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Primer, kit and detection method for detecting chicken green shin character linkage SNP locus genotype

A genotype and kit technology, which is applied in the field of detecting the genotype of the linked SNP locus of the chicken green shank trait, can solve the problems of complicated SSCP operation, long time-consuming and high cost, and achieve the effects of short cycle, simple operation and low cost.

Active Publication Date: 2015-01-21
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, a variety of methods have been developed for searching molecular genetic markers, the most common ones are restriction endonuclease fragment length polymorphism analysis (PCR-RFLP), single-strand conformation polymorphism (SSCP) and direct sequencing technology, etc., but the SSCP operation is cumbersome and time-consuming, and the result is easy to cause misjudgment; while the cost of direct sequencing technology is relatively high

Method used

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  • Primer, kit and detection method for detecting chicken green shin character linkage SNP locus genotype
  • Primer, kit and detection method for detecting chicken green shin character linkage SNP locus genotype
  • Primer, kit and detection method for detecting chicken green shin character linkage SNP locus genotype

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Embodiment 1

[0042] In this embodiment, the primers used to detect the genotype of the chicken green shank trait linked SNP locus include primer pair P:

[0043] Upstream primer P-F: 5'-AATCCAACCAACCAACCAA-3',

[0044] Downstream primer P-R: 5'-TTGCTGTTACACCATCATCT-3'.

Embodiment 2

[0046] In this embodiment, the kit used to detect the genotype of the linked SNP site of the chicken green shank trait includes: primer pair P20 μL, 2×Taq PCR Master Mix (containing 22mM Tris-HCl (pH8.4), 55mM KCl, 1.65mM MgCl2 , 220μM dGTP, 220μM dATP, 220μM dTTP, 220μM dCTP, 22U recombinant Taq DNA polymerase / ml) 1mL, 10U / μL endonuclease BamHI 50μL, 1×NEBuffer3.1 (100mM NaCl, 50mM Tris-HCl, 10mM MgCl 2 , 100μg / ml BSA) 200μL, sterilized ultrapure water 500μL and DNA Marker (600bp, 500bp, 400bp, 300bp, 200bp and 100bp) 30μL;

[0047] The primer pair P is:

[0048] Upstream primer P-F: 5'-AATCCAACCAACCAACCAA-3',

[0049] Downstream primer P-R: 5'-TTGCTGTTACACCATCATCT-3'.

Embodiment 3

[0051] The schematic diagram of the technical process for detecting the genotype of the chicken green shank trait linkage SNP locus in this embodiment is as follows figure 1 As shown, the specific method includes the following steps:

[0052] (1) Preparation and storage of samples

[0053] 24 Gushi chickens, 24 silky silky chickens, 24 Hessex A-series chickens, 24 silky-feathered silky chickens and Hessex A-series orthogonal F1 generations and cross-crosses from Henan Agricultural University Breeding Farm F1 generation 24, wing vein blood collection, add 1 / 3 anticoagulant, use proteinase K method to extract blood DNA, DNA samples are stored in 4 ℃ refrigerator for future use;

[0054] (2) Using the whole genome DNA of the chicken to be detected as a template, PCR amplification is performed on P with primers to obtain the amplified fragment;

[0055] The primer pair P is:

[0056] Upstream primer P-F: 5'-AATCCAACCAACCAACCAA-3',

[0057] Downstream primer P-R: 5'-TTGCTGTTACA...

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Abstract

The invention discloses a primer, a kit and a detection method for detecting chicken green shin character linkage SNP locus genotype, and belongs to the technical field of biological detection. Aiming at an SNP locus specific for the chicken green shin character, a primer pair (P) is designed at DNA segment near the SNP locus; and BamHI enzyme digestion is performed on an amplified product after PCR amplification. If the mutation site is G and a BamHI enzyme digestion sequence GGATCC presents, the segment of the PCR product after enzyme digestion is 263bp; and if the mutation site is T and no BamHI enzyme digestion sequence GGATCC presents, the segment of the PCR product after enzyme digestion is 317 bp. Compared with SSCP and a direct sequencing method, the detection method is simple to operate, low in cost and short in cycle, can greatly increase accuracy for determining the SNP locus genotype, is in no need of special instruments, and can be popularized easily. Experiments demonstrate that the method can effectively determine the genotype of the chicken green shin character SNP locus, can be used for marker-assisted selection of the chicken green shin characters, and provide effective molecular marker for establishing green shin chickens.

Description

technical field [0001] The present invention specifically relates to a primer for detecting the genotype of the SNP site linked to the trait of chicken green shank, and a kit containing the primer, and also relates to a method for detecting the genotype of the linked SNP site of the trait of chicken green shank, which belongs to biological detection technology field. Background technique [0002] With the standardized management of urban sanitation and strict environmental control requirements, the sales volume of live poultry in farmers' markets in large and medium-sized cities will gradually decrease, and the share of broiler products marketed in the form of white striped chicken and split chicken will continue to increase. For the chilled fresh chicken market, it is extremely important to distinguish high-quality chickens from fast broiler chickens. Local chickens in our country often have the characteristics of green shanks, so people often judge them by the color of wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 康相涛杨朋坤蒋瑞瑞韩瑞丽李转见任俊晓王顺红田亚东刘小军孙桂荣李国喜闫峰宾王彦彬
Owner HENAN AGRICULTURAL UNIVERSITY
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