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在用散弹 (shotgun)法测定水稻 (OryzasativaL .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HMWDNA .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HMWDNA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～ 3kb ,3～ 12kb) ,并且效果优于在TE中进行操作
Chloroplast and mitochondrial DNA contamination problems are very serious during the whole genome sequencing of rice (Oryzasativa L. ssp. Indica) by shotgun assay. Pulsed field electrophoresis (PFGE) was used to purify genomic DNA in rice and the results Indicating that it can effectively remove chloroplast and mitochondrial DNA and reduce the contamination rate from 3% to 0.2% .Meanwhile, DNA yield of rice green and yellow seedlings was compared with that of HB and NIB HMW) DNA.Finally, a set of methods for preparing rice genomic DNA was proposed, including the cultivation of yellowing seedlings, the separation, embedding and lysis of the nucleus, the purification of pulsed field electrophoresis and the recovery of rice HMW DNA The genomic DNA of rice obtained by this method has high purity (no chloroplast and mitochondrial DNA contamination), complete genome (less mechanical shearing and less degradation) and high recovery rate (less DNA loss during operation) .In addition, (1 5 ~ 3kb, 3 ~ 5kb) of DNA fragment for shotgun library and gradient library construction were obtained by sonication of rice HMW DNA at 38 ℃ in low-melting point (LMP) 12kb), and the effect is better than that in TE