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Kit for detecting 15bp insertion mutation in ninth exon of bovine coagulation factor XI gene

A technology of insertion mutation and coagulation factor, applied in the fields of genetics and molecular biology, to achieve high sensitivity, good specificity, and improved accuracy

Inactive Publication Date: 2012-11-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects that false negative results are prone to occur in the existing detection methods, and provide a kit that can accurately and efficiently detect 15 nucleotide insertion mutations in exon 9 of the bovine blood coagulation factor XI gene

Method used

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  • Kit for detecting 15bp insertion mutation in ninth exon of bovine coagulation factor XI gene
  • Kit for detecting 15bp insertion mutation in ninth exon of bovine coagulation factor XI gene
  • Kit for detecting 15bp insertion mutation in ninth exon of bovine coagulation factor XI gene

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Synthesis of PCR primer combination for detecting 15bp insertion mutation in exon 9 of bovine coagulation factor XI gene

[0032] According to the position of the 15bp insert in exon 9 of the bovine coagulation factor XI gene ( figure 1 ), artificially synthesized the following primers:

[0033] Primer pair 1:

[0034] a) Upstream primer wild-F5’-CTGCTGTGCAGTGTTCTGCC-3’;

[0035] b) The downstream primer wild-R5'-GGAGCGTCTATGGGACTTGA-3'; and

[0036] Primer pair 2:

[0037] a') Upstream primer mutant-F5'-TGCAAGCCCTTTCTTCTGAT-3';

[0038] b') Downstream primer mutant-R5'-AATGGCATATATTCTGCACA-3'.

[0039] The above primer combination corresponds to the position in exon 9 of the bovine coagulation factor XI gene as figure 2 Shown. The upstream primer (wild-F) of primer pair 1 spans the insert region, but does not contain the insert, so it can only specifically bind to the wild-type allele sequence to specifically amplify the wild-type allele; while the mutant type There is...

Embodiment 2

[0041] Example 2 Kit for detecting 15bp insertion mutation in exon 9 of bovine coagulation factor XI gene

[0042] The kit for detecting the 15bp insertion mutation in the 9th exon of the bovine coagulation factor XI gene includes: the primer combination in Example 1, namely primer pair 1 and primer pair 2. In addition, it can also include dNTPs, Taq DNA polymerase, Mg 2+ , One or more of the PCR reaction buffer. In order to facilitate the analysis of the test results, a positive control of known genotype can be further included.

Embodiment 3

[0043] Example 3 Using PCR primer combination to detect the specificity analysis of genetic defect of 15bp insertion mutation in exon 9 of bovine coagulation factor XI gene

[0044] 1.1 Sample source

[0045] The source of cattle used in this embodiment: six ordinary cattle (Bos taurus) from Xuelong Pasture in Liaoning were randomly selected and numbered 1 to 6 respectively.

[0046] 1.2 DNA extraction

[0047] 1.2.1 DNA extraction from cattle blood

[0048] The DP318 kit from Tiangen Biochemical Biotechnology Company was used to extract genomic DNA from blood clots. The specific steps are as follows:

[0049] 1) Take out the sample, after thawing, cut 200μL into a 2mL centrifuge tube;

[0050] 2) Add 600μL of cell lysate CL and shake well;

[0051] 3) Centrifuge at 10000rpm for 1min, discard the dark red supernatant;

[0052] 4) Repeat steps 2 and 3 once;

[0053] 5) Add 200μL of buffer GS and fully suspend the blood clot particles with a vortexer;

[0054] 6) Add 20μL proteinase K, 220μL bu...

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Abstract

The invention provides a kit for detecting 15bp insertion mutation in a ninth exon of a bovine coagulation factor XI gene. The kit comprises a primer combination shown as SEQ ID NO. 1-4. Based on the position of a 15bp insertion fragment in the ninth exon of the bovine coagulation factor XI gene, two pairs of primers across a 15bp insertion fragment area are designed, based on which a PCR (Polymerase Chain Reaction) detection system is established. The first pair of primers (Seq ID No. 1 and 2) only specifically amplify a wild type allele, and the second pair of primers (Seq ID No.3 and 4) only specifically amplify a mutant allele; the length difference between two PCR products is 94bp, different genotypes can be sensitively distinguished by shorter-timer electrophoresis by using low-concentration agarose gel, and thus carriers of genetic defects are accurately screened out.

Description

Technical field [0001] The present invention relates to the fields of genetics and molecular biology, in particular to a kit for detecting 15bp insertion mutation in the 9th exon of bovine coagulation factor XI gene. Background technique [0002] Coagulation is the process by which blood changes from a liquid state to a non-flowing gel state. It is a natural self-protection mechanism of the organism and can prevent blood from being lost from broken blood vessels. Except for platelets, all substances involved in the coagulation process are collectively called coagulation factors. There are currently more than 20 known coagulation factors, including traditional factors I-XIII, as well as PK, HMWK, etc. When vascular endothelial cells are traumatized and proteins such as tissue factor come into contact with the blood, coagulation will start immediately. First, platelets flow to the wound site and accumulate to form an embolism, which is called primary hemostatic reaction; at the sa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张毅王雅春李强张沅吴蒙徐仙洲俞英孙东晓张胜利
Owner CHINA AGRI UNIV
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