以小麦根尖分生组织细胞核及染色体制备高分子量DNA

为了克服以叶片为材提取HMW DNA的局限性，优化了一套简便、实用的提取方法。该法以细胞周期同步化处理的根尖分生组织为材，利用机械匀浆释放细胞核或中期染色体制备悬浮液，再以蔗糖密度梯度离心和流式分选技术分别分离细胞核和染色体，经去蛋白、酶切，透析获得HMW DNA。经检测，来自200条根尖（10个胶块）的HMW DNA的浓度约为4~20 ng/µL，而连接、转化后的BAC克隆平均插入片段超过100 kb。证明该法适用于提取HMW DNA以构建BAC文库。

High Molecular Weight DNA Preparation from Nuclei or Chromosomes of Root Meristem of Wheat

A simple and practical method was optimized to overcome the limitations of current method by which HMW DNA was prepared from leaf powder. Root tips of synchronized meristem were homogenized to make preparing suspension containing intact metaphase chromosomes and nuclei, which were further isolated by sucrose gradient centrifugation and flow sorting, respectively. Finally, HMW DNA was prepared with deproteinization, digestion and dialysis of nuclei and chromosomes. The results showed that concentration of the HMW DNA from 200 root tips (10 plugs) was about 4–20 ng/µL (4 ng/µL in chromosomes DNA and 20 ng/µL in nuclei DNA). The average insert fragment size was more than 100 kb after linage, transformation and insert fragment examination by pulse field gel electrophoresis for HMW DNA from nuclei. The similar results were obtained by HMW DNA prepared from chromosomes. It is confirmed that the method is applicable to prepare HMW DNA for construction of BAC library.