论文部分内容阅读
目的以蠕形螨的DNA为模板,对蠕形螨简单重复序列锚定PCR(ISSR-PCR)反应体系优化并对ISSR引物进行筛选,为ISSR分析奠定基础。方法用自然沉降法收集山羊蠕形螨,用基因组DNA提取试剂盒提取山羊蠕形螨DNA,并以此为模板,以(CA)8RG(R为1分子的嘧啶)为引物,利用正交实验法,从Mg2+浓度、引物用量、dNTPs、DNA模板用量和TaqDNA聚合酶以及退火温度对蠕形螨ISSR-PCR反应体系进行优化,并以优化的反应体系筛选ISSR-PCR的引物。结果 ISSR-PCR 25μL最佳反应体系包括:0.4 mmol/L Mg2+、30 pmol引物、30～60 ng模板DNA、2u Taq酶,最佳退火温度为49℃、循环35次。筛选出10条条带清晰、多态性好、重复性高的引物。结论筛选出蠕形螨ISSR-PCR最佳反应体系和理想的引物,为蠕形螨的分子生物学研究奠定了基础。
OBJECTIVE To optimize the reaction system of Demodex mites Simple Sequence Repeated Sequence PCR (ISSR-PCR) and screen the ISSR primers using the DNA of Demodex mites as a template to lay a foundation for ISSR analysis. Methods Demodex mites were collected by natural sedimentation method. Demodex mites DNA was extracted by genomic DNA extraction kit and used as a template. Using (CA) 8RG (R as one molecule of pyrimidine) as primer, orthogonal experiment Method was used to optimize the ISSR-PCR reaction system of Demodex from Mg2 + concentration, primer amount, dNTPs, DNA template dosage, Taq DNA polymerase and annealing temperature. The primers of ISSR-PCR were screened by optimized reaction system. Results The optimal reaction system of ISSR-PCR 25μL consisted of 0.4 mmol / L Mg2 +, 30 pmol primer, 30-60 ng template DNA and 2u Taq enzyme. The optimal annealing temperature was 49 ℃ and 35 cycles. Ten primers with clear bands, good polymorphism and high reproducibility were screened out. Conclusion The optimal ISSR-PCR reaction system and ideal primers for Demodex mites were screened, which laid the foundation for the molecular biology study of Demodex mites.