多枝赖草基因组Mb级DNA制备和酶切方法的优化  

Optimized Method of Preparing and Digesting Megabase-size DNA of Leymus multicaulis Genomes

作　　者：徐粤宇[1] 周玉雷[2] 赵茂林[3] 王志平[4] 王克武[4] 张艳[1] 

机构地区：[1]首都师范大学,北京100037 [2]甘肃农业大学,甘肃兰州730070 [3]北京市农林科学院,北京100097 [4]北京市土肥工作站,北京100029

出　　处：《华北农学报》2007年第6期24-29,共6页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金(3037090530571135);北京市自然科学基金(5032009)

摘　　要：Mb级DNA的制备和酶切是构建YAC,PAC,BAC,BIBAC和TAC文库与大尺度物理图谱的基础。以浓度0.4%(m/m)NaCl人工模拟盐池中生长的多枝赖草再生叶为材料提取细胞核,经钢丝细胞筛过滤,多次回收滤渣和离心上清中的细胞核,相差显微镜调浓度达108个/mL后,经LMP包埋,蛋白酶K降解核蛋白,脉冲电泳纯化回收后,在LMP胶块中2 Mb以上DNA的浓度约为250 ng/μL。在胶中经Hind III部分酶切再用脉冲电泳回收不同大小范围的DNA,电洗脱浓缩除盐后浓度约为25 ng/μL。用此方法得到的核DNA不但无细胞器DNA等的污染,而且浓度高,可直接用于各种人工染色体文库的构建。Preparation and digestion of Megabase-size DNA with high quality is the basis for construction of genomic library with large DNA inserts such as yeast artificial chromosome （YAC）, bacterial artificial chromosome （BAC）, binary BAC library（ BIBAC）, transformation-competent artificial chromosome （TAC）, and for long-range physical mapping. This method of embedding the nuclei is optimized to prepare Megabase-size DNA of Leymus muhicaulis genomes with high yield and purity. Generally, it includes isolation of purified nuclei by differential centrifugation, embedment of the nuclei in low-melting-point agarose pulgs, and digestion with proteinase K to release Megabase-size DNA. To purify the high molecular weight DNA with pulsed field gel electrophoresis can avoid the contaminants of organellar and small molecular weight DNA. Selecting digested DNA fragments is between 9 - 300 kb after partial digestion. It proves that the method is suitable for construction of genomic library of Leymus multicaulis.

关 键 词：多枝赖草 Mb级DNA 基因组文库 脉冲交变电场电泳 酶切 

分 类 号：Q781[生物学—分子生物学] Q243