蠕形螨ISSR-PCR的反应体系优化及引物筛选

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。

Condition optimization and primer screening for ISSR-PCR of Demodex

Objective To optimize conditions and screening for primers for inter-simple sequence repeat-anchored(ISSR-PCR) of Demodex,using genome DNA as the template,preparing for the genetic diversity research of Demodex with ISSR mark.Methods After mites were rinsed and precipitated in double distilled water(DDW),the genome DNA of Demodex was extracted with a kit.Then experiments were carried out using DNA of Demodex as the template,and(CA)8RG as the primer.The orthogonal array was conducted,and the involved factors were as follows: concentrations of Mg2+,dNTPs,primers,template DNA and Taq DNA polymerase and return temperature.An optimal reaction system of ISSR-PCR of Demodex was established,through which an optimal primer was obtained.Results An optimal 25 μL of ISSR-PCR amplification system contained 2.0 mmol/L of Mg2+,0.4 mmol/L of dNTPs,30-60 ng of template DNA,30 pmol of primers,and 2U of Taq DNA polymerase.The optional return temperature for the primer was 49 ℃,and the cycle index was 35.10 primers were selected,with clear bands,good polymorphism and high repetition.Conclusion The optimal conditions of ISSR-PCR and primers of genome DNA of Demodex have been screened,which are the basis of molecular biological research of Demodex mites.