添加重组Tth-单链结合蛋白的聚合酶链反应在基因型测定中的价值

The value of supplement of recombinational Tth-single strand binding protein in polymerase chainreaction for genotyping

目的探讨Tth一单链结合蛋白（SSB）作为添加剂在用于单核苷酸多态性（SNP）分型的聚合酶链反应（PCR）测定中提高特异性的价值。方法先构建Tth—SSB质粒，在表达纯化出Tth．SSB后，将Tth-SSB添加到检测细胞色素P450超家族（CYP2C19％3）636G〉A位点的PCR体系中；并摸索其扩增条件和最佳用量，然后用优化试验条件的PCR对30份脑缺血患者外周血标本进行SNP分型及直接测序，以验证添加Tth—SSB的PCR进行SNP分型的准确性。结果质粒表达获得的Tth．SSB蛋白经纯化后，纯度达85％，将其添加到PCR体系中最适用量为1斗g，与未添加的PCR结果相比，可消除非特异性条带，减低二聚体。添加Tth—SSB的PCR测定30份标本中CYP2C19＊3位点分型为G／G纯合子26份，G／A杂合子4份，无A／A纯合子，与平行测序结果的符合率达100％。结论Tth—SSB作为一种添加剂，能通过消除非特异性条带提高反应特异性，从而显著提高PCR对SNP分型的准确性o（中华检验医学杂志．2012,35：233-236、

Objective To explore the value of Tth-single strand binding protein （SSB） used as an additive to improve the polymerase chain reaction （PCR） specificity for single nucleotide polymorphisms （SNP） alleles genotyping. Methods Tth-SSB plasmid was constructed and the protein was expressed, then the expressed Tth-SSB was added into PCR system detecting cytoehrome P450 Protein （CYP2C19 ＊ 3, 636G〉A）genotype to determine the optimal usage and condition. Then, the genotypes of 30 cerebral ischemia patients were tested with established methods and compared with direct sequencing to verify the accuracy of Tth-SSB as an additive into PCR for SNP genotyping. Results The purity of Tth-SSB was 85% and optimal dosage was 1 Ixg. The protein could improve the specificity and reduce the dimer when Tth-SSB was added into the PCR system. Thirty patients genotyping results as follow： 26 patients belong to G/G homozygote, 4 patients belong to G/A heterozygote, no body belong to A/A homozygote. The coincidence acquire 100% with parallel sequencing. Conclusion As an additive, Tth-SSB could significantly improve the accuracy of genotyping by eliminating non-specific bands. （Chin J Lab Med, 2012,35：233-236）