不同预处理方法影响昆虫干标本DNA提取效果

Effects of Different Pretreatment on DNA Extracted from Dried Specimen of Insects

为从昆虫干标本中获取高质量DNA,以利于后期PCR扩增目的基因,本研究对DNA提取前昆虫干标本的预处理方法进行探讨,分别测定其纯度和质量,并用不同引物进行了扩增效果对比。结果显示:经过预处理的样品DNA平均含量均高于CK的52.283 ng/滋L,最高为0.9%Na Cl处理3 h,平均含量达122.632 ng/滋L;米象4种预处理方法提取的DNA利用引物Lco1490/Hco2198及UEA7/UEA10均能扩增出650 bp长度条带,仅有0.9%Na Cl溶液浸泡3 h处理的米象可通过引物J173/J1331扩增出1 000 bp的长片段条带。研究表明,经过预处理后,一定程度上减少了基因组DNA的断链,主要对样品DNA的质量浓度影响显著;以9%Na Cl溶液3 h预处理后的昆虫干标本,利用磁珠法所提DNA的质量及PCR扩增效果最佳。

In order to obtain high quality DNA from dried specimens of insects for PCR amplifying target gene,we studied different methods of pretreatment that affected DNA extraction from dried specimen of insects, and further measured its purity and quality as well as compared amplification effect with different primers in this research. Results showed that the average content of DNA samples with different pretreatments were higher than52.283 ng/μL of the control without pretreatment, and reached 122.632 ng/μL with 0.9% NaCl for 3 hours; DNA from the insect of sitophilus oryzae with 4 pretreatments enabled to amplify 650 bp gene fragment with primer Lco1490/Hco2198 and UEA7/UEA10, particularly, the treatment of 0.9% NaCl for 3 h can amplify 1 000 bp gene fragment with primer J173/J1331. The research suggested that pretreatment for dried specimens may reduce the chain scission of genomic DNA in a certain extent, which affect the mass concentration of DNA samples significantly. The pretreatment by 0.9% NaCl for 3 hours combining with paramagnetic particle method would be the best for the DNA quality and PCR amplification.