以病毒的230 kb DNA分子为模板的DNA序列测定

目的：为确认Tn3-gpt已插入小鼠巨细胞病毒(mouse cytomegalovirus，MCMV)基因组的特定位置，改进大分子病毒DNA的测序方法，建立以230kb DNA为模板直接进行DNA序列测定的方法。方法：待测MCMV的Tr3-gpt插入突变株感染NIH3T3细胞后，从培养液中制备病毒颗粒并从中纯化得到基因组DNA，以此DNA为模板，合成的寡核苷酸为引物，用热循环方法按Promega公司的DNA Cycle Sequencing System试剂盒的方法进行测序。结果：获得纯度较高可直接作为模板进行测序的：DNA，测序所得放射自显影图片条带清晰明亮，间隔正常，分辨率较高，可顺利读出插入位点的MCMV的DNA序列，证实Tn3-gpt插入在特定的位置。结论：长达230kb的大分子病毒DNA可直接作为模板，用热循环测序方法进行测序，无需切割成小片段后才进行测序。

DNA sequencing using 230 kb DNA of virus as template

Aim: To verify that the Tn3-gpt was inserted in specific site of MCMV (mouse cytomegalovirus)genome and predigest the method of large fragment DNA sequencing, we established the method of DNA sequencing using 230 kb DNA of MCMV as template directly. Methods: The NIH 3T3 cells were infected with MCMV containing Tn3-gpt insertion. After incubation the virions were prepared and 230 kb DNA of virus genome was purified from media. DNA sequencing was carried out by DNA cycle sequencing method using 230 kb DNA as template and synthetic oligonucleotide as primer. Results: The pure 230 kb genome DNA was obtained, which can be used as template directly. In the obtained gel picture the bands are clear and intensity, spaces between bands are even and easy to read. It was verified that the Tn3-gpt was inserted in specific site of MCMV genome. Conclusion:It is not necessary to cut large DNA fragment to small fragment in DNA sequencing. We could use 230 kb DNA as template directly.