【摘要】 以光裸星虫为试验材料,通过单因素方法对影响其ISSR-PCR扩增效果的因子(模板DNA、Mg2+、dNTPs、引物、TaqDNA聚合酶、甲酰胺和二甲基亚砜)浓度进行研究。确立适合光裸星虫ISSR-PCR的反应体系:25μL体积含1×PCR buff-er、模板DNA用量为10-50 ng、TaqDNA聚合酶为0.75-1.75 U、Mg2+浓度为1.5-2.5 mmol/L、dNTP浓度为0.20 mmol/L,引物浓度为0.8-1.2μmol/L,甲酰胺和二甲基亚砜的添加并不能优化光裸星虫ISSR-PCR反应结果。利用所建立的体系对广西北海群体的20个光裸星虫个体基因组DNA进行ISSR-PCR扫描,结果表明,优化后的体系均能扩增出清晰、多态性高的条带。更多还原

【Abstract】 Factors which affected the ISSR-PCR analysis in the study of genetic diversity of Sipunculus nudus,such as concentration scope of template DNA,Taq DNA polymerase,Mg2+,dNTPs,primers,formamide and dimethyl sulfoxide were studied.The optimal ISSR-PCR of Sipunculus nudus were determined.Each 25 μL amplification reaction system consisted of 1×PCR buffer,10-50 ng template DNA,0.75-1.75 U Taq DNA polymerase,1.5-2.5 mmol/L Mg2+,0.20 mmol/L each of dNTP,0.8-1.2 μmol/L primer.The addition of formamide and dimethyl sulfoxide could not optimize the band type.The ISSR fingerprint were detected in 20 Sipunculus nudus individuals by using the optimal ISSR-PCR reaction system,which indicated that it could amplify clearly and polymorphic band.更多还原