紫茎泽兰高分子量核DNA制备方法的研究

Isolation of the HMW-DNA from Crofton Weed(Ageratina adenophora)

以紫茎泽兰暗培养幼嫩叶片为材料,在细胞核提取缓冲液中加入PVP、抗坏血酸和氨基甲酸钠3种强抗氧化剂,较好地去除了多糖多酚等次生代谢物,经低熔点琼脂糖包埋细胞核及蛋白酶K原位裂解DNA胶块,以获得大分子量DNA;最后分别采用2种不同的限制性内切酶(BamHⅠ和HindⅢ)和5个浓度梯度(0U、0.2U、0.4U、0.6U、0.8U)进行酶切,并对不同的回收方法进行比较分析。结果表明:经脉冲电泳检测,本方法提取的DNA片段长度>600kb,基本无细胞器DNA和蛋白质污染;酶切条件以0.4U酶浓度HindⅢ的效果最好,酶切后的DNA片段主要集中在100～300kb之间,DNA片段大小满足构建BAC文库的要求;对回收方法进行比较分析,电洗脱法回收的大片段DNA浓度远远大于琼脂糖酶切法,大于8mg.L-1,这完全能够满足构建大片段基因组文库对DNA浓度的要求。

An essential element for genomic library is the preparation of high molecular weight （HMW） DNA. In this study,new method was developed to isolate HMW-DNA from crofton weed for BAC library. The leaves of the seedling grew under lightless were used as materials to isolate DNA. PVP,ascorbic acid and sodium diethyldithiocarbamate were added to exclude the polyphenol and polysaccharides and other secondary interfacing metabolites. Then,the intact nuclei was embedded in low melting point （LMP） agar- ose and digested with protein K in situ to release the HMW-DNA. Then, partial digestion of the isolated HMW-DNA was conducted to decide the optimal digestion system with two enzymes （BamH I and Hind Ⅲ） and five unit gradients （0 U,0.2 U,0.4 U,0.6 U,0.8 U）. Finally,the efficiency of two methods used to recover the digested DNA was compared. The result indicated that the HMW-DNA prepared by this method with the size more than 600 kb and without protein and organelle DNA,was suitable for construc- tion of crofton weed BAC library. The optimal digestion is 0.4 U of HindⅢ ,at 37℃ for 20 min,resultingin the digested DNA fragment with the main range from 100 kb to 300 kb. Comparison tion method,the electroelution method is better to recover the digested DNA fragment tion of 8 mg.L-1 ,and it meet the requirement of the large-fragment genomic library and purity. with with Agrose-diges- the concentra- high integrity