骨肉瘤基因组DNA的提取及部分酶切分析

Genomic DNA Extraction from osteosarcoma and partial digestion analysis

目的比较提取骨肉瘤基因组DNA,建立基因组DNA的最佳部分酶切条件并制备其部分酶切产物。方法采用经典的基因组DNA提取法和改良玻璃棒缠绕法提取骨肉瘤基因组DNA,以限制内切酶Sau3A I部分酶切,采用玻璃珠(SilverBeads)DNA胶回收试剂盒与蔗糖梯度离心回收分离目的片段DNA。结果提取基因组DNA片段大于150 kb,37℃,Sau3A I 1∶4(u/μg)部分酶切骨肉瘤基因组DNA 30 min获得18 kb^23 kb基因片段的富集,制备目的片段DNA。结论经改良玻璃棒缠绕法提取骨肉瘤基因组DNA具有纯度、产量高,方法简单,无蛋白和RNA污染,片段足够长,可被Sau3AI部分酶切,玻璃珠(Sil-ver Beads)DNA胶回收试剂盒和蔗糖梯度离心回收分离目的片段DNA纯度高,无小的DNA片段混入,可满足构建入噬菌体载体(EMBL3)基因组文库。为构建骨肉瘤的入EMBL3载体基因组文库做好了前期工作。

Objective To develop a simple,speedy,reliable and inexpensive method for extracting high molecular weight genomic DNA,we compared several methods to extract genomic DNA from osteosarcoma. To extablish the best partial digestion condition of genomic DNA, cained 18 kb-23 kb DNA gene fragments on the condition suited to be ligated to λ EMBL3, prepare the products of partial digestion genomic DNA.Methods Using conventional phenol-chloroform extrraction method and improved silver stick twist method,we extracted genomic DNA from osteosarcoma, by controlling digesting temperature, time and the quantity of Sau3AI, to establish the optimal enzyme conditions to generate a certain size range of DNA fragments （18 kb-23 kb） which may be ligated to λ EMBL3 ,and determine the size distribution of the digestion products by removing a small aliquot of the DNA and analyzing by electrophoresis in 0.5% agarose gel. Size fractionation was accomplished by sucrose gradient centrifugation or agarose gel electrophoresis followed by Silver Beads Agarose Gel DNA Purification kit.Results The high molecular weight DNA demonstrated an A260/280 ratio range from 1.8 to 2.0. The genomic DNA was analyzed by electrophoresis in 0.5 % agarose gel. It suggested that the genomic DNA was more than 150 kb. Genomic DNA was digested in proportion of DNA to restriction enzyme at 37E for 30 minutes, the optimal proportion is 1：4（u/μg）, the size range of fragments （18 kb-23 kb） were abundant generated. Partial digestion products selected were high purity and had no small DNA fragments interfused. Conclusion The genomic DNA extracted by improved silver stick twist method is high purity, high molecular weight （ 〉 150 kb） and can be digested by Sau3AI. DNA fragments selected by sucrose gradient centrifugation or agarose gel electrophoresis followed by Silver Beads Agarose Gel DNA Purification kit may meet the need of construction of EMBL3 genomic library of csteosarcoma, which helps to accomplish primary work for constructing csteosarcoma genomie DNA library.