Inbreeding heredity quality monitoring SNP (single nucleotide polymorphism) rapid detection method and SNP locus and primer of locus
A technology for quality monitoring and detection methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The effect of shortening the experimental period, increasing the throughput, and reducing the cost
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Embodiment 1
[0038] Example 1: SNP panel design
[0039] 1. Determine the inbred lines included in the SNP panel
[0040] 13 inbred mouse strains were selected for routine SNP detection (as shown in Table 1). The 13 inbred mouse strains were all provided by Nanjing University-Nanjing Biomedical Research Institute (NBRI) (public sale). During the screening process It was found that C57BL / 6J and C57BL / 6N, C57BL / 10J, C57BLKS / J, B6(Cg)-Tyr c-2J Genetic backgrounds are similar; meanwhile, DBA / 1 and DBA / 2 genetic backgrounds are similar. Therefore, C57BL / 6J was selected as a representative of C57BL, DBA / 1 was selected as a representative of DBA, and finally 8 inbred lines were determined to establish a SNP detection panel for C57BL / 6 inbred lines (Table 2). In follow-up experiments, the C57BL / 6 strain SNPpanel can be used for routine SNP detection to distinguish from A / J, 129S1 / SvIm, BALB / C, CBA, DBA, FVB, NOD strains; it can also be used for C57BL / 6N, C57BL / 10, C57BLKS, B6(Cg)-Tyr c-2JRout...
Embodiment 2
[0065] Embodiment 2: SNP site primer design
[0066] After the SNP site screening is completed, the 100bp sequences of the upstream and downstream sequences of the SNP site are pulled from the mouse genome sequence using programming tools; 5' end primer design: design 20-30bp primers at the upstream of the SNP site, and The ends of the primers are the two mutated bases of the SNP, and a 20bp sequence that recognizes different signals, such as FAM and HEX signals, is added to the 5' end of the primer; the downstream primer is designed at the 3' end, and the length is about 18-29bp.
[0067] The Tm (°C) was between 55-65°C, and the GC% was between 34%-60%. A total of 288 primers were designed, and the specific primer sequences are shown in the table below. For each sample, 96 sites were arranged for PCR amplification detection, and 3 primers for each site were simultaneously amplified by PCR. If one of the signals was generated in the system, it indicated that the sample t...
Embodiment 3
[0078] Example 3: KASP method for genotyping
[0079] 1. Extract the DNA of the sample to be tested as a template
[0080] Mouse tail DNA of inbred mice was extracted using oKtopureTM high-throughput DNA extractor from LGC Company. The extraction steps refer to the instrument instructions. Can effectively improve DNA extraction throughput, 3,500 samples / day; extraction speed: 20-30mg initial sample volume, 8×96≤1.5h, 80-100mg initial sample volume, 8×96≤2h.
[0081] 2. DNA concentration requirements
[0082] Each reaction of most KASP assays requires 5-50ng DNA; the DNA concentration requirement varies according to the genome size: DNA concentration requirement of the species to be tested = 5ng / uL × genome size of the species to be tested / human genome size.
[0083] 3. Preparation of PCR reaction system
[0084] The preparation of the reaction system was automatically carried out using the IntelliQube instrument of LGC Company. KASP genotyping reaction syste...
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