CN1990879A - Method of identifying buffalo embryo sex using nest-type PRC - Google Patents
Method of identifying buffalo embryo sex using nest-type PRC Download PDFInfo
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- CN1990879A CN1990879A CN 200510048301 CN200510048301A CN1990879A CN 1990879 A CN1990879 A CN 1990879A CN 200510048301 CN200510048301 CN 200510048301 CN 200510048301 A CN200510048301 A CN 200510048301A CN 1990879 A CN1990879 A CN 1990879A
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Abstract
The invention relates to a method for identifying sex of buffalo embryo by using PCR. It designs three pairs of primers according to the buffalo SRY gene sequence result: SRY-HMG box specific nest primer and reference gene GAPDH primer, and the GAPDH primer is used to amplify the common GAPDH gene for male and female buffalo (glyceraldehyde dehydrogenase gene) to check if the embryo sample is lost; SRY-HMG box primer is used to amplify the specific SRY conservative gene for male bufflalo to identify embryo sex. The designed specific primer can amplify relevant gene sequence steadily on basis of sequencing for buffalo SRY full sequence, and it can dramatically increase the sensitivity and feasibility for check.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to and utilize PCR (polymerase chain reaction) method amplification buffalo sry gene to carry out the method that embryo gender is identified.
Background technology
In the embryo biotechnology field, the sex control problem is the great theoretical problem that scientist endeavours to study for a long time always, at present, can realize that sex-controlled means mainly contain two kinds: the one, and the separating of X sperm and y sperm; The 2nd, the sex identification of body early embryo.Though the sperm separation efficiency is higher at present, has the cost height, is not suitable for promoting in the actual production.And the early embryo sex authenticate technology has the developing history of decades, and after PCR (polymerase chain reaction) technological invention, early embryo sex is identified full-fledged gradually especially, becomes the sex control technology that use value is arranged at present most.
The embryo gender authentication method has experienced such several stages: chromosome karyotype analysis method, X ligase enzyme activation analysis method, immunological method and present molecular biology method, after round pcr was used, it was the actual production field that this technology enters.Be commonly used at present identify that the detection gene of beef embryo sex has: the multiple complex sequences of Y-chromosome, sry gene core sequence, ZFX/ZFY gene order etc.Abroad, Herr CM etc. (1990) has at first successfully used round pcr to identify cattle and sheep embryo gender (HerrCM, Theriogenology, 1991,35 (1): 45-54), obtained 91.7% (11/12) accuracy rate; Peura etc. (1991) have taked to utilize 3 pairs of primers of pcr amplification to carry out beef embryo sex and have identified, wherein a pair of is ox DNA Auele Specific Primer, these primer amplification male and female all produce same band, whether be used for test sample loses, two pairs of primers are the cattle Y-chromosome Auele Specific Primer in addition, and what their increased is male special product.At home, Zhu Yuding etc. (1992) use the two-step pcr method, make sex identification rate of accuracy reached to 100% (hi-tech communication, 1992, (12): 12-14); Ceng Yitao etc. are by on the basis to the order-checking of ox Y-chromosome sex determination zone (SRY) gene core sequence, design a pair of SRY primer that is specific to ox, whether identified the sex achieving success of cow embryo by the existence that detects sry gene, success ratio is 100%[Chinese science (B), 1993,23 (4): 371-375], Xu Yaou etc. (1993) have designed a pair of primer according to ZFX/ZFY, utilize pcr amplification bovine blood DNA to identify sex, the segment that will increase is cut with Pst I enzyme, and the ZFY gene PCR amplification segment of bull has two restriction enzyme sites, and ZFX does not have, so again electrophoresis the former occur three segments (486bp, 356bp, 112bp).
Because the multiple complex sequences of Y-chromosome usually has homologous sequence on X chromosome, thereby qualification result has certain uncertainty.Amplification ZFX/ZFY gene also has weak point, cuts evaluation because the ZFX/ZFY gene need further be done enzyme, wastes time and energy, aborning can not widespread use; Amplification SRY core sequence method is because of SRY is male special, so have higher accuracy rate, but SRY is a single copy gene, the template of using is several blastomeres, and the sensitivity that conventional PCR once increases is limited, causes the amount owing to amplified production to influence the result who identifies very little sometimes.
As utilize the nest-type PRC technology, on to the basis of buffalo sry gene order-checking, design two pairs of primers, after using 20 circulations of external primer amplification, take out 1/25 product and use inner primer to carry out the pcr amplification second time, can reach very high evaluation efficient, can be used for the sex identification of single blastomere in theory.
Existing other Bos animal vias of report are crossed embryo gender and are identified the back calving at present.Identify but carry out embryo gender, yet there are no report at present both at home and abroad with nest-type PRC amplification buffalo sry gene.
Buffalo is one of main ox kind of China, and buffalo milk has very high nutritive value, but the present situation of just present China milk buffalo sees that its quantity and milk yield also do not satisfy the demand in market far away.And identify the transplanting female embryo by early embryo sex, and the milk ox head number that can increase sharply, thus the selection intensity of milk buffalo improved, and this is very great to accelerating milk buffalo development meaning already.
The objective of the invention is to utilize embryo gender authenticate technology this animal reproduction new technology that combines with technology in vitro fertilization to produce other buffalo embryo of foreseeability, to reach control breeding milk buffalo offspring's sex.Also provide technical support simultaneously for breeding milk buffalo embryo batch production, sex production.The maximum characteristics of producing this method of sex controllable in vitro embryo of buffalo be efficient, save time, laborsaving and cost saving, so this method is to accelerate high yield milk buffalo reproductive speed one of effective technical means the most at present.
Summary of the invention
The purpose of this invention is to provide a kind of primer that is specific to buffalo SRY sequence.Utilize this kind PCR primer when carrying out the detection of buffalo sry gene, not only sensitivity holds good security, and by introducing the GAPDH internal control gene, whether checking embryo sample is lost becomes possibility.The nest-type PRC primer of the present invention's employing is to design on the basis to the order-checking of buffalo sry gene in addition, and high specificity is fit to the small amount of sample amplification, is convenient to use aborning.
| The primer title | Sequence |
| Confidential reference items primer downstream, inner primer upstream, inner primer downstream, outer primer upstream, outer primer downstream, upstream confidential reference items primer | 5’AGG ACC ACA TCA AGC GAC CCA T 3’ 5’CGG GTA TTT GTC TCT GTG TAT GG 3’ 5’AGG CAG CTG GGC TAT GAG TGG AA 3’ 5’CGG GTA TTT GTC TCT GTG TAT GG 3’ 5’GGT GAA GGT CGG AGT CAA CGG 3’ 5’GGT CAT GAG TCC TTC CAC GAT 3’ |
Utilize the embryo gender authenticate technology in conjunction with technology in vitro fertilization, other buffalo embryo of mass production foreseeability satisfying the demand of market to controlled sex buffalo embryo, thereby reaches the speed of breeding of quickening breeding milk buffalo, and promotes the development of buffalo milk industry.
Particular content is as follows:
(1) reclaim blastaea when after fertilization the 6th, the 7th and the 8th day, use micrurgy instrument or manual cut to obtain a small amount of embryo's sample, the embryo after the sampling carries out freezing preservation and handles, and places liquid nitrogen container to store then.
(2) embryo's sample washs three times in the PBS of no calcium magnesium (phosphoric acid buffer), changes in the PCR pipe, adds cell pyrolysis liquid (Lysis Solution), places 5min in room temperature, directly carries out the PCR reaction as template behind 98 ℃ of inactivated proteases K.
(3) in new PCR pipe, add 24 μ L PCR Mix liquid and (comprise the PCR damping fluid, dNTP, Taq archaeal dna polymerase, deionized water) and upstream and downstream outer primer, upstream and downstream confidential reference items primer (each 50pM), the 94 ℃ of 2min that unwind in advance in the PCR instrument carry out following 20 circulations: 95 ℃ of 20s then; 62 ℃ of 30s; 72 ℃ of 30s, last 72 ℃ are extended 5min, stopped reaction.
(4) the PCR product of taking-up 2 μ L adds in the new PCR pipe, add 23 μ L PCR Mix liquid again and (comprise the PCR damping fluid, dNTP, the Taq archaeal dna polymerase, deionized water), upstream and downstream inner primer and upstream and downstream confidential reference items primer (each 50pM), the 94 ℃ of 2min that unwind in advance in the PCR instrument carry out following 30 circulations: 95 ℃ of 20s then; 61 ℃ of 30s; 72 ℃ of 30s, last 72 ℃ are extended 5min, stopped reaction.
(5) with amplified production in 2% agarose gel electrophoresis (100V, 30 minutes), observe down with ultraviolet lamp after gel placed EB (the own ingot of bromination) dyeing 20min, all samples that goes out all to occur band at 460bp and 160bp be male embryo all, have only 460bp occur band for female embryo, all occur band for losing sample.Compare with present sex identification technology, the invention has the advantages that:
(1) utilizing this nest-type PRC primer and GAPDH confidential reference items primer to carry out multiplex PCR identifies, can reach the evaluation accuracy rate more than 95%, and by the configuration premix PCR Mix liquid simplify the operation, effectively reduce pollution rate, improve sensitivity and accuracy in the embryo gender evaluation greatly, by this making property of technological approaches control Embryo Production batch production.
(2) this method the breeding of buffalo of suckling that combine with other various modern Embryo Production technology can be improved the efficient of breeding of milk buffalo, and the breeding that increases sharply milk buffalo quantity improves milk cows quality, quickens breed breeding work.
Therefore, using the sex authenticate technology to breed the efficient of milk cow will be than the doubling of asexuality control, thereby can save nearly half time, energy and expense.
Description of drawings
Fig. 1 is the result that sry gene nested primer and GAPDH confidential reference items primer increase to known sex buffalo genomic dna.
♀ is a cow; ♂ is a bull; B is a blank; The molecular weight standard of M:250bp gradient; 130bp is the electrophoretic band of the pcr amplification product of the distinctive sry gene of public buffalo; The 460bp place is the result of the total gene primer of male and female buffalo-GAPDH confidential reference items primer to the amplification of buffalo genomic dna.
Fig. 2 is sry gene nested primer and the GAPDH confidential reference items primer result to the buffalo embryo sex identification.
1,2,4,7,10 swimming lanes have the electrophoresis band of amplified fragments of the total gene of male and female buffalo-GAPDH gene of the peculiar gene of the public buffalo-sry gene amplified fragments of 130bp and 460bp simultaneously, so sex identification is a male embryo; 3,5,6,8 swimming lanes have only the segmental electrophoresis band of GAPDH gene amplification, so sex identification is a female embryo; 11 is blank; 12 positive contrasts; The molecular weight standard of M:250bp gradient; 130bp is the electrophoretic band of the pcr amplification product of the distinctive sry gene of public buffalo; 460bp is the electrophoretic band of the pcr amplification product of the total gene primer of male and female buffalo-GAPDH primer.
Embodiment
Embodiment 1: carry out sex identification by the buffalo genomic dna to known sex, detect the specificity of primer.
(1) gets the fresh water bovine blood, extract DNA with the imitative extraction process of phenol;
(2) add 2.5 μ L PCR damping fluids in the new EP pipe, 0.5 μ L dNTP (50 μ M), upstream and downstream outer primer and upstream and downstream confidential reference items primer GAPDH (each 50pM), 200ng genomic dna, 2U TaqDNA polysaccharase, adding deionized water to total reaction volume is 25 μ L.The 94 ℃ of 2min that unwind in advance in the PCR instrument carry out following 20 circulations: 95 ℃ of 20s then; 62 ℃ of 30s; 72 ℃ of 30s, last 72 ℃ are extended 5min, stopped reaction;
(3) the above-mentioned PCR product of taking-up 0.5 μ L adds in the new PCR pipe, add 2.5 μ L PCR damping fluids again, 0.5 μ L dNTP (50 μ M), upstream and downstream inner primer and upstream and downstream confidential reference items primer GAPDH (each 50pM), 2U Taq archaeal dna polymerase, adding deionized water to total reaction volume is 25 μ L.The 94 ℃ of 2min that unwind in advance in the PCR instrument carry out following 30 circulations: 95 ℃ of 20s then; 61 ℃ of 30s; 72 ℃ of 30s, last 72 ℃ are extended 5min, stopped reaction;
(4) with amplified production in 2% agarose gel electrophoresis (100V; 30min); observe down with ultraviolet lamp after gel placed EB (the own ingot of bromination) dyeing 20min; 2,4,5,6,8, No. 9 public buffalo genome DNA samples band all occurs at 460bp and 130bp; 1,3, No. 7 water cow genomic dna samples have only 460bp band to occur, and qualification result meets (seeing accompanying drawing 1) fully.
Embodiment 2: the buffalo embryo sex identification
(1) use micrurgy instrument or micropin to obtain a small amount of embryo's sample from blastaea cutting embryo sub-fraction.Embryo after the sampling carries out freezing preservation and handles, and places liquid nitrogen container to store then.
(2) the embryo's sample that obtains more than washs three times in the PBS of no calcium magnesium (phosphoric acid buffer), be respectively charged in the PCR pipe, add cell pyrolysis liquid (Lysis Solution) 10 μ L, after room temperature is placed 5min, 98 ℃ of insulation 10min are with inactivated proteases K, and this processing back sample directly carries out the PCR reaction as template.
(3) add 23 μ L PCR Mix liquid (comprising the PCR damping fluid, dNTP, Taq enzyme, deionized water) in the new PCR pipe, upstream and downstream outer primer and upstream and downstream confidential reference items primer (each 50pM), total reaction volume is 25 μ L.The 94 ℃ of 2min that unwind in advance in the PCR instrument carry out following 20 circulations: 95 ℃ of 20s then; 62 ℃ of 30s; 72 ℃ of 30s, last 72 ℃ are extended 5min, stopped reaction.
(4) the PCR product of taking-up 1 μ L adds in the new pipe, add 22 μ L PCR Mix liquid again and (comprise the PCR damping fluid, dNTP, the Taq enzyme, deionized water), upstream and downstream inner primer and upstream and downstream confidential reference items primer (each 50pM), the 94 ℃ of 2min that unwind in advance in the PCR instrument carry out following 30 circulations: 95 ℃ of 20s then; 61 ℃ of 30s; 72 ℃ of 30s, last 72 ℃ are extended 5min, stopped reaction.
(5) with amplified production in 2% agarose gel electrophoresis (100V, 30 minutes), observe down in ultraviolet lamp after gel placed EB (the own ingot of bromination) dyeing 20min, all samples that goes out all to occur band at 460bp and 130bp be male embryo all, have only 460bp occur band for female embryo, all occur band for losing sample (seeing accompanying drawing 2).
Claims (1)
1. utilize nest-type PRC (polymerase chain reaction) to carry out the buffalo embryo sex identification, it is characterized in that the following step composition:
(1) reclaim blastaea when after fertilization the 6th, the 7th and the 8th day, use micrurgy instrument or manual cut to obtain a small amount of embryo's sample, the embryo after the sampling carries out freezing preservation and handles, and places liquid nitrogen container to store then;
(2) embryo's sample washs three times in the PBS of no calcium magnesium (phosphoric acid buffer), changes in the PCR pipe, adds cell pyrolysis liquid (Lysis Solution), places 5 minutes in room temperature, directly carries out the PCR reaction as template behind 98 ℃ of inactivated proteases K;
(3) in new EP pipe, add 23 μ L PCR Mix liquid and (comprise the PCR damping fluid, dNTP, Taq enzyme, deionized water) and upstream and downstream outer primer, upstream and downstream confidential reference items primer (each 50pM), the 94 ℃ of 2min that unwind in advance in the PCR instrument carry out following 20 circulations: 95 ℃ of 20s then; 62 ℃ of 30s; 72 ℃ of 30s, last 72 ℃ are extended 5min, stopped reaction;
(4) the PCR product of taking-up 1 μ L adds in the new pipe, add 22 μ L PCR Mix liquid again and (comprise the PCR damping fluid, dNTP, the Taq enzyme, deionized water), upstream and downstream inner primer and upstream and downstream confidential reference items primer GAPDH (each 50pM), the 94 ℃ of 2min that unwind in advance in the PCR instrument carry out following 30 circulations: 95 ℃ of 20s then; 61 ℃ of 30s; 72 ℃ of 30s, last 72 ℃ are extended 5min, stopped reaction;
(5) with amplified production in 2% agarose gel electrophoresis (100V, 30min), place EB (the own ingot of bromination) dyeing to observe down in ultraviolet lamp after 20 minutes gel, all samples that goes out all to occur band at 460bp and 130bp be male embryo all, have only 460bp occur band for female embryo, all occur band for losing sample.
1, embryo gender authenticate technology according to claim 1 is characterized in that the outer primer in the step (3) is:
Upstream outer primer 5 ' AGG ACC ACA TCA AGC GAC CCA T 3 '
Downstream inner primer 5 ' CGG GTA TTT GTC TCT GTG TAT GG 3 '
2, the nest-type PRC that utilizes according to claim 1 carries out the buffalo embryo sex identification, it is characterized in that inner primer and the confidential reference items primer in the step (4) is:
Upstream inner primer 5 ' AGG CAG CTG GGC TAT GAG TGG AA 3 '
Downstream inner primer 5 ' CGG GTA TTT GTC TCT GTG TAT GG 3 '
Upstream confidential reference items primer 5 ' GGT GAA GGT CGG AGT CAA CGG 3 '
Downstream confidential reference items primer 5 ' GGT CAT GAG TCC TTC CAC GAT 3 '
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| CN101914548A (en) * | 2010-08-06 | 2010-12-15 | 中国农业科学院北京畜牧兽医研究所 | Bovine embryo sex development related gene, encoding protein and preparation method thereof |
| CN101215603B (en) * | 2007-12-26 | 2011-02-16 | 扬州大学 | Bubalus products molecule diagnosis kit |
| CN102586415A (en) * | 2011-11-14 | 2012-07-18 | 江苏迈健生物科技发展有限公司 | Method for collecting specimen for reverse transcription PCR (polymerase chain reaction) analysis of genetic expression of IVF (in-vitro fertilization) single preimplantation embryo |
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- 2005-12-27 CN CN 200510048301 patent/CN1990879A/en active Pending
Cited By (11)
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| CN101215603B (en) * | 2007-12-26 | 2011-02-16 | 扬州大学 | Bubalus products molecule diagnosis kit |
| CN101914548A (en) * | 2010-08-06 | 2010-12-15 | 中国农业科学院北京畜牧兽医研究所 | Bovine embryo sex development related gene, encoding protein and preparation method thereof |
| CN101914548B (en) * | 2010-08-06 | 2012-08-08 | 中国农业科学院北京畜牧兽医研究所 | Bovine embryo sex development related gene, encoding protein and preparation method thereof |
| CN102586415A (en) * | 2011-11-14 | 2012-07-18 | 江苏迈健生物科技发展有限公司 | Method for collecting specimen for reverse transcription PCR (polymerase chain reaction) analysis of genetic expression of IVF (in-vitro fertilization) single preimplantation embryo |
| CN105755127A (en) * | 2016-03-23 | 2016-07-13 | 华南农业大学 | Identification method of blastula gene knockout of non-human primate animals |
| CN109136348A (en) * | 2017-08-24 | 2019-01-04 | 江苏苏博生物医学科技南京有限公司 | A kind of the fluorescent marker amplification detection kit and its detection method in the gender source that can identify beef and its product |
| CN109402244A (en) * | 2018-12-20 | 2019-03-01 | 广西大学 | A kind of mammal embryo sex appraisal method |
| CN109402244B (en) * | 2018-12-20 | 2022-05-03 | 广西大学 | Sex identification method for mammalian embryo |
| WO2020258084A1 (en) * | 2019-06-26 | 2020-12-30 | 深圳华大智造科技有限公司 | Method for preparing nested multiplex pcr high-throughput sequencing library and kit |
| CN110720413A (en) * | 2019-11-22 | 2020-01-24 | 中国科学院南海海洋研究所 | Method for identifying female sea cucumber individual in ovarian development stage of holothuria fuscogongensis |
| CN110720413B (en) * | 2019-11-22 | 2021-04-06 | 中国科学院南海海洋研究所 | Method for identifying female sea cucumber individual in ovarian development stage of holothuria fuscogongensis |
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