CN103966349A - Screening method and application of multiple-allele PCR (Polymerase Chain Reaction) amplified fragment - Google Patents
Screening method and application of multiple-allele PCR (Polymerase Chain Reaction) amplified fragment Download PDFInfo
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- CN103966349A CN103966349A CN201410234407.0A CN201410234407A CN103966349A CN 103966349 A CN103966349 A CN 103966349A CN 201410234407 A CN201410234407 A CN 201410234407A CN 103966349 A CN103966349 A CN 103966349A
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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Abstract
The invention discloses a screening method and application of a multiple-allele PCR (Polymerase Chain Reaction) amplified fragment. The screening method comprises the steps of finding the published chromosome related data through NCBI (National Center for Biotechnology Information), and determining SNP (Single Nucleotide Polymorphism) aggregation suspected to exist; then, designing a primer for an SNP aggregation region suspected to exist, and carrying out PCR (Polymerase Chain Reaction); sequencing to detect whether a plurality of SNP sites really exist on a PCR amplified fragment; further carrying out correlation analysis on the SNP sites to determine whether the amplified fragment has multiple alleles so as to screen the multiple-allele PCR amplified fragment for animal individual identification, meat product tracing technology research and industrialization and the like.
Description
Technical field
The invention belongs to biology field, relate to a kind of screening method and application thereof of multiple alleles type pcr amplified fragment.
Background technology
Food safety affair takes place frequently in recent years, and government administration section and human consumer are also unprecedented to the concern of food safety, therefore researches and develops animal individual identity recognizing technology and meat product tracing technology, and particularly DNA recognition technology is imminent.
For this reason, the SNP bar codes technique that we trace to the source to animal individual identification and meat product has been carried out series of studies, and feel deeply if really realize the technical researches such as animal individual identification and real industrialization, need to make animal individual identity DNA identification and meat product DNA tracing technology and research and development thereof become more simple, must screen so the pcr amplified fragment that some contain multiple SNP site simultaneously, and pcr amplified fragment multiple alleles type, becomes SNP barcode by the effective SNP Sites Combination in these pcr amplified fragments.Multiple alleles type pcr amplified fragment, mainly for the gene intervening sequence screening on karyomit(e), does not only screen for functional gene.For gene intervening sequence, screening is not only easy to find a large amount of SNP sites, and the multiple alleles type pcr amplified fragment quantity of information that a large amount of SNP site forms is larger, and then making trace to the source DNA tracing technology and the research and development thereof of animal individual identity DNA identification and meat product become more simple, technology industrialization cost is lower; And that functional gene sequence is compared to gene intervening sequence on karyomit(e) is relatively conservative, to suddenly change relatively less, SNP screening efficiency is relatively not high, and the PCR fragment of amplification only contains a SNP site mostly, and quantity of information is relatively limited.For this reason, we have proposed " multiple alleles type pcr amplified fragment " concept, and have set up a kind of simple method for screening of multiple alleles type pcr amplified fragment.
Summary of the invention
The present invention seeks to the above-mentioned deficiency for prior art, a kind of screening method of multiple alleles type pcr amplified fragment is provided.
Another object of the present invention is to provide the application of the method.
Technical scheme of the present invention is as follows:
A kind of screening method of multiple alleles type pcr amplified fragment, to search by NCBI the karyomit(e) related data of having announced, the SNP that determines doubtful existence assembles, then design primer pcr amplification for the SNP aggregation zone of doubtful existence, whether amplified fragments really there is multiple SNP site by order-checking to detect pcr amplified fragment, and further by the correlation analysis in SNP site, determine whether amplified fragments has multiple alleles type, thereby filter out multiple alleles type pcr amplified fragment, for animal individual identification, meat product research and development and the industrialization of technology such as trace to the source.
Wherein, described multiple alleles type pcr amplified fragment refers to that pcr amplified fragment at least exists 2 or above SNP site, and allelotype quantity >=4 that are separated to of (in colony) amplified fragments.
Described karyomit(e) related data (data), refers to the doubtful SNP site on upper chromosomal DNA sequence and the DNA sequence dna of announcing of NCBI.
Described SNP assembles, and refer in a certain section (once sequencing length is advisable) of the DNA sequence dna that NCBI announces and have multiple doubtful SNP site, and the SNP site quantity of doubtful existence is The more the better.
The multiple alleles type pcr amplified fragment obtaining according to screening method of the present invention can be applicable to the aspects such as animal individual identification, the research and development of meat product tracing technology, molecular genetic marker screening, is preferably applied to the screening of the required multiple alleles type pcr amplified fragment in the aspects such as pig individual identity recognition technology research and development.
Beneficial effect: the present invention is the karyomit(e) related data (data) of having announced according to NCBI, come the doubtful hypervariable region of screening-gene group DNA, and design primer amplification acquisition multiple alleles type amplified fragments, for research and development and the industrialization etc. of animal individual identification, meat product tracing technology.Be different from the most of SNP screenings mainly for functional gene (functional gene sequence is relatively conservative) at present, multiple alleles type pcr amplified fragment screening of the present invention does not consider to screen object, and the pcr amplified fragment of screening is positioned at the gene intervening sequence on karyomit(e) mostly.Gene intervening sequence is compared to functional gene, and sequence is relatively not conservative, and has a large amount of hypervariable regions, and therefore SNP screening is more easy, and efficiency is relatively high.And the amplified fragments multiple alleles type of screening, is compared to the pcr amplified fragment that only has a SNP site, quantity of information is relatively large, is conducive to animal individual identification, meat product research and development and the industrialization of technology such as trace to the source.Example: if screening pcr amplified fragment genotype number more than 9,5 pairs of primers (in theory) just can be used for 9 so
5the identification of individual pig individuality (5.9 ten thousand), substantially can meet the pig individual identity identification on scale pig farm, if and the pcr amplified fragment of screening only contains a SNP site, reach so same individual identity recognition effect, need in theory 10 pairs of primers.Therefore, the screening of multiple alleles type pcr amplified fragment is conducive to research and development and the industrialization of the technology such as animal individual identification.Simultaneously, because the multiple alleles type pcr amplified fragment of screening is positioned at the gene intervening sequence on karyomit(e) mostly, belong to hypervariable region, therefore there is the total SNP site of some different varietiess in multiple alleles type pcr amplified fragment, or there is the SNP mutational site of different varieties simultaneously, these pcr amplified fragments that screen like this can be simultaneously produce meat for the individual identity identification of the multiple kinds of animal or meat traces to the source etc., thus degree of depth research and development and the industrialization of the technology such as realize that animal individual identification, meat product are traced to the source.
Brief description of the drawings
Fig. 1 be hybrid dna template taking 69 duroc samples as pcr template, the part order-checking peak figure of primer pair 1 extension increasing sequence.
Fig. 2 be primer pair 1 increase+470 and+502 two 5 kinds of genotype that SNP site combines in 69 duroc samples.
Figure 33 obtains SNP site and the effective SNP site for gene type in duroc to primer
The SNP site that Figure 43 screens in Du Luoke, long white, 3 pig kinds of Yorkshire primer amplification fragment, the SNP site wherein having is that three kinds are common, two kinds that have are common.
Embodiment
The polygene type pcr amplified fragment screening of embodiment 1 duroc genomic dna and individual identity identification
1.1 design of primers (taking No. 10 karyomit(e)s of pig as example)
1.1.1 the Nucleotide of NCBI database (
http:// www.ncbi.nlm.nih.gov/nuccore) middle searched key word Sus scrofa breed mixed chromosome10 (design primer on other karyomit(e)s, " 10 " are changed into corresponding karyomit(e) number).Open after the page clickable hyperlinks Sus scrofa breed mixedchromosome10 successively, Sscrofa10.2, Graphics.
In graphical interfaces, click configure button and eject tab, in the total Options under Tracks tab, only retain " √ " before sequence and SNP.Clicking configure button confirms.
1.1.2 click the place of dark (SNP is intensive) of blueness in SNP barcode by right key, in popup menu, select " Zoomto sequence ", be adjusted to scale by " Zoom bar " and show taking k as unit.On scale, pin left mouse button and mark SNP close quarters, in the menu ejecting while discharging left mouse button, click " set new marker forselection ".Mouse-pointing marker1, records the zero position numerical value of range.
1.1.3 click " Tools ", in popup menu, select after " sequence text view ", click " go to position ", the range zero position numerical value of input 1.1.2 step record, click " OK " key, in square brackets, show " Maker1 " flag sequence, copy the template (use Ctrl+C keying replication) of this sequence as design of primers.After replication sequence is preserved with notepad, can be by online or local software design primer.
1.1.4 click Marker1 region by right key, in popup menu, left button is clicked: " Zoom In ", until marker1 region is full of after its visibility window, then left button is clicked " BlAST and Primer Search/PrimerBlAST (visible Range) " in this menu.
1.1.5 ejecting new page fills according to following requirement:
Primer Parameters hurdle: under PCR product size project, Min item is inserted 500, Max item and inserted 1000.Primer Pair Specificity Checking Parameters hurdle: guarantee that Specificity check project is checked; In the drop-down menu of database item, select Genome (chormosomes from all organisms); Organism item is inserted Sus scrofa (taxid:9823).
Finally click Get Primers button.In Detailed primer reports column in the new page ejecting, can obtain several combination of primers, in conjunction with follow-up pcr amplification and direct Sequencing, screen effective primer.
Embodiment screens 3 pairs of primers (due to the most difficult differentiation of brood piglet born of the same parents, so embodiment has only enumerated the individual identity identification of 9 brood piglets born of the same parents, 9 piglets born of the same parents only need 3 pairs of primers just can realize individual identity identification) altogether.
The relevant information of 3 pairs of primers that table 1 screens
1.2 genomic dna template preparations
Sample is 69 pigs (kind pig farm, grand sight mountain, Hangzhou) pig hair (containing hair follicle), and prepares DNA profiling with DNA MiniExtract test kit (Nanjing Run Bang Bioisystech Co., Ltd).
Single sample DNA template preparation process: (several) pig mao mao capsule end inserts in the PCR pipe that 17.6 μ l DNAMiniExtract are housed down, 95 DEG C × 5min in PCR instrument, after 16 DEG C × 1min, adding concentration is 20mg/ml Proteinase K 2.4 μ l, 55 DEG C × 2hr, 95 DEG C × 10min, 16 DEG C × 1min, centrifuging and taking supernatant is made pcr amplification template.
Mixing sample DNA profiling preparation process: every pig is chosen 1 pig hair, hair follicle end mixes insertion down and is equipped with in the centrifuge tube of 352 μ l DNA MiniExtract, after 95 DEG C × 5min of water-bath, be cooled to normal temperature, adding concentration is 20mg/ml Proteinase K 48 μ l, 55 DEG C of water-bath 2hr, be cooled to normal temperature after centrifuging and taking supernatant make pcr amplification template.
1.3PCR amplification and primer preliminary screening
Reaction system: distilled water 7 μ l, Mix (Qi Boneng bio tech ltd, Nanjing) 10 μ l, the each 0.1 μ l of the forward and reverse primer of 5 μ M, DNA profiling 1.0 μ l.
Reaction conditions: 95 DEG C × 2min; 95 DEG C × 30s, 62 DEG C × 30s, 72 DEG C × 50s, 35 circulations; 72 DEG C × 10min.
PCR product is tentatively confirmed object band (electrophoretic band must be single) with 1% sepharose, amplified production direct Sequencing, and the necessary clean background of order-checking peak figure, reads and there is no ambiguity.
The preliminary screening of 1.4 object fragment authenticity validations and doubtful multiple alleles type pcr amplified fragment (taking the SNP site of the 1st pair of amplified fragments primer in table 1 as example explanation)
First carry out pcr amplification with 69 pig hair mixing sample DNA profilings, amplified fragments sequencing result and design of primers template are compared in Align X, confirm, after object fragment verity, in order-checking peak figure, to search cover peak (Fig. 1).Fig. 1 is the part order-checking peak figure of mixing sample DNA profiling extension increasing sequence, wherein: occur+470 ,+499 and+530 3 SNP sites, front indicates+502 SNP sites for confirming in follow-up single sample sequencing result) and, be confirmed whether as doubtful multiple alleles type pcr amplified fragment.
After treating that doubtful multiple alleles type pcr amplified fragment is determined, single sample DNA template increases, the sequencing result of 69 pig samples obtains 9 SNP sites altogether, + 82 (SNP site mark, with the 1st the base position at forward primer place in amplified production count+1) ,+129 ,+206 ,+219 ,+470 ,+499 ,+502 ,+530 ,+622.
The SNP site that the 3 pairs of primer amplification fragments obtain and for effective SNP site of gene type as shown in Figure 3.Wherein serve as a contrast with gray shade for effective SNP site of gene type, the numeral in bracket shows the heterozygosity in this SNP site.In the present embodiment, the 3 pairs of primer related datas are from the analytical results of 69 duroc samples.
1.5DNA fragment gene somatotype
9 SNP sites that obtain are through correlation analysis, selected+82 ,+470 and+502 totally 3 SNP sites, for multiple alleles type pcr amplified fragment gene type, and obtain 9 kinds of genotype of amplified fragments.9 kinds of genotype (genotype taking+82 ,+470 and+502 3 SNP sites orders are arranged in order) as ACTCTC, CCTTCC, ACTTTC, CCTCCC, CCTTTC, ACTTCC, CCCCCC, ACTCCC and ACCCCC.Fig. 2 has provided and has been separated in 69 duroc samples, by+470 5 kinds of genotype that form with+502 two SNP Sites Combination (wherein+470 is associated with+499 two SNP sites).
In other two pairs of primer amplification fragments for the relevant information in the SNP site of gene type as Fig. 3.
1.6 individual identity identifications (the SNP barcode establishment of brood piglet born of the same parents)
3 pairs of primers obtain 26 SNP sites altogether, through correlation analysis, retain 11 SNP sites (in Fig. 3, lining is with the SNP site of grey impact) for the gene type of amplified fragments and the individual identity of 9 piglets born of the same parents identification SNP barcode.SNP barcode establishment, with primer pair 1 ?3 order be arranged in order (wherein the SNP site of every pair of primer with 5 ’ ?to 3 ’ ?order be arranged in order).
The screening of the PCR screening amplified fragments in the total SNP of 1.7 different varietiess site
With above-mentioned 3 pairs of primers increase respectively long white (82 samples), Yorkshire Pigs (83), the SNP site that the 3 pairs of primers obtain in Du Luoke, long white, 3 pig kinds of Yorkshire as shown in Figure 4.In primer pair 1 amplified fragments+470 ,+502 ,+499 and+622 (indicating with wave underline) are Du Luoke, long white, the total SNP site of Yorkshire, can be preferentially for individual identity identify, the research and development of the technology such as meat product is traced to the source; Other SNP site or be that common (Du Luoke, the long white total lower stroke of fine rule in SNP site indicate for 2 kinds of kinds, Du Luoke, the total SNP site of Yorkshire indicate with lower stroke of thick line, long white, Yorkshire does not have common SNP site), or only belong to a kind.In primer pair 2, only have 2 total SNP site or SNP sites of kind only in a kind, to exist.3 of primer pairs screen SNP site in duroc, and in other 2 kinds, all do not screen SNP site, can not be as the total primer that 3 kind individual identities are identified and meat product is traced to the source.Therefore, primer pair 1 and 2 can be used as total primer, traces to the source, wherein taking primer pair 1 as best for Du Luoke, long individual identity identification or meat product white, Yorkshire Pigs.
Claims (5)
1. the screening method of a multiple alleles type pcr amplified fragment, it is characterized in that described multiple alleles type pcr amplified fragment refers to that pcr amplified fragment at least exists 2 or above SNP site, and allelotype quantity >=4 that amplified fragments is separated in colony, described screening method is to search by NCBI the karyomit(e) related data of having announced, the SNP that determines doubtful existence assembles, then design primer pcr amplification for the SNP aggregation zone of doubtful existence, whether amplified fragments really there is multiple SNP site by order-checking to detect pcr amplified fragment, and further by the correlation analysis in SNP site, determine whether amplified fragments has multiple alleles type, thereby filter out multiple alleles type pcr amplified fragment.
2. the screening method of multiple alleles type pcr amplified fragment according to claim 1, the karyomit(e) related data that it is characterized in that described announcement is the doubtful SNP site on upper chromosomal DNA sequence and the DNA sequence dna of announcing of NCBI.
3. the screening method of multiple alleles type pcr amplified fragment according to claim 1, is characterized in that described SNP assembles, and refers in a certain section of the DNA sequence dna that NCBI announces and has multiple doubtful SNP site.
4. the application of the screening method of multiple alleles type pcr amplified fragment claimed in claim 1 in animal individual identity DNA identification or animal meat product dna tracing technology, molecular genetic marker.
5. application according to claim 4, the application of the screening method that it is characterized in that multiple alleles type pcr amplified fragment claimed in claim 1 in pig individual identity DNA identification and DNA source tracing of pork products.
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| CN112094840A (en) * | 2019-06-17 | 2020-12-18 | 南京尧顺禹生物科技有限公司 | Rapid preparation method of trace animal sample genome DNA template for genome segment amplification |
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Application publication date: 20140806 |