CN111653311B - Multiplex methylation specific PCR primer design method and system - Google Patents
Multiplex methylation specific PCR primer design method and system Download PDFInfo
- Publication number
- CN111653311B CN111653311B CN202010479453.2A CN202010479453A CN111653311B CN 111653311 B CN111653311 B CN 111653311B CN 202010479453 A CN202010479453 A CN 202010479453A CN 111653311 B CN111653311 B CN 111653311B
- Authority
- CN
- China
- Prior art keywords
- primer
- screening
- primers
- multiplex
- reference sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000013461 design Methods 0.000 title claims abstract description 97
- 238000000034 method Methods 0.000 title claims abstract description 54
- 238000007855 methylation-specific PCR Methods 0.000 title claims abstract description 42
- 238000012216 screening Methods 0.000 claims abstract description 151
- 230000003321 amplification Effects 0.000 claims abstract description 45
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 45
- 239000000539 dimer Substances 0.000 claims abstract description 42
- 230000011987 methylation Effects 0.000 claims abstract description 35
- 238000007069 methylation reaction Methods 0.000 claims abstract description 34
- 230000008569 process Effects 0.000 claims abstract description 24
- 238000011156 evaluation Methods 0.000 claims description 51
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 230000027455 binding Effects 0.000 claims description 15
- 108091029430 CpG site Proteins 0.000 claims description 13
- 238000004590 computer program Methods 0.000 claims description 5
- 238000012544 monitoring process Methods 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000007619 statistical method Methods 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 8
- 230000007067 DNA methylation Effects 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000001973 epigenetic effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108091029523 CpG island Proteins 0.000 description 2
- 238000010357 RNA editing Methods 0.000 description 2
- 230000026279 RNA modification Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 238000012226 gene silencing method Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000012070 whole genome sequencing analysis Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 201000010374 Down Syndrome Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 108050002584 Septin 9 Proteins 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 206010044688 Trisomy 21 Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000012164 methylation sequencing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
Abstract
The invention discloses a method and a system for designing multiplex methylation specific PCR primers, wherein the method comprises the steps of setting parameters according to user requirements, designing and screening target methylation sites, pairing the screened methylation site primer pairs, checking compatibility, selecting the compatible multiplex primer combination with the largest quantity, evaluating the designed multiplex primer combination, and determining whether the primers need to be redesigned. The invention can realize the design of multiplex methylation specific PCR primers of a very long sequence, can effectively reduce the occurrence of secondary structures such as dimer/hairpin and the like between the interiors of single pair primers and between multiple pairs of primers, and the nonspecific amplification in genome, has strong specificity, improves the operability and accuracy of the multiplex methylation specific PCR experimental test process, and simultaneously greatly improves the working efficiency.
Description
Technical Field
The invention relates to the field of DNA methylation detection, in particular to a method and a system for designing a multiplex methylation specific PCR primer.
Background
The relationship of human phenotype to human genome mutation has become increasingly clear since the development of research in the human genome project and the like. About 8800 mutations (8470 single nucleotide mutations, 360 ten thousand short stretch insertions or deletions) and 6 structural variations have been identified in 2015. In 2001 human medical genetics on-line database (OMIM TM ) There have been 13005 entries relating to the research results of human genome mutations and human diseases. Human genome mutation has been increasingly used in the fields of human life such as drug guidance, drug resistance detection, prenatal detection, and tumor detection.
Epigenetic is a branch of genetics that studies heritable changes in gene expression without changing the nucleotide sequence of the gene. Epigenetic factors include DNA methylation (DNA methylation), genomic imprinting (genomic imprinting), gene silencing (gene silencing), nucleolus dominance, dormant transposon activation, RNA editing (RNA editing), and the like. Research shows that the epigenetic biological marker can also be used as an index of diseases to indicate the risk of the diseases or the health state of human bodies. DNA methylation refers to covalent bonding of a methyl group at the cytosine 5' carbon of a genomic CpG dinucleotide under the action of a DNA methyltransferase. Numerous studies have shown that DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression. DNA methylation markers have been widely used in the medical and health fields. Luming et al used certain CpG islands on fetal chromosome 21 to show different methylation patterns than corresponding CpG islands located on maternal chromosome 21 and detected whether the fetus had trisomy 21 (pre-foci) in advance of fetal birth (CN 101535502A). In addition, septin9 gene methylation detection has been identified as a gold standard for bowel cancer screening (CN 201810326484.7, CN108048570A, CN105861672A, CN201610948298.8, CN 201710285809.7). With the widespread development of genome-wide methylation sequencing and genome-wide methylation profiles in humans and other organisms, more and more methylation sites are identified as clinically or biologically phenotype-specific sites. Cancer and tumor Gene mapping (TCGA) programs have completed genome-wide methylation maps for more than about 11000 samples, 30 cancers, with a large number of clinically significant methylation sites for each cancer that can be used to guide tumor detection and prevention.
The detection method of the methylation locus comprises whole genome sequencing, whole genome methylation map, polygene methylation capturing sequencing, fluorescent quantitative PCR, methylation specific PCR, methylation amplified product sequencing and the like. However, whole genome sequencing, whole genome methylation profile, polygene methylation-captured sequencing methods are directed to the detection of more than 1000 methylation sites; the methods such as fluorescent quantitative PCR, methylation specific PCR, and methylation amplified product sequencing are aimed at detecting 1-2 target methylation sites, and the current DNA methylation specific PCR primer design software MethPrimer and Methyl Primer Express tools are all aimed at single-site primer design. Therefore, the detection of a plurality of methylation sites at present needs to design a large number of primers for a plurality of target fragments, and the detection is possible only through repeated screening of a plurality of experiments, so that the workload is high and the time consumption is long. The multiplex methylation specific PCR can reduce the experimental amount, improve the working efficiency, reduce the sample amount requirement and improve the technical feasibility. However, since unmethylated C is converted to T after genomic DNA is converted by sulfite, genome complexity is reduced, resulting in reduced primer complexity, increased primer dimer, secondary structure, and nonspecific amplification, further increasing difficulty in multiplex methylation-specific PCR primer design, and conventional common multiplex PCR primer design software cannot be applied to multiplex methylation-specific PCR primer design. At present, a system and a method for specifically amplifying in genome, which can design multiple PCR primers for multiple methylation sites and ensure the secondary structures such as non-dimer/hairpin between the interiors of single pairs of primers and between multiple pairs of primers, are lacking.
Disclosure of Invention
In view of the above, the invention provides a design method and a system for multiple methylation specific PCR primers, which can effectively reduce the secondary structures such as primer/hairpin and the like between the interiors of single pairs of primers and between multiple pairs of primers, reduce the non-specific amplification in chromosomes between the interiors of single pairs of primers and between multiple pairs of primers, reduce the intensity and difficulty in the test process of multiple methylation specific PCR experiments, and improve the working efficiency so as to achieve the purpose of efficiently and accurately detecting multiple methylation sites.
The technical scheme of the invention is realized as follows:
in a first aspect, the present invention provides a multiplex methylation specific PCR primer design method comprising the steps of:
s000-parameter configuration, setting a parameter bureau, and generating a primer design environment;
s100-primer design and screening, namely, designing a primer for a target methylation site, and screening the designed primer;
s200-multiple primer combination calculation, namely pairing the primer pairs in the plurality of primers, checking compatibility between every two primer pairs, and selecting the compatible multiple primer combination with the largest quantity;
s300-primer evaluation and design, evaluating the designed multiplex primer combination and determining whether the primer needs to be redesigned.
On the basis of the above technical solution, preferably, the step S000 includes the steps of:
s001, acquiring target site information and creating target sites;
s002, acquiring and establishing primer demand parameter information, and binding the primer demand parameter information into a target site;
s003, acquiring a reference sequence, and binding the reference sequence to a target site after CT conversion;
s004, obtaining a shielding site defined by a user;
s005, acquiring a shielding area defined by a user;
s006, generating a primer design environment comprising a site sequence, local environment parameters and global environment parameters according to the parameter content acquired in the steps S001-S005, and transmitting the environment parameters to the step S100.
On the basis of the above technical solution, preferably, the step S100 includes the following steps:
s101-primer design, namely receiving a primer design environment from the step S000, and designing and generating corresponding primers;
S102-CG site screening, checking whether the primer received from the step S101 contains CG site and terminal C site, and deleting unqualified primer;
S103-Dimer screening, namely screening primers with a Dimer structure;
s104-screening the primer with the Hairpin structure;
s105-screening of shielding sites, and screening primers with tail ends positioned at the shielding sites defined by users;
s106, screening a shielding region, and screening primers with the tail ends positioned in a shielding region defined by a user;
s107-non-specific screening inside the primer, screening the primer, wherein a plurality of non-specific regions possibly amplified in the reference sequence inside the primer, and transferring the information of the potential binding sites of the qualified primer and the single primer in the reference sequence to the step S200.
On the basis of the above technical solution, preferably, the step S200 includes the following steps:
s201-single primer pair specificity evaluation, namely calculating the qualified primers screened in the step S100 and potential binding sites thereof in a reference sequence, evaluating the specificity of each pair of primers, screening out a primer pair with highest specificity, and attributing all primers of other sites except the corresponding site of the primer pair to a first other primer set list and transmitting the first other primer set list to the step S202;
s202-inter-primer pair Dimer evaluation, namely performing Dimer evaluation between primers in the first other primer set list obtained in the step S201 and the highest specific primer pair generated in the step S201 one by one, screening out primer pairs without a Dimer structure, obtaining a second other primer set list, and transmitting the second other primer set list to the step S203;
s203-non-specific evaluation among the double primer pairs, namely performing non-specific evaluation among the primers in the second other primer set list obtained in the step S202 and the highest specific primer pair generated in the step S201 one by one, screening out primer pairs which have no non-specific amplification with the highest specific primer pair in a reference sequence, obtaining a third other primer set list and transmitting the third other primer set list to the step S204;
s204, evaluating compatibility of multiple primers, if the number of corresponding positions of the primers in the third other primer set list is greater than 1, continuing to send the third other primer set list to the step S201 for cyclic operation; if the number of the corresponding sites of the primer pairs in the third other primer set list is equal to 1, sending the third other primer set list to the step S201 for screening the highest specificity primer pair, combining all the circulated highest specificity primer pairs into a multi-primer pair compatible with the whole synthesis, and sending to the step S300; if the number of corresponding sites of the third other primer set list primer pair is equal to 0, the combination of the multiple primer pairs compatible with the whole of the highest specificity primers of all loops is directly sent to the step S300.
Based on the above technical solution, preferably, in the step S300, the compatible multiplex primer combination transferred in the step S200 is evaluated, and if the number of primer pairs is too small or the necessary sites are not included, the sites and primer parameters are modified, and then transferred to the step S000 for redesign; if the multiplex primer combination meets the requirements, but some target sites are not included, the target sites which are not included are input again to step S000, and the primers are redesigned for the sites to form a new set of multiplex primer combinations.
In a second aspect, the present invention provides a computer apparatus programmed to perform the steps of the multiplex methylation specific PCR primer design method of the first aspect of the invention; or a storage medium of the computer device has stored therein a computer program programmed to perform the multiplex methylation specific PCR primer design method according to the first aspect of the invention.
In a third aspect, the present invention provides a computer readable storage medium having stored therein a computer program programmed to perform the methylation specific PCR primer design method of the first aspect of the present invention.
In a fourth aspect, the invention provides a multiplex methylation specific PCR primer design system, which comprises a reference sequence CT conversion and parameter setting module, a primer design and screening module, a multiplex primer combination module and a reporting module, and is specifically as follows:
the reference sequence CT conversion and parameter setting module is used for carrying out C- > T conversion on the reference sequence, setting parameters such as target sites, primer length and the like according to user requirements and generating a primer design environment;
the primer design and screening module is used for carrying out primer design on the target site and screening qualified primers according to the user requirements;
the multiplex primer combination module is used for uniformly inspecting primers of a plurality of methylation sites and screening a multiplex primer group without non-specific amplification and secondary structure in a genome;
and the report module is used for recording and monitoring all the execution processes, generating a process execution report file according to the requirements of the user, including a primer design report, a screening report, a multiple combination report and the like, and providing assistance and support for the user to further screen primers and change parameters.
On the basis of the above technical solution, preferably, the reference sequence CT conversion and parameter setting module includes a reference sequence CT conversion sub-module, a parameter setting sub-module, and an environment generating sub-module, and specifically includes the following steps:
the reference sequence CT conversion submodule is used for converting C- > T of a reference sequence, converting unmethylated C of a genome into T, keeping methylated C unchanged, and carrying out reverse complementation after carrying out non-CpG site C- > T conversion on a negative strand of the reference sequence because positive and negative strands exist in the genome, wherein the non-CpG site C- > T conversion is needed to be carried out on a positive strand of the reference sequence;
the parameter setting submodule is used for setting parameters of a primer design and screening process in advance, wherein the parameters comprise a target site name, a position on a reference sequence, a primer design length, the number of primers generated, GC content, annealing temperature and a region and site which should be skipped in primer screening;
and the environment generation sub-module is used for generating a target region sequence and a parameter format required by primer design according to the set parameters, and the primer design environment can be transmitted into the primer design and screening module after the primer design environment is generated.
On the basis of the above technical scheme, preferably, the primer design and screening module comprises a primer design, CG locus screening, dimer screening, hairpin screening, screening of screening loci, screening of screening areas, and primer pair internal nonspecific screening sub-module, and the specific steps are as follows:
the primer design submodule is used for carrying out primer design according to the primer design environment transmitted by the reference sequence CT conversion and parameter setting module;
a CG locus screening submodule for evaluating whether the designed primer contains CG locus which possibly causes unbalanced amplification and removing primer pairs which possibly have unbalanced amplification;
a primer screening submodule for evaluating whether the designed primer contains a primer secondary structure which possibly causes the conditions of low amplification efficiency and invalid primer Dimer, and removing the primer pair which possibly causes the conditions of low amplification efficiency or invalid primer Dimer;
the Hairpin screening submodule is used for evaluating whether the designed primer contains a Hairpin secondary structure possibly causing low amplification efficiency or not and removing primer pairs possibly causing low amplification efficiency;
a screening sub-module of screening the screening sites, which is used for evaluating whether the designed primer contains the screening sites defined by the user or not and removing the primer pair containing the screening sites defined by the user;
a screening submodule for evaluating whether the designed primer contains a user-defined screening region or not and removing the primer pair containing the user-defined screening region;
the primer pair internal nonspecific screening submodule is used for evaluating whether a plurality of areas (namely nonspecific amplification) can be amplified in the converted reference sequence in the designed primer, and removing the primer pair possibly containing the nonspecific amplification characteristics.
On the basis of the above technical solution, preferably, the multiple primer combination module includes a single primer pair specificity evaluation, a double primer pair space primer evaluation, a double primer pair space non-specificity evaluation and a multiple primer compatibility evaluation submodule, and specifically includes the following steps:
the single primer pair specificity evaluation submodule is used for evaluating the binding sites of the forward and reverse primers contained in the single primer pair in the reference sequence and giving an evaluation value;
the double-primer pair Dimer evaluation submodule is used for evaluating whether a Dimer secondary structure which possibly causes low amplification efficiency and invalid primer Dimer occurrence exists between two primer pairs or not and recording compatibility between the two primer pairs;
the dual-primer pair nonspecific evaluation submodule is used for evaluating whether a plurality of areas are amplified in a converted reference sequence between two primer pairs (namely nonspecific amplification), and recording the compatibility condition between the two primer pairs;
and the multi-primer compatibility evaluation submodule is used for evaluating compatibility among primers by using the double-primer pair Dimer evaluation submodule and the double-primer pair nonspecific evaluation submodule according to the evaluation value given by the single-primer pair specificity evaluation submodule from small to large, and calculating the compatible multi-primer combination with the largest number in the target site by using a statistical method such as a shortest distance method.
Compared with the prior art, the method and the system for designing the multiplex methylation site amplification primer have the following beneficial effects:
(1) The invention provides a new design idea of methylation specific PCR primer by defining a reference sequence and carrying out methylation CT conversion necessary for methylation site detection, so that the design of the methylation primer based on the reference sequence with ultra-long length (> 1 Mb) is possible;
(2) The CG locus screening submodule can avoid loci which cannot be confirmed in methylation conversion state in the primer, so that the problem of differential amplification of the primer is avoided, the primer is not influenced by the methylation state on a reference sequence, and target loci are amplified comprehensively and specifically;
(3) The Dimer screening submodule and the Hairpin screening submodule can avoid the secondary structure in the primer pair, ensure the amplification efficiency of the qualified primer, and reduce the problems of Dimer and primer efficiency reduction in the experimental verification process;
(4) Because of the complexity of PCR amplification, the partial region and the locus of the reference genome may influence the effect of molecular experiment amplification or subsequent steps of a user, such as sequencing and library establishment, and the like;
(5) The system provides a primer pair internal non-specific screening function on a full reference sequence, no methylation software can realize the function at present, the function can fundamentally prevent the non-specificity of the primer in the experimental process, the experimental workload of primer verification is reduced by 90%, and the working efficiency is greatly improved;
(6) The invention provides a multi-primer compatibility evaluation method, which utilizes a preferable primer method and a Dimer and non-specificity evaluation method among a plurality of primer pairs to simulate the reaction process of the plurality of primer pairs in the actual molecular biology experiment process, reduces the secondary structure among the plurality of primer pairs and the non-specificity amplification in a reference sequence, reduces the abnormal amplification in the actual molecular biology experiment, and improves the amplification efficiency in the experiment process.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a block diagram showing the construction of a multiplex methylation specific PCR primer design system according to the present invention.
FIG. 2 is a flow chart of the method of operation of the multiplex methylation specific PCR primer design system of the present invention.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical aspects of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
The embodiment provides a multiplex methylation specific PCR primer design system, which comprises four modules, namely a reference sequence CT conversion and parameter setting module, a primer design and screening module, a multiplex primer combination module and a reporting module, as shown in fig. 1.
1. And the reference sequence CT conversion and parameter setting module is used for carrying out C- > T conversion on the reference sequence, setting parameters such as target sites, primer length and the like according to user requirements and generating a primer design environment. It may specifically comprise the following 3 sub-modules:
(1) The CT conversion submodule of the reference sequence converts C- > T of the reference sequence, converts unmethylated cytosine of a genome into thymine, and the methylated cytosine is unchanged, and because positive and negative chains exist in the genome, the positive chain of the reference sequence needs to be converted into non-CpG sites C- > T, and the negative chain of the reference sequence needs to be converted into non-CpG sites C- > T and then is reversely complemented;
(2) A parameter setting submodule, namely, setting parameters of a primer design and screening process in advance, wherein the parameters comprise a target site name, a position on a reference sequence, a primer design length, the number of primers generated, GC content, annealing temperature and a region and site which should be skipped in primer screening;
(3) And the environment generation submodule generates a target region sequence and a parameter format required by primer design according to the CT converted reference sequence and preset parameters, and can transfer the primer design environment into the primer design and screening module.
It should be understood that the region and site that should be skipped during primer screening in the parameters of the primer design and screening process described above are regions and sites that are required to be masked during the primer screening process that are custom-defined by the user in view of the complexity of PCR amplification, so as to avoid the designed primers from affecting the PCR amplification effect or subsequent sequencing library building and other experiments.
2. And the primer design and screening module is used for carrying out primer design on the target site and screening qualified primers according to the user requirements. It may specifically comprise the following 7 sub-modules:
(1) A primer design sub-module, namely, performing primer design according to the primer design environment transmitted by the environment generation sub-module;
(2) CG site screening submodules, i.e., evaluating whether the primers after design contain CG sites which may cause amplification imbalance, and removing primer pairs which may have amplification imbalance;
(3) A Dimer screening submodule, namely, whether a designed primer contains a Dimer secondary structure possibly causing low amplification efficiency and invalid primer Dimer occurrence or not is evaluated, and a primer pair possibly causing low amplification efficiency or invalid primer Dimer occurrence is removed;
(4) The Hairpin screening submodule is used for evaluating whether the designed primer contains a Hairpin secondary structure possibly causing low amplification efficiency or not and removing primer pairs possibly causing low amplification efficiency;
(5) A screening sub-module of screening the screening sites, which is used for evaluating whether the designed primer contains the screening sites defined by the user or not and removing the primer pair containing the screening sites defined by the user;
(6) A screening submodule for evaluating whether the designed primer contains a user-defined screening region or not and removing the primer pair containing the user-defined screening region;
(7) The primer pair internal nonspecific screening submodule is used for evaluating whether a plurality of areas (namely nonspecific amplification) can be amplified in the converted reference sequence in the designed primer, and removing the primer pair possibly containing the nonspecific amplification characteristics.
It should be understood that the qualified primer is obtained by screening six screening conditions including CG site screening, oligomer screening, hairpin screening, screening site screening, screening region screening and primer pair interior nonspecific screening one by one, wherein the oligomer sub-module, the Hairpin screening sub-module, the screening site screening sub-module and the screening region screening sub-module can be replaced by other selectable modules, and more condition screening sub-modules can be added after the screening region screening sub-module according to actual demands of users.
3. And the multiplex primer combination module is used for uniformly inspecting primers of a plurality of methylation sites and screening out multiplex primer groups without non-specific amplification and secondary structure in genome. It may specifically comprise the following 4 sub-modules:
(1) The single primer pair specificity evaluation submodule is used for respectively evaluating the binding sites of the forward and reverse primers contained in each single primer pair in the reference sequence and giving an evaluation value;
(2) The double-primer pair Dimer evaluation submodule is used for evaluating whether a Dimer secondary structure which possibly causes low amplification efficiency and invalid primer Dimer occurrence exists between two primer pairs or not and recording compatibility between the two primer pairs;
(3) The dual-primer pair nonspecific evaluation submodule is used for evaluating whether a plurality of areas are amplified in a converted reference sequence between two primer pairs (namely nonspecific amplification), and recording the compatibility condition between the two primer pairs;
(4) And the multi-primer compatibility evaluation submodule is used for evaluating compatibility among primers by using the double-primer pair Dimer evaluation submodule and the double-primer pair nonspecific evaluation submodule according to the evaluation value given by the single-primer pair specificity evaluation submodule from small to large, and calculating the compatible multi-primer combination with the largest number in the target site by using a statistical method such as a shortest distance method.
4. And the reporting module is used for recording and monitoring all the execution processes, generating a process execution reporting file according to the requirements of the user, and providing help and support for the user to further screen the primer and change the parameters.
It should be understood that the process execution report file includes text, graphics or list reports formed by each link in the primer design and screening process, and specifically includes: the execution environment generation sub-module generates an environment parameter concrete list; executing a primer design report generated by the primer design sub-module, wherein the primer design report comprises a primer design failure reason; executing a primer screening report generated by each screening sub-module, and recording screening result details; executing a multi-primer pair compatibility report generated by the multi-primer compatibility evaluation sub-module; and progress monitoring of all execution processes.
In addition, it should be noted that the above-described system embodiments are merely illustrative, and do not limit the scope of the present invention, and in practical applications, one skilled in the art may select some or all modules according to actual needs to achieve the purpose of the embodiment, which is not limited herein.
The system provided by the embodiment of the invention realizes the design of the multiplex methylation specific PCR primer, and with reference to FIG. 2, FIG. 2 is a flow chart of a working method of the primer design system.
In this example, the multiplex methylation specific PCR primer design method comprises the steps of:
s000-parameter configuration, setting a parameter bureau, and generating a primer design environment;
s100-primer design and screening, namely, designing a primer for a target methylation site, and screening the designed primer;
s200-multiple primer combination calculation, namely pairing the primer pairs in the plurality of primers, checking compatibility between every two primer pairs, and selecting the compatible multiple primer combination with the largest quantity;
s300-primer evaluation and design, evaluating the designed multiplex primer combination and determining whether the primer needs to be redesigned.
Further, the step S000 includes the steps of:
s001, acquiring target site information and creating target sites;
s002, acquiring and establishing primer demand parameter information, and binding the primer demand parameter information into a target site;
s003, acquiring a reference sequence, and binding the reference sequence to a target site after CT conversion;
s004, obtaining a shielding site defined by a user;
s005, acquiring a shielding area defined by a user;
s006, generating a primer design environment comprising a site sequence, local environment parameters and global environment parameters according to the parameter content acquired in the steps S001-S005, and transmitting the environment parameters to the step S100.
It should be understood that the execution of the reference sequence CT conversion sub-module may implement the process of step S003, the execution parameter setting sub-module may implement the processes of steps S001, S002, S004 and S005, and the execution environment generation sub-module may implement the process of step S006.
Further, the step S100 includes the steps of:
s101-primer design, namely receiving a primer design environment from the step S000 by executing a primer design submodule, and performing primer design generation by using Primer3.0 software;
S102-CG site screening, namely, checking whether the primer received from the step S101 contains CG site and terminal C site or not by executing a CG site screening submodule, and deleting unqualified primer;
S103-Dimer screening, namely screening and removing primers with a Dimer structure by executing a Dimer screening submodule;
s104-screening the Hairpin, and screening and removing the primer with the Hairpin structure by executing a Hairpin screening submodule;
s105-screening sites, namely screening and removing primers with the tail ends positioned at the screening sites defined by a user by executing a screening site screening submodule;
s106, screening a shielding region, namely screening and removing primers with the tail ends positioned in a shielding region defined by a user by executing a shielding region screening submodule;
s107-primer interior nonspecific screening, wherein primers which possibly amplify a plurality of nonspecific regions in a reference sequence in the primer interior are screened and removed by executing a primer pair interior nonspecific screening submodule, and finally the obtained qualified primers and potential binding site information of single primers in the reference sequence are transmitted to the step S200.
Further, the step S200 includes the steps of:
s201-single primer pair specificity evaluation, namely calculating the qualified primers screened in the step S100 and potential binding sites thereof in a reference sequence by executing a single primer pair specificity evaluation submodule, evaluating the specificity of each pair of primers, screening out the primer pair with the highest specificity, and attributing all primers of other sites except the corresponding site of the primer pair to a first other primer set list and transmitting the first other primer set list to the step S202;
s202-double primer pair-to-primer pair Dimer evaluation, namely performing Dimer evaluation between primers in the first other primer set list obtained from the step S201 and the highest specific primer pair generated in the step S201 one by executing a double primer pair Dimer evaluation submodule, screening out primer pairs without a Dimer structure, obtaining a second other primer set list and transmitting the second other primer set list to the step S203;
s203-non-specific evaluation among the double primer pairs, namely performing non-specific evaluation among the primers in the second other primer set list obtained from the step S202 and the highest specific primer pair generated in the step S201 one by executing a double primer pair non-specific evaluation submodule, screening out primer pairs which are not subjected to non-specific amplification in a reference sequence with the highest specific primer pair, obtaining a third other primer set list and transmitting the third other primer set list to the step S204;
s204, evaluating compatibility of multiple primers, if the number of corresponding positions of the primers in the third other primer set list is greater than 1, continuing to send the third other primer set list to the step S201 for cyclic operation; if the number of the corresponding sites of the primer pairs in the third other primer set list is equal to 1, sending the third other primer set list to the step S201 for screening the highest specificity primer pair, combining all the circulated highest specificity primer pairs into a multi-primer pair compatible with the whole synthesis, and sending to the step S300; if the number of corresponding sites of the third other primer set list primer pair is equal to 0, the combination of the multiple primer pairs compatible with the whole of the highest specificity primers of all loops is directly sent to the step S300.
It will be appreciated that the highest specific primer pair is the only primer pair with the highest specific binding capacity to the region of a certain target methylation site.
Further, in the step S300, the compatible multiplex primer combination transferred in the step S200 is evaluated, and if the number of primer pairs is too small or the necessary sites are not included, the sites and the primer parameters are modified and adjusted, and then the step S000 redesign is performed; if the multiplex primer combination meets the screening requirement, but some target sites are not included, re-inputting the target sites which are not included into the step S000, and re-designing primers for the sites to form a new set of multiplex primer combination; if the multiplex primer combination meets the screening requirements and contains all target sites, i.e., meets all the requirements of the user, the multiplex methylation specific PCR primer design process ends.
The present embodiment also provides a computer device programmed to perform the steps of the multiplex methylation specific PCR primer design method of the present embodiment described above; or a storage medium of the computer device stores therein a computer program programmed to perform the multiplex methylation specific PCR primer design method described in the present embodiment.
The present embodiment also provides a computer-readable storage medium having stored therein a computer program programmed to perform the methylation specific PCR primer design method of the present embodiment described above.
The method and the system for designing the multiplex methylation specific PCR primer can realize the design of the methylation specific primer of the ultra-long sequence, effectively reduce the two-stage structures such as a dimer/hairpin and the like between the interiors of the single pair of primers and between the plurality of pairs of primers, reduce the non-specific amplification in the genome between the interiors of the single pair of primers and between the plurality of pairs of primers, reduce the intensity and the difficulty in the test process of the multiplex methylation specific PCR experiment, greatly improve the working efficiency and achieve the purpose of efficiently and accurately detecting a plurality of methylation sites.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (9)
1. The design method of the multiplex methylation specific PCR primer is characterized by comprising the following steps of:
s000, setting a parameter bureau to generate a primer design environment;
s100, designing a primer for a target methylation site, and screening the designed primer;
s200, pairing the screened primer pairs, checking compatibility between every two primer pairs, and selecting the compatible multiple primer combination with the largest quantity;
s300, evaluating the designed multiple primer combination, and determining whether the primer needs to be redesigned;
the step S200 includes the steps of:
s201, calculating the qualified primers screened in the step S100 and potential binding sites thereof in a reference sequence, evaluating the specificity of each pair of primers, screening out the primer pair with the highest specificity, and classifying all primers of other sites except the corresponding site of the primer pair into a first other primer set list and transmitting the first other primer set list to the step S202;
s202, performing Dimer evaluation between primers in the first other primer set list acquired from the step S201 and the highest specific primer pair generated in the step S201 one by one, screening out primer pairs without Dimer structures, obtaining a second other primer set list, and transmitting the second other primer set list to the step S203;
s203, carrying out nonspecific evaluation on primers in the second other primer set list obtained from the step S202 and the highest specific primer pair generated in the step S201 one by one, screening out primer pairs which are not subjected to nonspecific amplification in a reference sequence with the highest specific primer pair, obtaining a third other primer set list, and transmitting the third other primer set list to the step S204;
s204, if the number of the corresponding positions of the primers in the third other primer set list is greater than 1, continuing to send the third other primer set list to the step S201 for circulation operation; if the number of the corresponding sites of the primer pairs in the third other primer set list is equal to 1, sending the third other primer set list to the step S201 for screening the highest specificity primer pair, combining all the circulated highest specificity primer pairs into a multi-primer pair compatible with the whole synthesis, and sending to the step S300; if the number of corresponding sites of the third other primer set list primer pair is equal to 0, the combination of the multiple primer pairs compatible with the whole of the highest specificity primers of all loops is directly sent to the step S300.
2. The method for designing a multiplex methylation specific PCR primer according to claim 1, wherein the step S000 comprises the steps of:
s001, acquiring target site information and creating target sites;
s002, acquiring and establishing primer demand parameter information, and binding the primer demand parameter information into a target site;
s003, acquiring a reference sequence, and binding the reference sequence to a target site after CT conversion;
s004, obtaining a shielding site defined by a user;
s005, acquiring a shielding area defined by a user;
s006, generating a primer design environment comprising a site sequence, local environment parameters and global environment parameters according to the parameter content acquired in the steps S001-S005, and transmitting the environment parameters to the step S100.
3. The method for designing multiplex methylation specific PCR primer according to claim 1, wherein the step S100 comprises the steps of:
s101, acquiring a primer design environment from the step S000, and designing and generating corresponding primers;
s102, checking whether the primer obtained from the step S101 contains a CG site and a terminal C site, and deleting the unqualified primer;
s103, screening primers with a Dimer structure;
s104, screening primers with a Hairpin structure;
s105, screening primers with tail ends positioned at shielding sites defined by users;
s106, screening primers with tail ends positioned in a shielding area defined by a user;
s107, screening primers of which the interior of the primers possibly amplify a plurality of nonspecific regions in the reference sequence, and transmitting information of potential binding sites of the qualified primers and single primers in the reference sequence to the step S200.
4. The method for designing multiplex methylation-specific PCR primer according to claim 1, wherein: in the step S300, the compatible multiplex primer combination transferred in the step S200 is evaluated, and if the number of primer pairs is too small or the necessary sites are not included, the sites and primer parameters are modified, and then the step S000 is transferred to redesign; if the multiplex primer combination meets the requirements, but some target sites are not included, the target sites which are not included are input again to step S000, and the primers are redesigned for the sites to form a new set of multiplex primer combinations.
5. A computer device, characterized by: the computer device programmed to perform the steps of the multiplex methylation specific PCR primer design method of any one of claims 1-4; or a storage medium of the computer device has stored therein a computer program programmed to perform the multiplex methylation specific PCR primer design method of any one of claims 1 to 4.
6. A multiplex methylation specific PCR primer design system based on the multiplex methylation specific PCR primer design method of claim 1, comprising:
the reference sequence CT conversion and parameter setting module is used for carrying out C- > T conversion on the reference sequence and setting parameters according to the requirements of a user to generate a primer design environment;
the primer design and screening module is used for carrying out primer design on the target site and screening qualified primers according to the user requirements;
the multiplex primer combination module is used for uniformly inspecting primers of a plurality of methylation sites and screening a multiplex primer group without non-specific amplification and secondary structure in a genome;
and the reporting module is used for recording and monitoring all the execution processes, generating a process execution reporting file according to the requirements of the user, and providing help and support for the user to further screen the primer and change the parameters.
7. The multiplex methylation specific PCR primer design system of claim 6, wherein the reference sequence CT conversion and parameter setting module comprises:
the reference sequence CT conversion submodule is used for converting C- > T of a reference sequence and comprises reverse complementation after non-CpG site C- > T conversion of a positive chain of the reference sequence and non-CpG site C- > T conversion of a negative chain of the reference sequence;
the parameter setting submodule is used for setting parameters of a primer design and screening process in advance, wherein the parameters comprise a target site name, a position on a reference sequence, a primer design length, the number of primers generated, GC content, annealing temperature and a region and site which should be skipped in primer screening;
and the environment generation sub-module is used for generating a target region sequence and a parameter format required by primer design according to the set parameters, and transmitting the target region sequence and the parameter format into the primer design and screening module.
8. The multiplex methylation specific PCR primer design system of claim 6, wherein the primer design and screening module comprises:
the primer design submodule is used for carrying out primer design according to the primer design environment transmitted by the reference sequence CT conversion and parameter setting module;
a CG locus screening submodule for evaluating whether the designed primer contains CG locus which possibly causes unbalanced amplification and removing primer pairs which possibly have unbalanced amplification;
a primer screening submodule for evaluating whether the designed primer contains a primer secondary structure which possibly causes the conditions of low amplification efficiency and invalid primer Dimer, and removing the primer pair which possibly causes the conditions of low amplification efficiency or invalid primer Dimer;
the Hairpin screening submodule is used for evaluating whether the designed primer contains a Hairpin secondary structure possibly causing low amplification efficiency or not and removing primer pairs possibly causing low amplification efficiency;
a screening sub-module of screening the screening sites, which is used for evaluating whether the designed primer contains the screening sites defined by the user or not and removing the primer pair containing the screening sites defined by the user;
a screening submodule for evaluating whether the designed primer contains a user-defined screening region or not and removing the primer pair containing the user-defined screening region;
and the primer pair internal nonspecific screening submodule is used for evaluating whether a nonspecific region can be amplified in the converted reference sequence in the designed primer or not and removing the primer pair possibly containing nonspecific amplification characteristics.
9. The multiplex methylation specific PCR primer design system of claim 6, wherein the multiplex primer combination module comprises:
the single primer pair specificity evaluation submodule is used for evaluating the binding sites of the forward and reverse primers contained in the single primer pair in the reference sequence and giving an evaluation value;
the double-primer pair Dimer evaluation submodule is used for evaluating whether a Dimer secondary structure which possibly causes low amplification efficiency and invalid primer Dimer occurrence exists between two primer pairs or not and recording compatibility between the two primer pairs;
the dual-primer pair nonspecific evaluation submodule is used for evaluating whether a nonspecific region is amplified in a converted reference sequence between two primer pairs and recording the compatibility condition between the two primer pairs;
and the multi-primer compatibility evaluation submodule is used for evaluating compatibility among primers by using the double-primer pair Dimer evaluation submodule and the double-primer pair nonspecific evaluation submodule according to the evaluation value given by the single-primer pair specificity evaluation submodule from small to large, and calculating the compatible multi-primer combination with the largest number in the target site by using a statistical method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010479453.2A CN111653311B (en) | 2020-05-29 | 2020-05-29 | Multiplex methylation specific PCR primer design method and system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010479453.2A CN111653311B (en) | 2020-05-29 | 2020-05-29 | Multiplex methylation specific PCR primer design method and system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111653311A CN111653311A (en) | 2020-09-11 |
| CN111653311B true CN111653311B (en) | 2023-05-12 |
Family
ID=72348128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010479453.2A Active CN111653311B (en) | 2020-05-29 | 2020-05-29 | Multiplex methylation specific PCR primer design method and system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111653311B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4253537A1 (en) * | 2020-11-26 | 2023-10-04 | FUJIFILM Corporation | Method of designing primer for amplicon methylation sequence analysis, production method, designing device, designing program and recording medium |
| CN113223623B (en) * | 2021-04-20 | 2022-12-20 | 成都大熊猫繁育研究基地 | Method for predicting specificity of microsatellite PCR product based on genome information |
| CN114596968B (en) * | 2022-05-10 | 2022-07-29 | 至本医疗科技（上海）有限公司 | Multiplex PCR primer design method and device |
| CN117501371A (en) * | 2022-05-27 | 2024-02-02 | 京东方科技集团股份有限公司 | Method, device and equipment for identifying source primer of nonspecific amplified sequence |
| CN115064208B (en) * | 2022-07-08 | 2023-01-03 | 北京阅微基因技术股份有限公司 | Second-generation STR/SNP primer design method and system based on maximum band edge weight group |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2886659A1 (en) * | 2013-12-20 | 2015-06-24 | AIT Austrian Institute of Technology GmbH | Gene methylation based colorectal cancer diagnosis |
| CN107447004A (en) * | 2017-08-11 | 2017-12-08 | 北京呈诺医学科技有限公司 | The method for detecting specificity of DNA methylation PCR detection primers or probe |
| WO2018066910A1 (en) * | 2016-10-06 | 2018-04-12 | (주)지노믹트리 | Multiple detection method of methylated dna |
| CN109735608A (en) * | 2019-01-24 | 2019-05-10 | 深圳因合生物科技有限公司 | A kind of multiple PCR primer design method |
-
2020
- 2020-05-29 CN CN202010479453.2A patent/CN111653311B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2886659A1 (en) * | 2013-12-20 | 2015-06-24 | AIT Austrian Institute of Technology GmbH | Gene methylation based colorectal cancer diagnosis |
| WO2018066910A1 (en) * | 2016-10-06 | 2018-04-12 | (주)지노믹트리 | Multiple detection method of methylated dna |
| CN109952381A (en) * | 2016-10-06 | 2019-06-28 | 基因特力株式会社 | Method for Multiple detection methylate DNA |
| CN107447004A (en) * | 2017-08-11 | 2017-12-08 | 北京呈诺医学科技有限公司 | The method for detecting specificity of DNA methylation PCR detection primers or probe |
| CN109735608A (en) * | 2019-01-24 | 2019-05-10 | 深圳因合生物科技有限公司 | A kind of multiple PCR primer design method |
Non-Patent Citations (3)
| Title |
|---|
| Long-Cheng Li，et,al..PCR Primer Design.Humana Totowa,NJ,2007,(第1版),371-383. * |
| Radoslav S. Davidović*,et,al.Methylation-specific PCR: four steps in primer design.Central European Journal of Biology.2014,第9卷(第12期),1127-1139. * |
| Zhiyong Shen,et,al..MPprimer: a program for reliable multiplex PCR primer design.BMC Bioinformatics.2010,第11卷(第143期),1-7. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111653311A (en) | 2020-09-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111653311B (en) | Multiplex methylation specific PCR primer design method and system | |
| US11667959B2 (en) | Systems and methods to detect rare mutations and copy number variation | |
| US10876171B2 (en) | Systems and methods to detect rare mutations and copy number variation | |
| Kint et al. | Evaluation of bisulfite kits for DNA methylation profiling in terms of DNA fragmentation and DNA recovery using digital PCR | |
| US10894974B2 (en) | Systems and methods to detect rare mutations and copy number variation | |
| Garrett-Bakelman et al. | Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution | |
| Rizzo et al. | Key principles and clinical applications of “next-generation” DNA sequencing | |
| RU2620959C2 (en) | Methods of noninvasive prenatal paternity determination | |
| ES2688458T5 (en) | Varietal nucleic acid count for genomic copy number information | |
| US10407720B2 (en) | Methods for full-length amplification of double-stranded linear nucleic acids of unknown sequences | |
| US11913065B2 (en) | Systems and methods to detect rare mutations and copy number variation | |
| US20220389489A1 (en) | Systems and methods to detect rare mutations and copy number variation | |
| CN113728391A (en) | Method for context-based compression of genomic data of immunooncology biomarkers | |
| Robinson | Discrimination of Monozygotic Twins using DNA methylation levels of one CpG site at Chromosome 3 | |
| Rapley | Molecular cloning and DNA sequencing | |
| Groß | Development of novel SNP panels for the application of massively parallel sequencing to forensic genetics | |
| Oligny | 15Molecular genetics and diagnostic techniques | |
| Lohret et al. | 25 Gene Expression and Discovery: Application of | |
| Zeenath et al. | EVOLVING GENOME TECHNOOGY AND HUMAN HEALTH |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A method and system for designing multiplex methylation specific PCR primers Granted publication date: 20230512 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: WUHAN IGENEBOOK BIOTECHNOLOGY Co.,Ltd. Registration number: Y2024980007417 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |