运用ABI PRISM 310型基因分析仪测定重组人蛋白激酶CK2 α和β亚基cDNA序列

Sequencing of cDNAs encoding human protein kinase CK2 α and β subunits in recombinant plasmids with ABI PRISM 310 Genetic Analyzer

目的：测定重组质粒中编码人蛋白激酶ＣＫ２α和β亚基ｃＤＮＡ序列，筛选含完全正确插入片段的重组质粒。方法：随机选择人蛋白激酶ＣＫ２α和β重组质粒各四个阳性克隆。使用二氯罗丹明荧光标记终止底物循环测序试剂盒，分别加入首尾正反或／和中段正反引物，运用ＡＢＩＰＲＩＳＭ３１０型基因分析仪进行ＤＮＡ测序。结果：在ＣＫ２α和β阳性克隆中各有两个含完全正确的插入片段的重组质粒，其他各两个重组质粒的插入片段中均存在至少一个碱基突变。２４个测序反应的平均精确度为（９８．７±０．４）％；Ｎ平均出现率为（１．４±０．４）％。

Objective:In order to screen the recombinant plasmid containing correct insert fragment, cDNAs encoding human protein kinase CK2 α and β subunits in recombinant plasmids were sequenced. Methods: Every four positive clones in recombinant plasmids of human protein kinase CK2 α and β subunits were selected at random. The cDNA inserts were sequenced with the start or end or/and middle region of forward or reverse primers by using dRhodamine Terminator Cycle Sequencing Ready Reaction Kit on ABI PRISM 310 Genetic Analyzer. Results: The results of sequencing showed that two of every four α and β clones possessed correct sequence of encoding human protein kinase CK2 α and β subunits,the two other α and β clones contained at least one mutant base.The average accuracy of 24 sequencing reactions is (98.7±0.4)%,N base average rate is (1.4±0.4)%. Conclusion: Recombinant plasmids containing completely correct sequence of human protein kinase CK2 α and β subunits cDNA were screened successfully by DNA sequencing.