide area network, Metropolitan Area Network (MAN) or local area network.The embodiment of the present invention
The method of processing PCR primer can be executed by server, can also be executed, be can also be by server and end by terminal
End is common to be executed.
For example, can directly integrate method institute of the invention at the terminal for the terminal for carrying out processing PCR primer
The function of the processing PCR primer of offer, or installation is for realizing the client of method of the invention.For another example, the present invention is mentioned
The method of confession can operate in server in the form of Software Development Kit (Software Development Kit, SDK)
Etc. in equipment, the interface of the function of processing PCR primer, terminal or other equipment are provided in the form of SDK and are connect by provided
The function of processing PCR primer can be realized in mouth.
As shown in Figure 1, the method specifically comprises the following steps the present invention provides a kind of method for handling PCR primer,
The sequence of step can change in the flow chart according to different requirements, and certain steps can be omitted.
Obtain target primer data information to be processed；
According to target primer data information to be processed, coordinate data of the target primer region on reference genome is obtained；
Division is split to target primer region；
The index object area completed is divided according to segmentation and refers to genome, obtains the DNA sequence dna of target primer region；
According to the DNA sequence dna and custom parameter of target primer region, processing obtains candidate drugs；
Candidate drugs are dispensed into different groupings, and generate PCR primer design report.
Specifically, the target primer data information specifically includes:
Target gene title, No. rs of the site ncbi database snp, transcript number and hot spot region.
Described is split division to target primer region, specifically:
According to customized expanding fragment length range, longer target area is divided into lesser target primer region.
The custom parameter, specifically includes:
Expanding fragment length range, primer G/C content range, primer annealing temperature range, returns and draws primer length range
Object quantity and maximum annealing temperature difference.
In embodiments of the present invention, candidate drugs are dispensed into different groupings in step, and generate PCR primer design
Among report, further comprise the steps of:
Obtain design of primers parameter information, primer grouping information, primer sequence and primer attribute.
That is, as shown in Fig. 2, to realize the full-automatic of the full exon of gene, the site snp or other hot spot regions
Design of primers, the present invention include with lower module, that is, method and step:
Step 1, inputs the target of design primer, and input type includes target gene title, the site ncbi database snp
No. rs, transcript number, hot spot region, run getTar.py program, obtain coordinate of the target area on reference genome；
Specifically, the format of input file is as shown in the table, can be with polymorphic type, including passes through Gene Name (such as EGFR) and obtain
It takes exon region (Exon) or protein encoding regions (CDS), exon target is obtained by transcript number (such as NM_005228)
Region and encoding histone target area (CDS), design of primers target is obtained by genomic coordinates (chr:123456-123457)
Region and by the automatic acquisition target area of No. rs (such as rs13035) acquisitions design of primers target area batch in reference gene
Position in group:
| query | type |
| rs13035 | snp |
| chr:123456-123457 | Coordinate |
| EGFR | Gene Exon Only |
| BRAF | Gene CDS Only |
| NM_005228 | Transcript CDS only |
| NM_005228 | Transcript Exon only |
Step 2, according to customized expanding fragment length range, longer target area is divided into lesser mesh by segmentation
Mark region；
That is, customized design of primers parameter.Set the primer quantity returned, range 1-999999；Setting is drawn
Object length, setting range 15-40；Set primer G/C content, setting range 15%-85%；Primer melting temperatures are set, if
Determining range is 50-70 DEG C；The maximum consecutive identical base quantity of setting, setting range 3-6；, set expanding fragment length, setting
Range is 60-400.Setting maximum divides pool quantity, setting range 1-5.It can be wanted according to actual experiment in present example
It asks, the customized above various parameters.
The target area divided is corresponded to the mankind with reference on genome, gets the DNA sequence of target area by step 3
Column；
Step 4, by expanding fragment length range, primer length range, primer G/C content range, primer annealing temperature model
It encloses, return to the custom parameters such as primer quantity, maximum annealing temperature difference and write in a document files, with this document and step
The DNA sequence dna obtained in rapid three runs primer software as input parameter, and the candidate that acquisition meets custom parameter condition draws
Object；
Step 5, using the obtained candidate drugs of step 4 as input file, the primer run based on blast software is special
Opposite sex detection, screening obtain the candidate drugs of specific amplification；
Step 6 reads the candidate drugs for the specific amplification that screening obtains, and runs drawing based on pooler software
Object dimer screens to obtain the candidate drugs of primer free dimer；In the design of full exon multiple PCR primer, because needing to cover
Whole exon regions can have the case where overlap (amplified fragments are overlapped mutually), and candidate obtained in step draws in reading
Object runs the overlap detection based on pooler software, if there is overlap situation, then automatically by candidate drugs point
It is attached in different groupings, avoids the occurrence of multiple PCR primer overlap.
Step 7, reads design of primers parameter information, primer grouping information, primer sequence, primer attribute, and integration is output to
In a document files to get arrive final multiple PCR primer design result.
It in other words, in embodiments of the present invention, is the complete of the full exon of realization gene, the site snp or other hot spot regions
Automatic design of primers, the present invention include with lower module:
Step 1: the target of input design primer, input type includes target gene title, ncbi database snp/rs
Number, transcript number, hot spot region, run getTar.py program, obtain coordinate of the target area on reference genome；Input
The format of file is as shown in the table, can be with polymorphic type, including passes through Gene Name (such as EGFR) acquisition exon region (Exon)
Or protein encoding regions (CDS), exon target area and encoding histone target obtained by transcript number (such as NM_005228)
Region (CDS) obtains design of primers target area by genomic coordinates (chr:123456-123457) and by No. rs
(such as rs13035) obtains the automatic position for obtaining target area on reference genome of design of primers target area batch:
| query | type |
| rs13035 | snp |
| chr:123456-123457 | Coordinate |
| EGFR | Gene Exon Only |
| BRAF | Gene CDS Only |
| NM_005228 | Transcript CDS only |
| NM_005228 | Transcript Exon only |
Step 2: customized design of primers parameter.Set the primer quantity returned, range 1-999999；Set primer
Length, setting range 15-40；Set primer G/C content, setting range 15%-85%；Set primer melting temperatures, setting
Range is 50-70 DEG C；The maximum consecutive identical base quantity of setting, setting range 3-6；, expanding fragment length is set, model is set
It encloses for 60-400.Setting maximum divides pool quantity, setting range 1-5.The present invention can be required according to actual experiment, customized
The above various parameters.
Step 3: target area is divided, according to customized expanding fragment length range, operational objective region segmentation journey
Longer target area is divided into lesser target area by sequence, segmentation, such as the expanding fragment length range set is 80-200,
And target area length is 2000, will be automatically segmented into 10 or more lesser target areas；
Step 4: the target area divided is corresponded to the mankind with reference on genome (hg19), target area is got
DNA sequence dna, the format of file is fasta；
Step 5: operation primer3 software, obtains all candidate drugs for meeting custom parameter condition, each region
The primer for returning to setting quantity is saved in candidate drugs destination file；
Step 6: circulation reads each pair of primer using the obtained candidate drugs of step 5 as input file, run with blast
Based on software, found out using heuristic search with reference on genome to candidate drugs to relevant sequence, it is such as only unique
Corresponding sequence, then primer specific, if any more than two corresponding sequences, then primer is non-specific.Screening obtains specific amplification
Candidate drugs then return to previous step such as without specific primer, return to the candidate drugs of another set setting quantity；
Preferably, step 6 further includes steps of
1, it removes the low complex degree in primer sequence and has the region of concatenation polyisomenism, by k every in primer sequence
Combinatorics on words is made into a table, lists the word group of our all possibility of concern, by these after screening remaining high score word
Group is organized into the structure of fast search tree.
2, step 1 is repeated to the word group in each primer sequence, and obtains all corresponding high score word groups, scan database
In sequence, see whether with remaining high score word group exactly match situation, by these exactly match places be extended to
High score ordered pair.
3, the enough high high score ordered pairs of all scores are listed, high score ordered pair their score of assessing that these stay is
It is no it is meaningful, multiple high score ordered pairs region in a database sequence is combined into a longer parallelism；
4, show primer sequence and it is all before find may relevant database sequence have gap area parallelism, list
Expected mark E is finally obtained mostly concerned with primer lower than the database sequence of the threshold value required by us in previous step
Reference sequences relevant range；
5, whether the reference sequences relevant range that judgement obtains is unique, special if unique, is otherwise non-specific.
Step 7: according to step 5 the selection result, read the candidate drugs for the specific amplification that screening obtains, operation with
Primer dimer based on pooler software detects program, obtains dimer situation that may be present between primer；
Further, step 7 the following steps are included:
1. reading input candidate drugs sequence, sequence inputting format is as follows:
| Input content | Description of contents |
| >IRF1_4-F | Normal chain Primer |
| TGGCAAGATCCACACGAAAA | Normal chain primer sequence |
| >IRF1_4-R | Negative strand primer title |
| GGAAAGTGGGGTCCTTCGG | Negative strand primer sequence |
2. each primer of circulation reading, each primer and other all primers compositions match, it is double to calculate formation between primer
Gibbs free energy needed for chain (Δ G kcal/mol), calculation formula is as follows: Δ G=Δ H-T Δ S, such as the Ji between primer
Buss free energy is greater than -5.0, then has that a possibility that primer dimer is lower, and such as less than -5.0 there are primer dimers
Possibility is larger.This result is recorded in primer dimer testing result file, such as primer AMPL000727 and primer
The possible implementations of existing dimer are as follows between rs4885963:
| Input content | Description of contents |
| AMPL000727-F versus rs4885963-R | There may be the titles of dimer primer pair |
| DG=-6.68 | Gibbs free energy numerical value |
| 5'-GGATTGAGGGCCAGGTCATG-3' | Primer sequence |
| x|xxx||||||||||xxx| | " | " indicates that complementary pairing, " X " indicate not complementary pairing |
| 3'-ATCTATCCCGGTCCACACCA-5 | Primer sequence |
Step 8:, because needing to cover whole exon regions, can exist in the design of full exon multiple PCR primer
The case where overlap (amplified fragments are overlapped mutually), candidate drugs obtained in step in reading, operation are with pooler software
The overlap detection on basis.
Further, step 8 comprises the steps of:
1, with algorithm used in step 6, each primer pair will be found and compared to the position on reference genome；
2, circulation reads the location information of each primer pair on reference genome, with the location informations of other primer pairs into
Row compares, and judges whether there is the phenomenon that region is overlapped mutually.Judging result is recorded in following formatted file:
Overlapping amplicons:
EGFR-001_2-F:EGFR-001_2-R(chr7:56375642+56375763)/EGFR-001_1-F:E GFR-
001_1-R(56375577+56375696)
EGFR-002_2-F:EGFR-002_2-R(chr7:56378410+56378520)/EGFR-002_1-F:E GFR-
002_1-R(56378355+56378462)
EGFR-002_3-F:EGFR-002_3-R(chr7:56378468+56378584)/EGFR-002_2-F:E GFR-
002_2-R(56378410+56378520)
EGFR-002_4-F:EGFR-002_4-R(chr7:56378502+56378622)/EGFR-002_3-F:E GFR-
002_3-R(56378468+56378584)
EGFR-002_5-F:EGFR-002_5-R(chr7:56378551+56378662)/EGFR-002_3-F:E GFR-
002_4-R(56378502+56378622)
EGFR-002_6-F:EGFR-002_6-R(chr7:56378613+56378726)/EGFR-002_5-F:E GFR-
002_5-R(56378551+56378662)
EGFR-002_7-F:EGFR-002_7-R(chr7:56378668+56378789)/EGFR-002_6-F:E GFR-
002_6-R(56378613+56378726)
EGFR-002_8-F:EGFR-002_8-R(chr7:56378726+56378838)/EGFR-002_7-F:E GFR-
002_7-R(56378668+56378789)
8 overlaps found
Step 9: reading candidate drugs sequence, the associated primer dimer of primer and primer amplification are obtained according to primer I D
The information that region is overlapped mutually.Automatically it will be present primer dimer and primer pair that primer amplified region is overlapped mutually be assigned to not
With grouping in, avoid in same reaction system, the case where primer dimer and primer amplified region are overlapped mutually.
Step 10: judging whether number of packet is greater than maximum defined in step 2 and divides pool quantity, such as it is not more than, then will
Final primer information is written in final destination file；Such as larger than, then auto-returned step 5, from candidate drugs file
Remaining other primer for meeting parameter in step 2 is read, repeats step 5 to step 10 step.It is final to obtain needed for experiment
Design primer still has part primer to lack because design section complexity is too low or setting parameter is unreasonable, then it is fixed to need again
Rerun routine after adopted parameter.
Specifically, embodiment one:
Step 1: input type includes target gene title MEN1, the full exon of MEN1 gene is obtained in reference genome
On coordinate；Input following content:
| query | type |
| MEN1 | Gene Exon Only |
Operational objective region obtains program, obtains 10 sections of target areas, as a result as follows:
| chr11 | 64570982 | 64572293 | MEN1-001 |
| chr11 | 64572500 | 64572675 | MEN1-002 |
| chr11 | 64573101 | 64573247 | MEN1-003 |
| chr11 | 64573698 | 64573845 | MEN1-004 |
| chr11 | 64574477 | 64574575 | MEN1-005 |
| chr11 | 64574645 | 64574696 | MEN1-006 |
| chr11 | 64575018 | 64575157 | MEN1-007 |
| chr11 | 64575357 | 64575576 | MEN1-008 |
| chr11 | 64577116 | 64578218 | MEN1-009 |
| chr11 | 64578280 | 64578771 | MEN1-010 |
Step 2: customized design of primers parameter: it sets primer and returns to quantity as 20, primer length minimum 18, primer
Length maximum length is 27, and primer G/C content minimum is 20%, and primer G/C content peak is 80%, and (primer is molten by minimum Tm
Solving temperature) value is 58 DEG C, maximum Tm value is 61 DEG C, and maximum consecutive identical base quantity is 4, and minimum expanding fragment length is defaulted as
100, maximum expanding fragment length is 275.Maximum divides pool quantity to be defaulted as 2.
Step 3: being 100-275 according to customized amplified fragments magnitude range, operational objective area dividing program will
Altogether at 37 lesser target areas, partial segmentation result is as follows for 10 target area segmentations；
| chr11 | 64570944 | 64571122 | MEN1-001_1 |
| chr11 | 64571066 | 64571245 | MEN1-001_2 |
| chr11 | 64571174 | 64571334 | MEN1-001_3 |
| chr11 | 64571283 | 64571459 | MEN1-001_4 |
| chr11 | 64571403 | 64571572 | MEN1-001_5 |
| chr11 | 64571520 | 64571695 | MEN1-001_6 |
| chr11 | 64571635 | 64571820 | MEN1-001_7 |
| chr11 | 64571735 | 64571920 | MEN1-001_8 |
| chr11 | 64571854 | 64572027 | MEN1-001_9 |
| chr11 | 64571977 | 64572157 | MEN1-001_10 |
| chr11 | 64572098 | 64572283 | MEN1-001_11 |
| chr11 | 64572164 | 64572325 | MEN1-001_12 |
| chr11 | 64572459 | 64572622 | MEN1-002_1 |
| chr11 | 64572527 | 64572708 | MEN1-002_2 |
| chr11 | 64573064 | 64573246 | MEN1-003_1 |
| chr11 | 64573101 | 64573286 | MEN1-003_2 |
| chr11 | 64573656 | 64573838 | MEN1-004_1 |
| chr11 | 64573777 | 64573955 | MEN1-004_2 |
| chr11 | 64574434 | 64574604 | MEN1-005_1 |
| chr11 | 64574584 | 64574756 | MEN1-006_1 |
Step 4: comparing the target area divided to the mankind with reference on genome (hg19), target area is got
DNA sequence dna, the format of file is fasta；
Step 5: the target area DNA sequence dna in read step five, calls primer3 software Design primers, is met
All candidate drugs of custom parameter condition, each design section return to 20 pairs of candidate drugs altogether, are saved in candidate drugs knot
Fruit file carries out subsequent screening step as input primer；
Step 6: 20 pairs of candidate drugs for obtaining each design section using step 5 are recycled and are each set as input file
Region is counted, 20 pairs of candidate drugs of each design section is read in circulation, calls blast software, looked for using heuristic search
The mankind refer on genome with candidate drugs to the highest sequence of phase knowledge and magnanimity out, and such as only unique corresponding sequence, then primer is special
Different, if any more than two corresponding sequences, then primer is non-specific.Screening obtains the candidate drugs of specific amplification, such as without special
Property primer, then return to previous step, return to other 20 pairs of candidate drugs；
Step 6 further includes steps of
1, it removes the low complex degree in primer sequence and has the region of concatenation polyisomenism, by k every in primer sequence
Combinatorics on words is made into a table, lists the word group of our all possibility of concern, by these after screening remaining high score word
Group is organized into the structure of fast search tree.
2, step 1 is repeated to the word group in each primer sequence, and obtains all corresponding high score word groups, scan database
In sequence, see whether with remaining high score word group exactly match situation, by these exactly match places be extended to
High score ordered pair.
3, the enough high high score ordered pairs of all scores are listed, high score ordered pair their score of assessing that these stay is
It is no it is meaningful, multiple high score ordered pairs region in a database sequence is combined into a longer parallelism；
4, show primer sequence and it is all before find may relevant database sequence have gap area parallelism, list
Expected mark E is finally obtained mostly concerned with primer lower than the database sequence of the threshold value required by us in previous step
Reference sequences relevant range；
5, whether the reference sequences relevant range that judgement obtains is unique, special if unique, is otherwise non-specific.
Step 7: reading the candidate drugs for the specific amplification that screening obtains according to step 6 the selection result, being obtained 37
To specific primer, pooler software is called, checks that primer dimer sieves situation, primer dimer situation is recorded in dimer
In testing result file.
Further, step 7 the following steps are included:
1. reading 37 pairs of candidate primer sequences of input, part list entries is as follows:
>MEN1-001_2-F
TGTCACCCACTCCTAACCCT
>MEN1-001_2-R
TCTGGGACACAAAACTCCGC
>MEN1-001_4-F
TCTGGAGCAGGACTGAAGTT
>MEN1-001_4-R
GCTCCTCCCAGCTTGTAGGA
>MEN1-001_6-F
GGCTGGGCCTTTAAAGACTG
>MEN1-001_6-R
CAAACCCTAGGTTCCCGGTC
>MEN1-001_8-F
CATGGGCTCAGAGTTGGGG
>MEN1-001_8-R
CGCCATCAAGCTGCAACTC
2. each primer of circulation reading, each primer and other all primers compositions match, it is double to calculate formation between primer
Gibbs free energy needed for chain (Δ G kcal/mol), calculation formula is as follows: Δ G=Δ H-T Δ S, such as the Ji between primer
Buss free energy is greater than -5.0, then has that a possibility that primer dimer is lower, and such as less than -5.0 there are primer dimers
Possibility is larger.Testing result shows between two pairs of primers that there are primer dimer situations.This result is recorded in primer two
In aggressiveness testing result file, there may be primer dimer feelings between MEN1-005_1-R primer and MEN1-006_1-F primer
Condition is as a result as follows:
MEN1-005_1-R versus MEN1-006_1-F
DG=-12.9
5'-CTGGGTGGCAGCCTGAATTA-3'
||||||||||||||||||||
3'-GACCCACCGTCGGACTTAAT-5'
Step 8:, because needing to cover whole exon regions, can exist in the design of full exon multiple PCR primer
The case where overlap (amplified fragments are overlapped mutually), candidate drugs obtained in step in reading, operation are with pooler software
The overlap detection on basis
Further, step 8 comprises the steps of:
1. will find each primer pair with algorithm used in step 6 and compare to the position on reference genome；
2. circulation reads the location information of each primer pair on reference genome, with the location informations of other primer pairs into
Row compares, and judges whether there is the phenomenon that region is overlapped mutually.There are there are overlap between 14 pairs of primers for inspection result discovery
Situation.
Step 9: reading candidate drugs sequence, the associated primer dimer of primer and primer amplification are obtained according to primer I D
The information that region is overlapped mutually.Automatically it will be present primer dimer and primer pair that primer amplified region is overlapped mutually be assigned to not
In same grouping, operation result shows that packet count is 2.
Step 10: primer number of packet be 2, customized largest packet quantity be 2, judge number of packet meet it is customized most
Big number of packet.Primer needed for the final acquisition full exon of MEN1 gene.The final primer in part is as follows as the result is shown, and first is classified as
Primer, secondary series are positive primer sequence, and third is classified as reverse primer sequences, and the 4th is classified as primer amplification fragment length,
5th is classified as chromosome number, and the 6th is classified as amplicon starting position, and the 7th is classified as Insert Fragment starting position, and the 8th is classified as insertion
Segment end position, the 9th is classified as amplicon end position, and the tenth is classified as group names, and destination file is as follows:
To achieve the above object, as shown in figure 3, the present invention also provides a kind of system for handling PCR primer, the system
Include:
First acquisition unit, for obtaining target primer data information to be processed；
Second acquisition unit, for obtaining target primer region in reference base according to target primer data information to be processed
Because of the coordinate data in group；
Divide division unit, for being split division to target primer region；
Third acquiring unit obtains target and draws for dividing the index object area completed according to segmentation and referring to genome
The DNA sequence dna of object area；
Processing unit, for the DNA sequence dna and custom parameter according to target primer region, processing obtains candidate drugs；
Generation unit for candidate drugs to be dispensed into different groupings, and generates PCR primer design report.
Further, the system further include:
First obtains module, for obtaining design of primers parameter information, primer grouping information, primer sequence and primer category
Property.
The present invention also proposes a kind of platform for handling PCR primer, as shown in Figure 4, comprising:
Processor, memory and processing PCR primer platform courses program；
Wherein the processing PCR primer platform courses program, the processing PCR primer platform are executed in the processor
Control program is stored in the memory, the processing PCR primer platform courses program, realizes that the processing PCR draws
The method and step of object, such as:
Obtain target primer data information to be processed；
According to target primer data information to be processed, coordinate data of the target primer region on reference genome is obtained；
Division is split to target primer region；
The index object area completed is divided according to segmentation and refers to genome, obtains the DNA sequence dna of target primer region；
According to the DNA sequence dna and custom parameter of target primer region, processing obtains candidate drugs；
Candidate drugs are dispensed into different groupings, and generate PCR primer design report.
Step detail is being described above, and details are not described herein again；
In the embodiment of the present invention, the platform internal processor of the processing PCR primer can be made of integrated circuit,
Such as can be made of the integrated circuit of single package, it is also possible to be encapsulated by multiple identical functions or different function integrated
Circuit is formed, including one or more central processing unit (Central Processing unit, CPU), microprocessor,
Digital processing chip, graphics processor and combination of various control chips etc..Processor is taken using various interfaces and connection
All parts by running or execute the program being stored in memory or unit, and are called and are stored in memory
Data, to execute the various functions and processing data of processing PCR primer；
Memory is mounted in the platform of processing PCR primer, and running for storing program code and various data
The access realized high speed in journey, be automatically completed program or data.
The memory includes read-only memory (Read-Only Memory, ROM), random access memory (Random
Access Memory, RAM), it is programmable read only memory (Programmable Read-Only Memory, PROM), erasable
Only except programmable read only memory (Erasable Programmable Read-Only Memory, EPROM), disposable programmable
Reading memory (One-time Programmable Read-Only Memory, OTPROM), electronics erasing type can make carbon copies read-only
Memory (Electrically-Erasable Programmable Read-Only Memory, EEPROM), CD-ROM
(Compact Disc Read-Only Memory, CD-ROM) or other disc memories, magnetic disk storage, magnetic tape storage,
Or it can be used in any other computer-readable medium of carrying or storing data.
The present invention also proposes a kind of computer-readable storage medium, as shown in figure 5, the computer-readable storage is situated between
Matter is stored with processing PCR primer platform courses program, and the processing PCR primer platform courses program realizes the processing PCR
The method and step of primer, for example,
Obtain target primer data information to be processed；
According to target primer data information to be processed, coordinate data of the target primer region on reference genome is obtained；
Division is split to target primer region；
The index object area completed is divided according to segmentation and refers to genome, obtains the DNA sequence dna of target primer region；
According to the DNA sequence dna and custom parameter of target primer region, processing obtains candidate drugs；
Candidate drugs are dispensed into different groupings, and generate PCR primer design report.
Step detail is being described above, and details are not described herein again；
In the description of embodiments of the present invention, it should be noted that in flow chart or described otherwise above herein
Any process or method description be construed as, indicate to include one or more for realizing specific logical function or mistake
Module, segment or the part of the code of the executable instruction of the step of journey, and the range packet of the preferred embodiment of the present invention
Include other realization, wherein sequence shown or discussed can not be pressed, including according to related function by it is basic simultaneously
Mode or in the opposite order, Lai Zhihang function, this should be managed by the embodiment of the present invention person of ordinary skill in the field
Solution.
Expression or logic and/or step described otherwise above herein in flow charts, for example, being considered use
In the order list for the executable instruction for realizing logic function, may be embodied in any computer-readable medium, for
Instruction execution system, device or equipment (such as computer based system, including the system of processing module or other can be from instruction
Execute system, device or equipment instruction fetch and the system that executes instruction) use, or combine these instruction execution systems, device or
Equipment and use.For the purpose of this specification, " computer-readable medium ", which can be, any may include, store, communicating, propagating
Or transfer program uses for instruction execution system, device or equipment or in conjunction with these instruction execution systems, device or equipment
Device.The more specific example (non-exhaustive list) of computer-readable medium include the following: there are one or more wirings
Electrical connection section (electronic device), portable computer diskette box (magnetic device), random access memory (RAM), read-only memory
(ROM), erasable edit read-only storage (EPROM or flash memory), fiber device and portable optic disk is read-only deposits
Reservoir (CDROM).
In addition, computer-readable medium can even is that the paper that can print described program on it or other suitable Jie
Matter, because can then be edited, be interpreted or when necessary with other for example by carrying out optical scanner to paper or other media
Suitable method is handled electronically to obtain described program, is then stored in computer storage.
The full exon of gene, snp may be implemented in step, system, platform and storage medium by means of the present invention
The full-automatic design of point, transcript and the multiple PCR primer of other hot spot regions, can greatly shorten the design of such primer
Time, but also all standing of target area may be implemented.
That is, the invention can be transcribed automatically according to Gene Name compared to the primer design method of current majority
This title, No. rs and other hot spot regions, obtains target area automatically, inquires without manually in database website corresponding
Design section；
The process automation of entire design of primers is run, centre is without manual intervention, it is only necessary to input Gene Name, turn
Record this title etc., so that it may full-automatic output specificity, high amplification efficiency multiple PCR primer, and primer can be grouped automatically,
Avoid the occurrence of overlap.The time that design of primers can greatly be saved, the mistake for avoiding manual intervention from generating mention
The quality and efficiency of high design of primers, and all standing accomplished the full exon of such as gene, change this target area.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (9)
1. a kind of method for handling PCR primer, which is characterized in that the method specifically comprises the following steps:
Obtain target primer data information to be processed；
According to target primer data information to be processed, coordinate data of the target primer region on reference genome is obtained；
Division is split to target primer region；
The index object area completed is divided according to segmentation and refers to genome, obtains the DNA sequence dna of target primer region；
According to the DNA sequence dna and custom parameter of target primer region, processing obtains candidate drugs；
Candidate drugs are dispensed into different groupings, and generate PCR primer design report.
2. a kind of method for handling PCR primer according to claim 1, which is characterized in that the target primer data
Information specifically includes:
Target gene title, No. rs of the site ncbi database snp, transcript number and hot spot region.
3. a kind of method for handling PCR primer according to claim 1, which is characterized in that described to target guiding region
Domain is split division, specifically:
According to customized expanding fragment length range, longer target area is divided into lesser target primer region.
4. a kind of method for handling PCR primer according to claim 1, which is characterized in that the custom parameter, tool
Body includes:
Expanding fragment length range, primer G/C content range, primer annealing temperature range, returns to primer number at primer length range
Amount and maximum annealing temperature difference.
5. a kind of method for handling PCR primer according to claim 1, which is characterized in that divide candidate drugs in step
It is attached in different groupings, and generates among PCR primer design report, further comprise the steps of:
Obtain design of primers parameter information, primer grouping information, primer sequence and primer attribute.
6. a kind of system for handling PCR primer, which is characterized in that the system specifically includes:
First acquisition unit, for obtaining target primer data information to be processed；
Second acquisition unit, for obtaining target primer region in reference genome according to target primer data information to be processed
On coordinate data；
Divide division unit, for being split division to target primer region；
Third acquiring unit obtains target guiding region for dividing the index object area completed according to segmentation and referring to genome
The DNA sequence dna in domain；
Processing unit, for the DNA sequence dna and custom parameter according to target primer region, processing obtains candidate drugs；
Generation unit for candidate drugs to be dispensed into different groupings, and generates PCR primer design report.
7. a kind of system for handling PCR primer according to claim 6, which is characterized in that the system further include:
First obtains module, for obtaining design of primers parameter information, primer grouping information, primer sequence and primer attribute.
8. a kind of platform for handling PCR primer characterized by comprising
Processor, memory and processing PCR primer platform courses program；
Wherein the processing PCR primer platform courses program, the processing PCR primer platform courses are executed in the processor
Program is stored in the memory, the processing PCR primer platform courses program, is realized as appointed in claim 1 to 5
The method and step of processing PCR primer described in one.
9. a kind of computer-readable storage medium, which is characterized in that the computer-readable storage medium is stored with processing
PCR primer platform courses program, the processing PCR primer platform courses program are realized such as any one of claims 1 to 5 institute
The method and step for the processing PCR primer stated.
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