在用散弹(shotgun)法测定水稻(Oryza sativa L.ssp. indica)基因组全序列的过程中,叶绿体和线粒体DNA的污染问题非常严峻.应用脉冲场电泳(PFGE)技术对水稻基因组DNA进行纯化,结果表明它能够有效去除叶绿体和线粒体DNA,使其污染率从3%降低到0.2%. 同时,比较了水稻绿苗和黄化苗的DNA得率,以及HB法和NIB法制备大分子质量(HMW)DNA的异同.最后提出一套制备水稻基因组DNA的方法,包括黄化苗培养; 细胞核的分离、包埋和裂解;脉冲场电泳纯化、回收聚集在低熔点(LMP)胶中的水稻HMW DNA.用该方法所得的水稻基因组DNA,纯度高(无叶绿体和线粒体DNA污染)、基因组完整(机械剪切和降解少)、回收率高(操作过程中DNA损失少). 另外,首次报道在融化的低熔点(LMP)胶中对水稻HMW DNA于38℃进行超声波处理,能够获得用于shotgun文库和梯度文库构建所需要的各种DNA片段(1.5～3 kb, 3～12 kb),并且效果优于在TE中进行操作.%The contamination of chloroplast and mitochondrial DNAs was a serious problem during genome sequencing of rice (Oryza sativa L.ssp.indica) by whole-genome shotgun strategy. Pulse field gel electrophoresis (PFGE) was utilized to purify rice genomic DNA, which could efficiently remove the organelles (chloroplast and mitochondrion) DNA and reduce the contamination ratio from 3% to 0.2%. At the same time, the rice DNAs yielded from yellow seedlings and green seedlings were compared, and the differences between HB method and NIB method in high molecular weight(HMW) DNA isolation were also studied. Finally, a set of methods for obtaining the whole and highly pure rice genomic DNAs were proposed, which included culturing rice yellow seedling; isolating, embedding and lysising rice nuclei, purifying and recovering rice genomic DNAs in low melting point (LMP) agarose gel by PFGE. Ultrasonic treatment on HMW DNAs in the melting LMP gel at 38℃ was reported at first time, it facilitated to obtain the desired DNA fragments for construction of shotgun library and gradient libraries.
展开▼
