| 摘要: |
| 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。 |
| 关键词: 线虫 固定 基因组DNA 18S核糖体RNA基因 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金资助项目(40176028) |
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| Isolation of fixed nematode individual genomic DNA and amplification of18S ribosomal RNA gene fragments |
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| Abstract: |
| 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 with 0.05 mol/L EDTA(pH8.0) and 5% formalin in seawater) using two different genomic DNA isolation methods (alkaline lysis and protease K treatment) were compared for their performances in the amplification of 18s ribosomal RNA gene fragments. It was found that absolute alcohol is the best fixing solution for preparing PCR template DNA, and 5% 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. |
| Key words: nematode fixation genomic DNA 18S ribosomal RNA gene |
