The value of supplement of recombinational Tth-single strand binding protein in polymerase chain reaction for genotyping / 中华检验医学杂志

ABSTRACT

ObjectiveTo 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.MethodsTth-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.ResultsThe 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 follow26 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.ConclusionAs an additive,Tth-SSB could significantly improve the accuracy of genotyping by eliminating non-specific bands.