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Primer, probe, kit and method for identifying homozygote and heterozygote of Herenbel short-tailed sheep

A short-tailed sheep, homozygous technology, applied in the field of molecular biology, can solve the problems of abnormal termination of DNA polymerase, low throughput, long time, etc., and achieve the effects of increased throughput, simple operation, and low cost

Inactive Publication Date: 2021-03-26
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional sequencing technology has the disadvantages of long time, high cost, low throughput, and the composition or secondary structure of DNA template sometimes causes abnormal termination of DNA polymerase

Method used

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  • Primer, probe, kit and method for identifying homozygote and heterozygote of Herenbel short-tailed sheep
  • Primer, probe, kit and method for identifying homozygote and heterozygote of Herenbel short-tailed sheep

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Design of Primers and Probes for Homozygous and Heterozygous Identification of Hulunbeier Short-tailed Sheep

[0032] The sequences of primers and probes for identification of homozygous and heterozygous in Hulunbeier short-tailed sheep are as follows:

[0033] Forward primer: 5'-TGCGCCCCTTCCTTTTCAG-3'

[0034] Reverse primer: 5'- GGGGGAGTCGGGGTGGATGTAG-3'

[0035] taqman probe 1: 5'HEX-TGGCTTGCCCCCCGGCA-BHQ1 3

[0036] taqman probe 2: 5'FAM-GCTTGCCCCAGGGCACCCA-BHQ1 3,

[0037] The taqman probes 1 and 2 are fluorescently labeled probes.

[0038] The design ideas of identification primers and probes are as follows:

[0039] Input the sequence interval (500 bp before and after the SNP site) where the SNP site is located into the Primer 5 software, and find that the GC value is about 55%, the TM value is about 60 ℃, and the primer-dimer and primer self-complementary sequences are not consistent. Primers greater than 4 were used as forward primers and reverse primers. ...

Embodiment 2

[0043] A kit for identifying homozygotes and heterozygotes of Hulunbeier short-tailed sheep, comprising the identification of forward primers in Example 1, reverse primers, fluorescently labeled taqman probes 1 and 2, PCR reaction mixture, Hulunbeier short-tailed sheep genomic DNA and Standard plasmids prepared by homozygous and heterozygous T / Brachyury genes of Hulunbeier short-tailed sheep, in which the PCR reaction mixture includes SNP Probe Master Mix (2X) (Novazyme) and RNase-Free ddH 2 O, the dosage is 10 ul and 4.6 ul respectively; the concentration of the standard plasmid is 50 ng / ul, and the dosage is 1 ul; the concentration of Hulunbuir short-tailed sheep genomic DNA is 10 ng / ul, and the dosage is 1 ul.

Embodiment 3

[0045] A method for identifying homozygotes and heterozygotes of Hulunbeier short-tailed sheep based on primers and taqman SNP fluorescent labeling probes in Example 1, comprising the steps of:

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Abstract

The invention discloses a primer, probe and kit for identifying homozygote and heterozygote of a Herenbel short-tailed sheep, and further provides a method for identifying the homozygote and the heterozygote of the Herenbel short-tailed sheep based on the primer and the probe. The method comprises the following steps that (1) blood genome DNA of the Herenbel short-tailed sheep is extracted and diluted; (2) taqman SNP fluorescent probe qPCR is performed on the DNA obtained in the step (1); and (3) product detection is performed. The homozygote and the heterozygote of the Herenbel short-tailed sheep can be quickly distinguished through the taqman SNP fluorescent probe qPCR. As one of the means for genotyping the Herenbel short-tailed sheep, the identification method is simple and convenientto operate, good in repeatability and short in reaction time, and has extremely high sensitivity and specificity.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer, a probe, a kit and a method for identifying homozygotes and heterozygotes of Hulunbeier short-tailed sheep based on a taqman SNP fluorescent probe. Background technique [0002] Hulunbeier short-tailed sheep is one of the meat sheep breeds grown in Hulunbuir area of ​​my country. The short and thick tail is the unique symbol of this breed. We used genome resequencing technology to screen the T / Brachyury gene related to vertebral development as a candidate gene, and determined that there was a stable T / Brachyury gene c. Trait correlation of economic value. In sheep molecular breeding, homozygous individuals have stable traits compared with heterozygous offspring. Therefore, in recent years, people tend to screen homozygous individuals as the main breeding objects. [0003] Traditional sequencing is mainly based on the Sanger sequencing method, whi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/156C12Q2600/124C12Q2531/113C12Q2561/101C12Q2563/107
Inventor 曹贵方杨光宋永利窦傲蕾马腾智达夫苏红刘默凝杨宏新霍雨佳李喜和何亭漪杨燕燕达来李秀男
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY