CN101724700B - Method for rapidly detecting third exon single base mutation of myostatin gene - Google Patents
Method for rapidly detecting third exon single base mutation of myostatin gene Download PDFInfo
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Abstract
The invention provides a method for rapidly detecting the third exon single nucleotide polymorphism (SNP) of a beef myostatin gene. In the method, aiming at the G-to-A mutation of a third exon of the myostatin gene, two groups of PCR primer pairs for respectively amplifying an upstream sequence and a downstream sequence of a G938A single base polymorphism site are designed, wherein the primer pairs are respectively provided with a mismatched primer for mutation site, and the 3' tail end of the primer is positioned on the mutation site. By utilizing the primers, different beef DNA samples can obtain different amplification results, thereby judging the polymorphism of the third exon of the myostatin gene. The method has low cost, and simple, rapid and accurate operation, is suitable for popularization and application, and is important to rapidly and accurately carry out marker-assisted selection of beef, accelerate the beef selecting and breeding process and research a rapid PCR detecting method of the field of molecular biology.
Description
Technical field
The present invention relates to biology field, be specifically related to the method for quick of a kind of detection ox myostatin gene the 3rd exon single nucleotide polymorphism (SNP).
Background technology
Myostatin (myostatin) claims that again (growthdifferentiation factor 8 GDF-8), is transforming growth factor-beta (transforminggrowth factor to Growth differentiation factor 8
TGF-β) member of superfamily, result of study shows, its function is the negative regulatory factor that Skeletal Muscle Growth is grown, so the sudden change of GDF-8 is the reason that causes the two fleshes of ox.Two flesh individualities of Belgian Blue ox lack 11bp (937-947) at exon 3, cause phase shift mutation, cause from the disappearance after 14 codons begin to stop the translation, as a result C-end only 7 amino acid be translated, 102 amino acid (274-375) are afterwards all lost, therefore the albumen that translates can not normally be brought into play its biological function.In the Piemonte ox, be positioned at 938 G of exon 3 → A sudden change, cause coded amino acid to become tyrosine by conservative halfcystine, the function of myostatin is lost (Meng He etc., 2004 all or almost all as a result; History is bright and beautiful etc., and 2005); The disappearance to the muscle growth gene inhibition that causes due to sudden change makes the adenomyosis with this mutated individual flourishing, and in Genetic Improvement of Beef Cattle, the detection of this gene pleiomorphism just seems particularly important.
At present for myostatin gene the 3rd exon polymorphism except direct Sequencing, set up 3 kinds of detection methods:
1) PCR-SSCP method
The method is the combination of round pcr and SSCP technology, its principle is to have specific conformation under non-Denaturing according to single stranded DNA, and when DNA sequence dna even during single sequence change, this specific conformation just is changed, when carrying out native polyacrylamide gel electrophoresis, the single stranded DNA that conformation equal in length is different shows as different electrophoretic mobilities, thereby indirectly gene pleiomorphism is detected analysis (Xue Yan etc., 2005; Wang Wei etc., 2006).The method need not to know having or not of polymorphism on sequence dna fragment to be detected, as long as target sequence is carried out pcr amplification, then the PCR product becomes single stranded DNA through denaturing treatment, then can judge gene polynorphisms through sscp analysis.Can detect all different mutational sites of the identical base sequence of DNA fragmentation length; But the detected result poor repeatability, exist false positive and false negative, can not the Direct Identification target site the polymorphic type of base, must the developing technology such as dye by means of isotropic substance or silver and detect analysis, length consuming time, complex operation, cost higher (Sheffield V C et al, 1993; Xue Yan etc., 2005).
2) polymerase chain reaction-restriction fragment length polymorphism (polymerase chainreaction-restriction fragment length polymorphism, PCR-RFLP) method
This technology is can identify specifically characteristics with cutting DNA specific site (4-6bp) according to restriction enzyme, utilize on DNA sequence dna the sudden change of base namely to replace or insert, lack and to produce or to eliminate a specific restriction enzyme site, thereby make enzyme cut the change of product generation clip size.So, after target site carries out pcr amplification, can be due to the existence in SNP site, produce or eliminated certain restriction endonuclease sites in target product, by selecting specific restriction enzyme pcr amplification product to be carried out enzyme is cut and electrophoresis detection, and the identification and analysis of realize target loci gene type polymorphism.
The PCR-RFLP method is applicable to the detection in some small sample known mutations site, have easy and simple to handle, the advantages such as specificity is high, good reproducibility, but the selection difficulty of restriction enzyme site is large, the price with restriction endonuclease of having or not of restriction enzyme site is also problem (Yang Weihua etc., 2002 of should emphasis in practice considering; Wang Wei etc., 2006).
3) create restriction enzyme site method (Created Restriction Site PCR, CRS-PCR)
Base alternative case design PCR primer according to the single base mutation site, near the base mutational site (as last bit base) is designed to primer 3 ' end, the artificial base mismatch of introducing in primer sequence, make a kind of mutant of primer 3 ' end and single base mutation form a restriction enzyme site after pcr amplification, can be come by corresponding restriction endonuclease cutting, and the another one genotype can not be cut open by selected restriction endonuclease, and pcr amplification product can carry out the Analysis and Identification of gene pleiomorphism after restriction enzyme digestion and electrophoresis like this.
Studies show that the CRS-PCR method is to detect the effective ways of known mutations site SNP, handiness is larger, can carry out genotypic evaluation, has widened the scope of application of RFLP method; But mispairing Primer selection leeway is less, difficulty is larger, after the selection of base mismatch simultaneously will consider to form restriction enzyme site, whether corresponding restriction enzyme is easily bought and Cost Problems, it is less that enzyme is cut rear short-movie section, the difference that long segment and non-enzyme are cut product is less, and it is more difficult that genotype is distinguished, and operates more loaded down with trivial details, (Zhao Chunjiang etc., 2003 grown consuming time; Zhang Zhongbin etc., 2004; Kwok S et al, 1990).
Therefore, the molecular method of setting up a kind of accurate, quick, economic detection ox myostatin gene extron polymorphism is very important.
Summary of the invention
The object of the present invention is to provide and a kind ofly carry out that ox myostatin gene the 3rd exon polymorphism is easy, quick, low-cost detection method.
The method in detection SNP provided by the invention site is used increase the respectively upstream sequence in mutational site and two groups of PCR primer pairs of downstream sequence, takes dual-PCR method that nucleic acid samples is increased, and detects amplified production.Wherein respectively there is one to be that 3 ' end of this primer is positioned on the mutational site for the dissimilar mispairing primer in mutational site in these two groups of PCR primers.That is to say, be used for the upstream primer of amplification SNP site downstream sequence primer pair, and the 3 ' end that is used for the downstream primer of amplification SNP site upstream sequence primer pair all is positioned at the mutational site, but for be the dissimilar base in this site.For the ease of detecting, the chain length of the amplified production of these two groups of primer pairs is not identical.
According to above-mentioned thinking, for ox myostatin gene the 3rd exon SNP pleomorphism site, the invention provides a kind of primer that detects ox myostatin gene the 3rd exon SNP site, it comprises increase the respectively upstream sequence of the single base polymorphic site of G938A and two groups of PCR primer pairs of downstream sequence, wherein respectively there is one to be that 3 ' end of this primer is positioned on the mutational site for the dissimilar mispairing primer in mutational site.
The present invention is directed to this site, upstream and downstream designed respectively a plurality of special primers pair, through multi-turns screen and repetition test, find that following two groups of primer pairs combination significantly is better than other primer pairs, the nucleotide sequence of these two groups of primer pairs is as follows:
Mispairing primer to the downstream, mutational site: MF 5 '-GCCAATTACTGCTCTGGCTAATA-3 ',
Its downstream primer: MR 5 '-ATGGCTGGAATCTTCCCGTAT-3 ';
Mispairing primer to the upstream, mutational site: WR 5 '-GATGCTGTCGTTACCCTCTAAC-3 ',
Its upstream primer: WF 5 '-CTTTTGCAGGAATACAACGTCAC-3 '.
Myostatin gene the 3rd exons mutation site is set to 3 ' end of mispairing primer, has adjusted simultaneously 3 ' end to 5 ' end the 5th and the 6 two base, and the mispairing of these two bases has increased the specific detection effect to myostatin gene polymorphic site.The present invention does not separately establish interior label primer again, uses but can be combined into interior label primer in the primer of design, and namely interior label primer is actual lies among above-mentioned primer.
The present invention further provides a kind of optimize PCR response procedures for above-mentioned pcr amplification, this response procedures is: 94 ℃ of 4min; 94 ℃ of 5s, 52～53 ℃ of 5s, 72 ℃ of 5s, 30 circulations; 72 ℃ of 4min are cooled to 4 ℃.
The present invention adopt two primer PCR technology in conjunction with PCR mispairing technology set up detection ox myostatin gene the 3rd exon polymorphism easy, fast, molecular biology for detection cheaply, for the marker assisted selection of utilizing molecule marker to carry out beef cattle provides effective technique means.
Three exons that the present invention is directed to the myostatin gene have designed three pairs of primers and take Nanyang cattle, Pi Nanniu, Piemonte ox, He Sitanniu as experiment material, the polymorphism of these three exons have been screened.To directly carrying out sequencing after each species blood sample genomic dna pcr amplification, measurement result shows, only have the 3rd exon to have the single base of G938A in the Pi Nanniu population with double-muscling shape and Piemonte ox polymorphic, so take the mutant of myostatin gene the 3rd exon as stencil design pair of primers M, again take wild-type as stencil design pair of primers W, 3 ' end of mispairing primer is designed on the mutational site, and the corresponding product length of primer MF and MR and WF and WR is respectively 185bp and 122bp.MR and WF combination simultaneously also can produce special amplified production, and this product can be used as the interior mark of PCR reaction, and its product length is 262bp.This amplified production is directly carried out 1.2% agarose gel electrophoresis to be detected, each swimming lane of detected result all can detect 262bp product band, 185bp and two product bands of 262bp can be detected for the sudden change homozygous individual, 122bp and two product bands of 262bp can be detected for wild homozygous individual, 122bp, 185bp and two product bands of 262bp can be detected for heterozygous individual, so, this amplified production is only carried out agarose gel electrophoresis both can obtain detected Id accurate judgement, primer sequence and relevant information see Table 2.
Check order individual pcr amplification result and sequencing result are met fully, illustrate that designed primer can be used for carrying out the corresponding analysis that detects to having this polymorphic hereditary population.
Use ox myostatin gene the 3rd exon pleiomorphism detecting method that primer of the present invention is set up, comprise sample collecting, genomic dna preparation, pcr amplification and gel electrophoresis four processes.For sample collecting, both can collecting semen, can gather again blood and hair follicle etc. and organize sample, and only need microcomponent's sample just can obtain to be fit to the DNA of pcr amplification.Prepare this link at genomic dna, the present invention adopts Proteinase K cracking process to prepare the hair follicle genomic dna, and this link only needs one hour, compares with the imitative extraction process of traditional phenol, and the method is simple to operate, with low cost, required time is short.The present invention combines two primer PCR technology and error-prone PCR technology, only needs a PCR reaction and agarose to detect and just can judge detected individual genotype.The present invention, by the technical parameter of change normal PCR and realizes completing pcr amplification about 50 minutes simultaneously with reference to two temperatures grads PCR technology.
In addition, the present invention's reagent used can be domestic reagent, and detecting instrument can be also conventional instrument.
The invention provides a kind of with low cost, simple to operate, quick, accurate and suitable detection method for ox myostatin gene the 3rd exon polymorphism of promoting, the polymorphism of utilizing myostatin is carried out the marker assisted selection important in inhibiting of beef cattle.
Description of drawings
Fig. 1 is for Nanyang cattle, Piemonte ox, the southern beef cattle of skin, 16 individual detected results of order-checking of 4 kinds of He Sitanniu.Wherein ck, w, m are respectively negative control, wild-type contrast and heterozygous mutation contrast, M is molecular weight marker, 1～4 is the He Sitanniu detected result, be wild homozygosity, 5～8 is that Pi Nanniu is individual, and No. 5 swimming lanes are that heterozygote is individual, other are that no mutant homozygote was is individual, 9～12 is that the Piemonte ox is individual, is the no mutant homozygote was individuality, and it is individual that 13～16 Nanyang cattle individualities are wild homozygote.This detected result conforms to fully with sequencing result;
Fig. 2 is the detected result for 13 individualities of skin south cows body.Wherein, 1,2 are respectively the homozygous and individual contrast of heterozygous of sudden change, and swimming lane 3,4,5,6,7,8,9,10,13,14,15 is the heterozygote individuality, and 11,12 be the wild-type homozygote, and this detects and does not find the no mutant homozygote was individuality.16 negative contrasts; M is provided by Guangzhou Dongsheng bio tech ltd for Marker, and band from bottom to up is followed successively by 100bp, 200bp, 300bp, 400bp, 500bp and 600bp; Three objective electrophoretic bands are respectively 122bp, 185bp and 262bp from the bottom to top.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be appreciated that these embodiment only are used for explanation the present invention, and can not limit protection scope of the present invention.
The detection in embodiment 1myostatin gene the 3rd exon SNP site
Materials and methods
1, sample collecting
This example adopts Piemonte ox and Nanyang cattle filial generation Pi Nanniu as research object, is used for the SNPs of colony and detects.In addition, selected three kinds with Typical Representative: Nanyang cattle, Piemonte ox and He Sitanniu are used for screening SNPs.Wherein, the sample of Pi Nanniu and Nanyang cattle picks up from cattle farm, Nanyang Henan Province Xinye County; The sample of Piemonte ox and He Sitanniu picks up from Changping plant of Institute of Animal Husbandry, China Academy of Agriculture Scinces.All test samples are hair follicle, and after gathering, refrigerator freezing is preserved, and the Detection of content that each sample is detailed sees Table 1.
Table 1: laboratory animal sample source and purposes
2, Proteinase K cracking process prepares the hair follicle genomic dna
Proteinase-K (20mg/mL) is diluted to 2g/L: get 10 μ L Proteinase K (20mg/ μ L), add 90 μ LddH
2O。
Preparation 1mL Proteinase-K lysate: get 100 μ L 10 * PCR buffer, add 20 μ LMgCl
2(25mM), add 10 μ L Proteinase Ks (2mg/mL), add 870 μ L ddH
2O。
Preparation process is as follows: 4 hairs with hair follicle are placed in 0.5ml centrifuge tube bottom, add the 20 ready-made Proteinase K lysates of μ l preparation.A simple cyclic program is set: 65 ℃ of 30min, 95 ℃ of 15min, 4 ℃ of 10min on the PCR instrument.After EP (end of program), carry out instantaneous centrifugally, carry out immediately pcr amplification or-20 ℃ of prolonged preservation are standby.
3、PCR
3.1 the design of primer
Table 2 has been listed primer involved in the present invention, and the length of the sequence of primer, length, annealing temperature and specific amplification products.Wherein MF and WR also can obtain the specific amplified product in the double PCR amplification procedure, key point of the present invention that Here it is: and need not establish separately interior mark, in be marked in the design process of dual primer and just lie in wherein, primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.
Table 2 primer M, W sequence and relevant information
Annotate: F represents upstream primer, and R represents downstream primer.Interior label primer amplified production length is 262bp.
3.2PCR reaction system
Through optimizing, the present invention adopts 20 μ l reaction systems, each reacted constituent composed as follows:
Table 3PCR reaction system
The present invention dNTP, Taq enzyme and 10 * PCR buffer used provided by Guangzhou Dongsheng bio tech ltd.
3.3PCR response procedures: 94 ℃, 4min → 94 ℃, 5S; 52 ℃, 5S; 72 ℃, 35 times → 72 ℃ of 5S circulations, 4min.
4, results and analysis
Adopt 2% agarose gel electrophoresis, 110V, after electrophoresis finishes (approximately 20min left and right), gel imaging system detects analysis.M is provided by Guangzhou Dongsheng bio tech ltd for Marker, and band from bottom to up is followed successively by 100bp, 200bp, 300bp, 400bp, 500bp and 600bp; Three objective electrophoretic bands are respectively 122bp, 185bp and 262bp from the bottom to top; Result shows, 16 individual detected results of order-checking of 4 kinds are conformed to fully with sequencing result, and all He Sitanniu and Nanyang cattle are the wild-type homozygous individual, and the southern ox of a scalp is heterozygous individual, all the other 4 are the sudden change homozygous individual, and 4 Piemonte ox individualities are the sudden change homozygous individual.Detected result as shown in Figure 1.
Adopt the above-mentioned method of setting up to carry out the detection analysis of this gene single base mutation of the southern cows body of skin, as shown in Figure 2, detected result to 13 Pi Nanniu individualities shows, electrophoresis result can be carried out genotype judgement clearly to tested individuality, wherein 1,2 be respectively the homozygous and individual positive control of heterozygous of sudden change, swimming lane 3,4,5,6,7,8,9,10,13,14,15 is the heterozygote individuality, and 11,12 be the wild-type homozygote, and this detects does not find that no mutant homozygote was is individual.16 negative contrasts.
Sequence table
＜110〉Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
＜120〉method for quick of myostatin gene the 3rd exon single base mutation
<130>KHP09113606.4
<160>4
<170>PatentIn version 3.5
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＜213〉artificial sequence
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gccaattact gctctggcta ata 23
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atggctggaa tcttcccgta t 21
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gatgctgtcg ttaccctcta ac 22
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Claims (6)
1. primer that detects ox Myostatin gene the 3rd exon SNP site, it comprises increase the respectively upstream sequence of the single base polymorphic site of G938A and two groups of PCR primer pairs of downstream sequence, wherein respectively there is one to be the mispairing primer for the mutational site, 3 ' end of this primer is positioned on the mutational site, and described primer is:
Mispairing primer to the downstream, mutational site: MF 5 '-GCCAATTACTGCTCTGGCTAATA-3 ',
Its downstream primer: MR 5 '-ATGGCTGGAATCTTCCCGTAT-3 ';
Mispairing primer to the upstream, mutational site: WR 5 '-GATGCTGTCGTTACCCTCTAAC-3 ',
Its upstream primer: WF 5 '-CTTTTGCAGGAATACAACGTCAC-3 '.
2. the detection kit that contains the described primer of claim 1.
3. method that detects ox Myostatin gene the 3rd exon SNP site, it adopts primer claimed in claim 1 or test kit claimed in claim 2 amplification ox DNA sample, and detects amplified production.
4. method as claimed in claim 3, it is characterized in that, after adopting the described primer of claim 1 to carry out pcr amplification, carrying out agarose gel electrophoresis detects, wherein, what present 185bp and 262bp two bands is the homozygous mutation type, presents the homozygous wildtype that is of 122bp and 262bp two bands, presents the heterozygote that is of 122bp, 185bp and 262bp three bands.
5. method as described in claim 3 or 4, is characterized in that, the hair follicle genome DNA sample of described ox DNA sample for adopting Proteinase K cracking process to prepare.
6. method as described in claim 3 or 4, is characterized in that, the PCR response procedures is: 94 ℃ of 4min; 94 ℃ of 5s, 52～53 ℃ of 5s, 72 ℃ of 5s, 30 circulations; 72 ℃ of 4min are cooled to 4 ℃.
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| CN102296073B (en) * | 2011-08-11 | 2014-04-23 | 中国农业科学院北京畜牧兽医研究所 | Specific target site for site-directed knockout of gene Myostatin by zinc finger nuclease |
| CN102649962B (en) * | 2012-04-18 | 2016-09-14 | 江苏师范大学 | The mononucleotide polymorphism site of cattle WNT10B gene and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101405404A (en) * | 2002-12-31 | 2009-04-08 | 迈特默菲公司 | Compositions, methods and systems for inferring bovine breed |
| CN101591696A (en) * | 2008-05-26 | 2009-12-02 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | German Mutton Merino myostatin gene SNP site |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101405404A (en) * | 2002-12-31 | 2009-04-08 | 迈特默菲公司 | Compositions, methods and systems for inferring bovine breed |
| CN101591696A (en) * | 2008-05-26 | 2009-12-02 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | German Mutton Merino myostatin gene SNP site |
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