单条固定线虫基因组DNA提取及18S rRNA基因PCR扩增

Isolation of fixed nematode individual genomic DNA and amplification of18S ribosomal RNA gene fragments

根据线虫18S核糖体RNA基因PCR扩增效果比较了丙酮、乙醇、乙醇+0.05mol/LEDTA(pH8.0)和5%海水福尔马林4种固定剂,碱裂解和蛋白酶K处理2种单条线虫基因组DNA提取方法的优劣。用乙醇固定的样品最适合制备PCR模板DNA,而5%海水福尔马林固定的样品能最完整地保持样品形态。蛋白酶K处理获得的DNA较碱裂解获得的更适合PCR扩增。结果有助于分子生物学方法在海洋线虫分类、多样性和生态学研究中的应用。

Based on the lengths and yields of PCR products,eight template DNAs were isolated from marine nematode individuals and stored respectively in four fixing solutions(acetone,absolute alcohol,alcohol with0.05mol/L EDTA(pH8.0)and5%formalin in seawater)using two different genomic DNA isolation methods(alkaline lysis and protease K treatment)were compared for their performances in the amplification of18s ribosomal RNA gene fragments.It was found that absolute alcohol is the best fixing solution for preparing PCR template DNA,and5%formalin in seawater is the best for keeping morphological characters of nematode individuals.The genomic DNA isolated with protease K treatment was better for PCR amplification than that isolated with alkaline lysis.Our results would help approach to taxonomical and ecological studies of free-living marine nematode.