家蚕微孢子虫(Nosema bombycis)染色体DNA的分离纯化

为了获取可供家蚕微孢子虫(Nosema bombycis)单染色体测序的基础材料,通过脉冲场凝胶电泳(PFGE)技术分离家蚕微孢子虫CQ1株系的染色体DNA,随后切取含有目的染色体DNA条带的琼脂糖胶块,分别利用电洗脱法、柱式胶回收试剂盒法及玻璃奶胶回收试剂盒法进行染色体DNA的分离纯化实验。结果显示3种方法均能回收到目的染色体DNA,但分离纯化的效率和样品质量存在差异:电洗脱法对胶块中的DNA损失较大,回收的染色体DNA含量低,而柱式胶回收试剂盒回收的染色体DNA断裂严重,因此这2种方法获得的样品质量均达不到染色体建库测序的要求;通过优化后的玻璃奶胶回收试剂盒法获得的染色体DNA相对完整且污染较少,质量检验分析133μL样品中的DNA质量浓度为8.495 ng/μL(DNA总量1.13μg),达到后续构建染色体测序文库的要求。玻璃奶胶回收试剂盒法不仅适用于家蚕微孢子虫染色体DNA的分离纯化,也可供基因组较小的物种制备单染色体建库测序材料参考。

Isolation and Purification of Nosema bombycis Chromosomal DNA

To obtain basic materials for Nosema bombycis single chromosome sequencing,in this study,pulsed field gel electrophoresis(PFGE) was used to isolate chromosomal DNA of N.bombycis CQ1 strain.Agarose gels containing the target chromosomal DNA band were excised and subjected to further isolation and purification by means of electroelution,column gel recovery kit and glassmilk gel recovery kit.The results showed that all three methods could recover the target DNA but differed in efficiency of isolation and purification and in quality of obtained sample.Electroelution method led to high loss of DNA in the gel and the content of chromosomal DNA was low,while large portion of chromosomal DNA recovered by column gel extraction kit was in small pieces.The quality of sample purified using these two methods could not satisfy the sequencing requirement for library construction of chromosome.The modified glassmilk gel recovery kit method could recover relatively complete and less polluted chromosomal DNA.Quality inspection indicated that the mass concentration of sample DNA was 8.495 ng/μL(the total DNA quantity of 133 μL sample was 1.13 μg),and the sample reached the requirement of subsequent chromosome sequencing and library construction.The glassmilk gel recovery kit method is not only applicable to isolate and purify chromosomal DNA of N.bombycis,it can also be referred in preparation of sequencing material for library construction of single chromosome of species with small genome.