Polymerase chain reaction (PCR) detection method for arachnomelia syndrome pathogenic locus of brown cattle
A syndrome and spider technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as introduction of pathogenic genes, achieve high-throughput detection, stable detection results, and cost-effective low effect
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Embodiment 1
[0032] The preparation of embodiment 1 test material and the extraction of DNA
[0033]The test materials used in this research come from two parts, one part is 55 brown cattle bulls that are currently being promoted and used in Xinjiang, my country, and the frozen semen of 50 bulls comes from the Tianshan Bull Breeding Bull Station, and the other 5 from the Xinjiang Livestock and Poultry Breeding and Improvement General Station , a total of 55 thin tubes with cold semen. The pedigree analysis showed that the 55 brown bulls contained European and American brown cattle to varying degrees (Table 1).
[0034] Table 155 Brown Cattle Bulls Containing External Blood of American and European Swiss Brown Cattle
[0035]
[0036] The other part comes from 10 cattle breeds (lines) bull control frozen semen samples (Table 2). Among them, one ROMEL of the four German Simmental cattle was a carrier of the known AS pathogenic gene of Simmental cattle spider leg syndrome (MOCS1 gene c.de...
Embodiment 2
[0041] Example 2 Screening of brown cattle AS pathogenic mutation PCR amplification primers
[0042] Primer3 and Oligo 6 were used to design primers for the sequence near c.363-364insG of the SUOX mutation (Genbank accession number: 509837) of the causative gene SUOX that causes brown cow spider leg syndrome. Two pairs of primers SUOX1 and SUOX2 were designed. Synthesized by Shanghai Handsome Company. The primer sequences and lengths of the amplified fragments are listed in Table 3:
[0043] Table 3 Primer Sequence and Amplified Fragment Length
[0044]
[0045] PCR using 20μL PCR system: DNA template 1μL, 10×PCR Buffer 2μL, 25mmol.L -1 MgCl 2 1.2μL, 2.5mmol.L -1 dNTP 2μL, forward and reverse primers diluted to 10mmol.L -1 Add 1.5 μL each, Taq DNA polymerase (5U.μL -1 ) 0.25 μL, add sterilized deionized water to 20 μL. Gradient PCR showed that both pairs of primers could obtain the expected fragments, and the annealing temperature was selected as 63°C. The PCR rea...
Embodiment 3
[0047] The direct sequencing of embodiment 3PCR product
[0048] Common PCR primer SUOX 2 in Table 3 was used for amplification, and the reaction system and conditions were the same as in Example 2. The PCR products were sequenced after recovery and purification, which was completed by Huada Gene Company. The sequencing peaks were viewed using Chromas software, and the sequences were multiple aligned using the BioEdit software ClustalW Multiple alignment.
[0049] The PCR product of the target fragment is recovered and purified and then sequenced. For the sequencing results, see image 3 . In this study, the sequencing results of all 55 brown cattle were all the same. The peak type of each base in the sequencing profile was single and clear, showing homozygosity, and no regular set of peaks (frameshift mutation) was found near the mutation site. That is, there is no insertion of ornithine (G) between ornithine (G) at position 127 and cytosine (C) at position 128 in the PCR ...
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