Primer group, kit and method for detecting cattle horn-free gene

A technology of primer sets and kits, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as inaccurate typing, time-consuming, and inability to achieve rapid detection, etc., and achieve improvement The effect of the hornless gene frequency

Active Publication Date: 2021-12-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Summarizing the existing detection methods, there are the following problems: 1. There is no unified detection technology that can detect the three hornless alleles at the same time; 2. The existing detection method based on Sanger sequencing technology has many steps and takes a long time. 3. For the Friesian hornless gene, the agarose gel electrophoresis detection of the reported PCR amplification products can only determine whether the hornless gene is carried, and cannot distinguish heterozygotes and homozygotes of the hornless allele. The effect of precise classification is not achieved

Method used

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  • Primer group, kit and method for detecting cattle horn-free gene
  • Primer group, kit and method for detecting cattle horn-free gene
  • Primer group, kit and method for detecting cattle horn-free gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 The design and optimization process of the primer group

[0091] According to the three kinds of unopened gene sequences, the layout primer groups I, II, III are designed. Among them, the primer group I is based on P 202ID The specific sequence of the 2 alleles of the site is optimized, using the differentiality of the repeating sequence; the primer group II is based on P 219ID The specific sequence of the 2 alleles of the site is optimized, and 6 bases are replaced by 7 bases at upstream 628 bp (resulting in 1 bp length difference); primer III is based on P 80kbID The specific sequence of the 2 alleles of the site is optimized, and 2bp is deleted in the repeating sequence.

[0092] The optimized design process of primer III is specifically described for example. Specifically, the inventors have the Sanger sequencing results of the Friesian unopened gene P80kbid site, and the original 80KB sequence represents the original 80KB sequence, square bracket "[]" represe...

Embodiment 2

[0099] Example 2 Terminal detection of three kinds of cattle angle genes

[0100] Collect 39 bulls freeze semen of different varieties, and extract genomic DNA by high salt method. Among them, the samples include 9 cows, varieties are Holinstein cattle and Juan Hao cow; bull cows 17, varieties include Angers Niu, Flewiki Niu, Charlottle Niu, Limuzi and Simmental Bull; There are 13 three rivers, Qinchuan cattle and Mongolia.

[0101] SNP is performed based on the principle of KASP competitive allele specific PCR, wherein the PCR reaction is completed on the microfluidic chip (Beijing Bo Otry Biotechnology Co., Ltd., the product number G020010), the method steps are:

[0102] 1) On a dedicated microfluidic chip, genomic DNA (5 ~ 10 ng) to be detected in the chip well is injected;

[0103] 2) For 4 sites, the PCR amplification reaction mixture is separately configured. In the reaction mixture of each site, a site-specific primer (Table 1), PCR premix, and double steamed. The configur...

Embodiment 3

[0115] Example 3 General detection verification KASP method accuracy

[0116] In order to verify the accuracy of KASP detection results, all 39-head sample PCR amplification, and detect the angular genotype by agarose gel electrophoresis or Sanger sequencing.

[0117] (1) PCR primer

[0118] In response to different unopened gene sites, the primers designed in the literature and Primer3.0 (http: / / bioinfo.ut.ee / primer3 / ) are used to detect, and the primer details are shown in Table 3.

[0119] Table 3 primer sequence and amplification fragment length

[0120]

[0121] (1) PCR amplification

[0122] PCR reaction system: Total volume 25 μL, of which 10 × buffer 2.5 μL, DNTP mixture (2.5 mM) 2 μL, TAQ enzyme (5 U / μL) 0.5 μL, 5 μm of the upper and lower proders (20 μm), 1 μL of genomic DNA template (about 50 ng), with double steamed water to make up 25 μL.

[0123] PCR reaction conditions: 5 min at 94 ° C; then 35 cycles, 94 ° C denaturation 30SEC, and the annealing temperature of ...

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Abstract

The invention relates to the technical field of gene mutation detection, and in particular relates to a primer group, kit and method for detecting a cattle horn-free gene. The primer group comprises primers as shown in SEQ ID NO. 1 to 9, and more preferably, the primer group also comprises primers as shown in SEQ ID NO. 10 to 12. According to the invention, a cattle Celic horn-free gene, a Mongolian horn-free gene and a Friesian horn-free gene can be detected at the same time, and the phenotype and / or genotype of the cattle horn-free character can be rapidly and accurately detected in batches. When the method is applied to the molecular breeding of the cattle horn-free character, the horn-free gene homozygote can be selected so as to efficiently improve the horn-free gene frequency of offspring cattle herds.

Description

Technical field [0001] The present invention relates to a gene mutation detection technologies, and particularly relates to a primer set, kit and method for detecting bovine polled gene. Background technique [0002] In nature, the horn is against predators, competition for spouses tool, and in the intensive large-scale farming conditions, there is an angle between the animal will not only hurt each other, but also pose a security threat to breeders. Currently large-scale cattle calves usually within a week of birth de corner, but the disadvantage is that the cattle brought to the stress response, affect the growth of animals and animal welfare, while the cost of manpower, material and financial resources. Natural bovine polled trait is advantageous in the production, development hornless bovine genetic detection methods, can be used in molecular breeding cattle polled trait. For example, when selecting cattle, identify and select homozygote polled, can quickly enhance gene frequ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/124C12Q2600/156C12Q2600/16C12Q2563/107C12Q2531/113Y02A50/30
Inventor 张毅颜泽王雅春张胜利肖炜
Owner CHINA AGRI UNIV
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