CN112899375A - PCR-RFLP method and primer for identifying homozygote heterozygote of Hulenbel short-tailed sheep - Google Patents
PCR-RFLP method and primer for identifying homozygote heterozygote of Hulenbel short-tailed sheep Download PDFInfo
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Abstract
The invention provides a PCR-RFLP method and a primer for identifying pure heterozygous individuals of a Renbell Brachyury, which utilize a specific recognition site of Bci T130I restriction endonuclease to perform genotyping, and amplify the Brachyury/T gene of the Renbell Brachyury by taking sample genome DNA as a template, wherein the length of the amplified gene is 203 bp. The upstream primer is positioned from the 95879504 site to the 95879486 site of the Brachyury/T gene, and the downstream primer is positioned from the 95879302 site to the 95879323 site; purifying the PCR product, and then performing enzyme digestion by using Bci T130I; the enzyme digestion product is observed by 1.5 percent agarose gel electrophoresis, and two obvious bands are homozygous Hurenbel short-tailed sheep, and three obvious bands are heterozygous Hurenbel short-tailed sheep. The invention has simple operation, no need of extra analysis steps in reaction, and can accurately classify pure and heterozygous genotypes according to the number of bands, and the enzyme digestion system and the time can not cause the star activity of the Bci T130I enzyme.
Description
Technical Field
The invention belongs to the field of sheep molecular breeding identification, and particularly relates to a PCR-RFLP identification method and primers for identifying homozygote heterozygotes of a Hulenbel short-tailed sheep.
Background
We screened T/Brachyury genes related to spinal development as candidate genes by using a genome re-sequencing technology, confirmed that stable T/Brachyury gene c.G334T mutation exists in a Renbell Brachyury population, and proved to be related to the character of short tail with economic value. In sheep molecular breeding: the homozygous individuals are stable in performance relative to the characters of heterozygous offspring, and the homozygous individuals in the population are screened and used as main breeding objects. In the conventional method, a sanger sequencing method and Taqman probe genotyping are mainly used as bases, and a primer combined on a template of a sequence to be determined is extended by using a DNA polymerase until a chain-terminating nucleotide is doped, wherein the base is associated with an SNP site with a commercial value-related trait. Each sequencing consists of a set of four separate reactions, each containing all four deoxynucleoside triphosphates (dNTPs) mixed with a limited amount of a different dideoxynucleoside triphosphate (ddNTP). The extended oligonucleotide selectively terminates at G, A, T or C due to the absence of the 3-OH group required for extension of the ddNTP. The termination point is determined by the corresponding dideoxy reaction. The relative concentrations of each of dNTPs and ddNTPs can be adjusted so that a set of chain-terminating products of several hundred to several kilobases is obtained. They have a common starting point but terminate in different nucleotides, and fragments of different sizes can be separated by high resolution denaturing gel electrophoresis, which can be followed by detection using X-ray film autoradiography or non-isotopic labeling.
The conventional methods have disadvantages of long useful time, high cost, low throughput, and abnormal termination of DNA polymerase sometimes caused by the composition or secondary structure of DNA template.
Disclosure of Invention
The invention provides a PCR-RFLP method and primer for identifying homozygote of the Renbell Brachyury, which overcomes the defects of more complicated operation, long time consumption and the like of the traditional sanger sequencing method, has extremely high sensitivity compared with the prior art and based on the strip quantity analysis of the restriction enzyme result, can detect the individual difference of the homozygote and heterozygote of the T/Brachyury gene c.G334T mutation of the Renbell Brachyury, and has the advantages of low cost, high flux, high speed, accurate result and no limitation of experimental environment.
The purpose of the invention is realized by the following technical scheme:
a primer used for identifying pure heterozygous individuals of a Renbell short-tailed sheep by a PCR-RFLP method is characterized in that: the primer sequences are as follows:
BrachyuryFX-F:5’-TGCGCCCCTTCCTTTTCAG-3’
BrachyuryFX-R:5’-GGGGGAGTCGGGGTGGATGTAG-3’。
a PCR-RFLP method for identifying pure heterozygous individuals of a Hulunebel short-tailed sheep is characterized in that: the operation steps are as follows:
step 1) using sample genome DNA as a template to amplify the Brachyury/T gene interval sequence to obtain an amplified gene segment, wherein the primer is the sequence primer of claim 1;
step 2) using an agarose gel recovery kit or a PCR product recovery kit to recover and purify the amplification product obtained in the step 1);
step 3) carrying out enzyme digestion on the purified product obtained in the step 2);
and 4) carrying out agarose gel electrophoresis on the enzyme digestion product obtained in the step 3) and observing the result.
The length of the amplified gene fragment in the step 1) is 203bp, and the PCR reaction conditions are as follows: 3min at 95 ℃; 15s at 95 ℃, 15s at 65 ℃ and 12s at 72 ℃ for 30 cycles, and 5min at 72 ℃.
The step 3) uses Bci T130I to perform enzyme digestion, wherein the enzyme digestion system is as follows: filling 1 mu g of the purified product obtained in the step 2), Bci T130I 1 mu l, 10 XK Buffer 2 mu l and sterile water to 20 mu l, wherein the enzyme cutting temperature is 37 ℃, the reaction time is 1-1.5 hours, and after the enzyme cutting is finished, quickly placing the enzyme cutting product on ice for cooling.
The reaction time of the enzyme digestion is 1.5 hours.
The concentration of the agar in the step 4) is 1 to 1.5 percent.
The primer BrachyuryFX-F, BrachyuryFX-R was purified for Page at a working concentration of 10 μ M.
The working concentration of the primer is 10 ng/. mu.l.
The concentration of the sample genome DNA is 80-120 ng/. mu.l.
The concentration of the genomic DNA of the sample should be 100 ng/. mu.l.
Drawings
FIG. 1 is a diagram showing the result of agarose gel electrophoresis after extraction of genomic DNA of a Hulenbel brachyury and PCR amplification using the extracted DNA as a template.
FIG. 2 is an electrophoretogram of the product after enzyme digestion, wherein the result of the comparison and sequencing shows that homozygote is two bands and heterozygote is three bands.
Fig. 3 is a recognition site map of BciT 130I.
Detailed Description
The concentration of the upstream and downstream primers of the specific primer used in the present invention was 10. mu.M, and the Bci T130I (EcoRII, Mva I) enzyme used was a restriction enzyme available from TaKaRa. The agarose gel recovery kit and the DNA polymerase for PCR according to the present invention are suitable for products sold in the market. Firstly, whole genome DNA extraction is carried out on blood of a sheep to be identified, quality identification is carried out on the extracted DNA, the identification means can adopt a trace ultraviolet spectrophotometer to read the concentration and OD value 260/280, the OD value 260/280 is required to be between 1.5 and 2.0, the concentration is about 100 ng/mul, and the extracted DNA can also be subjected to agarose gel strip analysis, and the strip is required to be single, bright and has no tailing. PCR was performed using the extracted genomic DNA as a template, and the primer sequences were:
BrachyuryFX-F:5’-TGCGCCCCTTCCTTTTCAG-3’
BrachyuryFX-R:5’-GGGGGAGTCGGGGTGGATGTAG-3’
the reaction conditions are as follows: 3min at 95 ℃; 15s at 95 ℃, 15s at 65 ℃ and 12s at 72 ℃ for 30 cycles, and 5min at 72 ℃. After the reaction is finished, taking the purified PCR product for enzyme digestion: mu.g of the purified product, 1. mu.l of Bci T130I (EcoRII, Mva I), and 2. mu.l of 10 XK Buffer were made up to 20. mu.l in sterile water. The digestion temperature was 37 ℃ and the reaction time was 1 hour and 30 minutes. After the completion of the digestion, the digested product was rapidly cooled on ice. And (3) carrying out 1.5% agarose gel electrophoresis on the enzyme-cut product, wherein if DNA polymerase used for PCR is a premixed Loding buffer type, the loading amount can be selected from 5 to 10 mu l, and if the DNA polymerase used for PCR is a non-premixed Loding buffer type, the loading amount is adjusted to 1 to 5 mu l. And after the electrophoresis is finished, carrying out ultraviolet observation and judging the number of bands obtained after enzyme digestion of the sample, wherein two bands are obviously homozygous Renbell short-tailed sheep, and three bands are obviously heterozygous Renbell short-tailed sheep.
Claims (10)
1. A primer used for identifying pure heterozygous individuals of a Renbell short-tailed sheep by a PCR-RFLP method is characterized in that: the primer sequences are as follows:
BrachyuryFX-F:5’-TGCGCCCCTTCCTTTTCAG-3’
BrachyuryFX-R:5’-GGGGGAGTCGGGGTGGATGTAG-3’。
2. a PCR-RFLP method for identifying pure heterozygous individuals of a Hulunebel short-tailed sheep is characterized in that:
the operation steps are as follows:
step 1) using sample genome DNA as a template to amplify the Brachyury/T gene interval sequence to obtain an amplified gene segment, wherein the primer is the sequence primer of claim 1;
step 2) using an agarose gel recovery kit or a PCR product recovery kit to recover and purify the amplification product obtained in the step 1);
step 3) carrying out enzyme digestion on the purified product obtained in the step 2);
and 4) carrying out agarose gel electrophoresis on the enzyme digestion product obtained in the step 3) and observing the result.
3. The PCR-RFLP method for identifying pure heterozygous individuals of the Hulenbel short-tailed sheep according to claim 2, characterized in that: the length of the amplified gene fragment in the step 1) is 203bp, and the PCR reaction conditions are as follows: 3min at 95 ℃; 15s at 95 ℃, 15s at 65 ℃ and 12s at 72 ℃ for 30 cycles, and 5min at 72 ℃.
4. The PCR-RFLP method for identifying pure heterozygous individuals of the Hulenbel short-tailed sheep according to claim 3, characterized in that: the step 3) uses BciT 130I to perform enzyme cutting, and the enzyme cutting system is as follows: filling 1 mu g of the purified product obtained in the step 2), Bci T130I 1 mu l, 10 XK Buffer 2 mu l and sterile water to 20 mu l, wherein the enzyme cutting temperature is 37 ℃, the reaction time is 1-1.5 hours, and after the enzyme cutting is finished, quickly placing the enzyme cutting product on ice for cooling.
5. The PCR-RFLP method for identifying pure heterozygous individuals of the Hulenbel short-tailed sheep according to claim 4, characterized in that: the reaction time of the enzyme digestion is 1.5 hours.
6. The PCR-RFLP method for identifying pure heterozygous individuals of the Hurenberg brachyury sheep according to any one of claims 2 to 5, characterized in that: the concentration of the agar in the step 4) is 1 to 1.5 percent.
7. The PCR-RFLP method for identifying pure heterozygous individuals of the Hulenbel short-tailed sheep according to claim 6, characterized in that: the primer BrachyuryFX-F, BrachyuryFX-R was purified for Page at a working concentration of 10 μ M.
8. The PCR-RFLP method for identifying pure heterozygous individuals of the Hulenbel short-tailed sheep according to claim 7, characterized in that: the working concentration of the primer is 10 ng/. mu.l.
9. The PCR-RFLP method for identifying pure heterozygous individuals of the Hulenbel short-tailed sheep according to claim 6, characterized in that: the concentration of the sample genome DNA is 80-120 ng/. mu.l.
10. The PCR-RFLP method for identifying pure heterozygous individuals of the Hurenberg brachyury sheep according to claim 9, characterized in that: the concentration of the genomic DNA of the sample should be 100 ng/. mu.l.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for detecting goat prolactin gene single nucleotide polymorphism |
| US20100092946A1 (en) * | 2007-02-12 | 2010-04-15 | Pina Fratamico | Genetic methods for speciating Campylobacter |
| CN112251519A (en) * | 2020-12-01 | 2021-01-22 | 内蒙古农业大学 | Specific primer, probe, kit and method for identifying homozygote and heterozygote of Hulenbel short-tailed sheep |
| CN112553349A (en) * | 2021-01-15 | 2021-03-26 | 内蒙古农业大学 | Identification primer, probe, kit and method for homozygote and heterozygote of Hulunbel short-tailed sheep |
-
2021
- 2021-04-01 CN CN202110356310.7A patent/CN112899375A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100092946A1 (en) * | 2007-02-12 | 2010-04-15 | Pina Fratamico | Genetic methods for speciating Campylobacter |
| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for detecting goat prolactin gene single nucleotide polymorphism |
| CN112251519A (en) * | 2020-12-01 | 2021-01-22 | 内蒙古农业大学 | Specific primer, probe, kit and method for identifying homozygote and heterozygote of Hulenbel short-tailed sheep |
| CN112553349A (en) * | 2021-01-15 | 2021-03-26 | 内蒙古农业大学 | Identification primer, probe, kit and method for homozygote and heterozygote of Hulunbel short-tailed sheep |
Non-Patent Citations (5)
| Title |
|---|
| DAFU ZHI等: "Whole Genome Sequencing of Hulunbuir Short-Tailed Sheep for Identifying Candidate Genes Related to the Short-Tail Phenotype", 《SHEEP GENOME SEQUENCING》 * |
| GUANG YANG等: "Development and Application of a High-Resolution Melting Analysis with Unlabeled Probes for the Screening of Short-Tailed SheepTBXTHeterozygotes", 《ANIMALS》 * |
| 曹墨菊主编: "《植物生物技术概论》", 31 December 2014 * |
| 杨光: "绵羊短尾性状T/Brchyury基因的克隆及在早期胚胎中表达的研究", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
| 马晓琴等: "麦洼牦牛乳酸脱氢酶-B基因多态性的 PCR-RFLP 分析", 《四川动物》 * |
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