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Method for screening excellent breeding cattle with normal CD18 gene and dedicated primer therefor

A technology for normal and breeding cattle, applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc. Promising effects

Inactive Publication Date: 2007-05-30
北京奶牛中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the molecular detection method of BLAD harmful gene is mainly PCR-RFLP method (polymerase chain reaction-restriction fragment length polymorphism) (Mukhopadhyaya PN, Mehta HH, RathodRN. A method for the simulation of normal, carrier and affected controls for PCR-RFLP screening of a genetic disease in dairy cattle. Mol Cell Probes. 2000, 14(6): 381-384; Norouzy A, Nassiry MR, Eftekhari Shahrody F. Identification of bovine leucocyte adhesion deficiency (BLAD) carriers in Holstein and BrownSwiss AI bulls in Iran .Genetika.2005.41(12):1697-1701.), however, the PCR-RFLP method requires the establishment of a positive control to ensure the accuracy of the test results, and the cost is high, so it is not suitable for large-scale detection

Method used

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  • Method for screening excellent breeding cattle with normal CD18 gene and dedicated primer therefor
  • Method for screening excellent breeding cattle with normal CD18 gene and dedicated primer therefor

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Embodiment 1

[0027] Example 1. Primer design and optimization of PGR reaction conditions for screening good breed cattle with normal CD18 gene

[0028] 1. Primer design

[0029] According to the nucleotide sequence of the bovine CD18 gene (GenBank number: Y12672), use Oligo6.0 software to design specific PCR primers for PCR-SSCP screening of fine breed cattle with normal CD18 gene, and the length of the PCR product is about 150-300bp for comparison Suitable for PCR-SSCP detection. Through the design, screening and optimization of a series of PCR primers, finally according to the nucleotide sequence shown in sequence 3 in the sequence listing, the sequence contains the mutation site that causes BLAD (the 32nd base, namely A55G:CD18 From the 55th base at the 5' end of the gene Exon2, which is base A for normal cattle and base G for BLAD cattle), a primer BLF (upstream primer) is designed: 5'-ACCTTCCGGAGGGCCAAGGG-3' (sequence 1 in the sequence listing) And primer BLR (downstream primer): 5'...

Embodiment 2

[0032] Example 2, PCR-SSCP Screening of Excellent Breed Cattle with Normal CD18 Gene and Its Sequencing Identification

[0033] 1. PCR-SSCP screening

[0034] Extract the genomic DNA of the fresh blood of 54 bulls and use it as a template, and carry out PCR amplification under the guidance of the specific primer pair BLF / BLR obtained in Example 1. The PCR reaction system is the same as in Example 1, and the reaction conditions are: Pre-denaturation at 94°C for 5min; denaturation at 94°C for 30s, annealing at 64°C for 30s, extension at 72°C for 30s, and 35 cycles; extension at 72°C for 7min. After the reaction, 1.5 μl of the PCR amplification products of the 54 bulls were placed in PCR tubes, and 6 μl of loading buffer (98% formamide, 10 mM EDTA pH8.0, 10% glycerol, 0.025% bromophenol blue and 0.025 xylene cyanine), after denaturing at 98°C for 10 minutes, immediately place it on ice for 5 minutes, apply samples, and perform 14% non-denaturing polyacrylamide gel electrophoresi...

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Abstract

The invention discloses a screening method and specific primer of superior cow with CD18 gene, which comprises the following steps: adopting detected cow genome DNA as mold; proceeding PCR augumentation for primer couple with nucleotide sequence in the list 1 and list 2; detecting the base at 32th from 5' end as A in the augumentation segment; regarding the cow as normal cow; testing N as heterozygote of A and G.

Description

technical field [0001] The invention relates to a method for screening fine breed cattle and special primers thereof, in particular to a method for screening fine breed cattle with normal CD18 gene by using single nucleotide polymorphism of bovine CD18 gene and special primers thereof. Background technique [0002] Bovine leukocyte adhesion deficiency (BLAD) is an autosomal recessive genetic disease controlled by a single gene, which is caused by a point mutation in the CD18 gene encoding neutrophil surface glycoprotein ( Shuster, D.E., Bosworth, B.T., Kehrli, M.E.Jr. Sequence of the bovine CD18-encoding cDNA: comparison with the human and murineglycoproteins. Gene. 1992, 114(2):267-271.). The CD18 gene has been located on chromosome 1. Shuster et al. conducted sequence analysis on the CD18 gene of a Holstein cattle with BLAD, and found that the 383rd base from the 5' end was mutated from A to G. This mutation is a missense mutation, resulting in asparagus Amino acid is mu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12P19/34
Inventor 李艳华张胜利韩广文薛建华朱玉林刘振君
Owner 北京奶牛中心
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