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PCR-SSCP primers for detecting mutation of DGAT1 gene and application of PCR-SSCP primers in yak milk quality identification

A PCR-SSCP and yak milk technology, applied in the field of molecular biology, can solve the problems of less research on the genetic mechanism of milk fat traits, and achieve the effects of sensitive response, strong specificity, and simple operation

Active Publication Date: 2017-12-08
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] High milk fat percentage is the main characteristic of yak milk. In recent years, there are many reports on the molecular genetics of yak milk protein, but there are few studies on the genetic mechanism of its milk fat traits.

Method used

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  • PCR-SSCP primers for detecting mutation of DGAT1 gene and application of PCR-SSCP primers in yak milk quality identification
  • PCR-SSCP primers for detecting mutation of DGAT1 gene and application of PCR-SSCP primers in yak milk quality identification

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Experimental program
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Effect test

Embodiment 1

[0023] Design primers for PCR-SSCP detection of yak milk quality trait candidate gene DGAT1, specifically:

[0024] DGAT1-F: 5'-CTCCGCCTTCTTCCACGAG-3';

[0025] DGAT1-R: 5'-GTCCAACACCCACGAGG-3'.

[0026] Primers were synthesized by Beijing Liuhe Huada Gene Company.

Embodiment 2

[0028] Preparation of PCR-SSCP detection kit for candidate genes of yak milk quality traits:

[0029] The PCR-SSCP detection kit of yak milk quality character candidate gene DGAT1 includes PCR reaction solution, DNA standard sample of DGAT1*A; Deionized water, 10% ammonium persulfate, loading denaturation buffer, TEMED (tetramethylethylene di Amine), 12% non-denaturing polyacrylamide gel Acr:Bis=37.5:1;

[0030] Wherein, the loading denaturation buffer includes 98% deionized formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 10mmol / LEDTA, pH8.0;

[0031] Reaction solution for PCR reaction: total volume 20 μL, including 0.8 μL of DNA template, 0.8 μL of upstream and downstream primers, 10 μL of TaKaRaPremix Taq polymerase, and 7.6 μL of sterilized ultrapure water.

[0032] The nucleotide sequence of the DNA standard sample of DGAT1*A is (SEQ ID NO.3 in the sequence listing):

[0033] CTCCGCCTTCTTCCACGAGGTCAGTGCACTGAGGGCGCGCCCTGCCCCTGGTGGGGGTGGGGGTGGGGCTCGCTGACGCCTCTCTC...

Embodiment 3

[0035] The method for detecting the candidate gene of yak milk quality character by using the kit of the present invention:

[0036] (1) Sample collection: 10ml of blood was collected from the jugular vein of the yak, anticoagulated with ACD, and stored at -70°C;

[0037] (2) Genomic DNA extraction: Genomic DNA was extracted from frozen blood samples by the phenol-chloroform method;

[0038] (3) Polymerase chain reaction:

[0039]PCR reaction system: total volume 20 μL, including 0.8 μL DNA template, 0.8 μL upstream and downstream primers, 10 μL TaKaRaPremix Taq polymerase, 7.6 μL sterilized ultrapure water; reaction conditions: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, Anneal at 62°C for 30s, extend at 72°C for 30s, a total of 35 cycles; finally extend at 72°C for 5min;

[0040] The primer sequences are:

[0041] DGAT1-F: 5'-CTCCGCCTTCTTCCACGAG-3';

[0042] DGAT1-R: 5'-GTCCAACACCCACGAGG-3'.

[0043] (4) SSCP detection of PCR products: ...

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Abstract

The invention discloses a PCR-SSCP primer pair for detecting mutation of a DGAT1 gene. The nucleotide sequence of a forward primer of the PCR-SSCP primer pair is shown as SEQ ID NO.1 in a sequence list, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO.2 in the sequence list; and the invention also provides the application of the primer pair in yak milk quality predication and identification as well as identification method and a corresponding kit. The primer pair provided by the invention has the beneficial effects that the pair of primers are designed in accordance with the sequence of an area from the 15th intron to the 17th exon of the DGAT1 gene of bos turus; genome DNA of a yak is subjected to PCR amplification and SSCP typing screening detection is implemented; the genotype and the allele of the yak DGAT1 gene are determined by analyzing a banding pattern; SNPs mutation is determined by conducting allele sequence alignment; and the influence of the mutation of the DGAT1 gene to a butter-fat percentage is analyzed, so that the butter-fat percentage of the yak is predicted; and the primer pair has the advantages of being sensitive in response, strong in specificity, simple to operate and high in accuracy, and rapid detection can be implemented.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a PCR-SSCP primer for detecting DGAT1 gene mutation and its application in methods for predicting and identifying yak milk quality. Background technique [0002] Yaks are distributed in the Qinghai-Tibet Plateau and its surrounding alpine pastoral areas. They provide local herdsmen with plateau-specific livestock products such as meat, milk, and plush. They are important local production and living resources. Yak milk is rich in nutrition and unique in flavor, and is the main raw material for processing plateau specialty milk products. The milk fat rate, milk protein rate and total solid matter content in yak milk are 5.45%-7.22%, 4.86%-5.40%, 16.91%-17.40%, respectively, which are higher than Holstein milk. [0003] Milk fat is an important part of milk. More than 95% of triglycerides in milk fat are synthesized by glycerol-3-phosphate acyltransferase (GPAT), 1-acylgl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 胡江高小莉刘秀石斌刚李少斌
Owner GANSU AGRI UNIV
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