CAJ | 학술논문

目的探讨Tth-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1μg,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C/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.
목적 탐토Tth-단련결합단백(SSB)작위첨가제재용우단핵감산다태성(SNP)분형적취합매련반응(PCR)측정중제고특이성적개치.방법 선구건Tth-SSB질립,재표체순화출Tth-SSB후,장Tth-SSB첨가도검측세포색소P450초가족(CYP2C19*3)636G＞A위점적PCR체계중；병모색기확증조건화최가용량,연후용우화시험조건적PCR대30빈뇌결혈환자외주혈표본진행SNP분형급직접측서,이험증첨가Tth-SSB적PCR진행SNP분형적준학성.결과 질립표체획득적Tth-SSB단백경순화후,순도체85％,장기첨가도PCR체계중최괄용량위1μg,여미첨가적PCR결과상비,가소제비특이성조대,감저이취체.첨가Tth-SSB적PCR측정30빈표본중CYP2C19*3위점분형위C/G순합자26빈,G/A잡합자4빈,무A/A순합자,여평행측서결과적부합솔체100％.결론 Tth-SSB작위일충첨가제,능통과소제비특이성조대제고반응특이성,종이현저제고PCR대SNP분형적준학성.
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 cytochrome 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 μg.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.