论文部分内容阅读
目的 5种方法提取湖北钉螺基因组DNA,PCR扩增线粒体基因组的COⅡ基因以比较其扩增效果,从而选择适合湖北钉螺大样本基因组抽提的最佳方法。方法将50只钉螺分为5组,每组10只,分别用OMEGA基因组DNA抽提试剂盒、天根海洋动物组织基因组DNA提取试剂盒、简化基因组抽提方法、CTAB法和饱和酚抽提法抽提单只湖北钉螺基因组DNA,从消化、抽提时间、抽提浓度、成本等方面评价各种方法的优劣;PCR扩增线粒体基因组COⅡ基因,测定目的条带相对浓度以衡量扩增效果。结果 5种方法均能提取基因组DNA,消化时间最短的为饱和酚法,其余均为3 h;抽提时间最短的为两种试剂盒和CTAB,最长的为简化法;抽提成本最高的为OMEGA试剂盒法,最低的为CTAB法。5种方法PCR均扩增出COⅡ基因,但目的条带浓度有一定差异,其中OMEGA公司和天根公司试剂盒及实验室简化法提纯的模板PCR扩增浓度较高。结论用OMEGA公司生产的基因组DNA抽提试剂盒抽提湖北钉螺基因组DNA效果好,但成本较高,适用于对小样本的抽提;简化法从提纯效果、成本等方面评价,较适用于大样本抽提。
Objective To extract the genomic DNA of snail from Hubei and amplify the COⅡ gene of mitochondrial genome by PCR to compare the amplification results. The best method for genomic extraction of snail from Hubei was selected. Methods 50 snails were divided into 5 groups with 10 animals in each group. The kit was extracted by OMEGA genomic DNA extraction kit and genomic DNA extraction kit from Tiangen marine animal tissue. The genomic DNA extraction kit, CTAB method and saturated phenol extraction A single snail genomic DNA was extracted from Hubei Province to evaluate the advantages and disadvantages of various methods in digestion, extraction time, extraction concentration and cost. The mitochondrial CO Ⅱ gene was amplified by PCR and the relative concentration of the target band was measured to measure the amplification effect . Results The genomic DNA was extracted by 5 methods, the saturated digestion time was the shortest, and the others were 3 h. The shortest extraction time was two kits and CTAB, the longest was the simplified method and the highest extraction cost For the OMEGA kit method, the lowest is the CTAB method. All of the five methods amplified the COⅡ gene by PCR, but there was a certain difference in the concentration of target bands. Among them, the template of OMEGA company and Tiangen company kit and the simplified method of laboratory purification had a higher concentration of PCR amplification. Conclusion Genomic DNA extraction kit produced by OMEGA has good effect on extracting snail genomic DNA from Hubei, but its cost is high, which is suitable for the extraction of small samples. The simplification method is more suitable for large scale purification and cost evaluation Sample extraction.