Preparation of Single-Stranded DNA for Pyrosequencing by Linear-after-the-Exponential-Polymerase Chain Reaction

To establish a simple method for preparing single-stranded DNA templates for pyrosequencing, the Linear-after-the-exponential (LATE)-PCR technology on the basis of Taq DNA polymerase without hot-start capacity was applied to amplify a 78-bp sequence (containing the SNP6 site), and the PCR-enhancing reagents (glycerol and BSA) were used to increase the efficiency and specialization, much more before the reagents A and B were designed to eliminate the impurity (limited primers, PPi, dNTPs and so on), and 1–2 μl LATE-PCR products with simply treatment can be used in pyrosequencing directly. Then, five SNPs related with human breast-cancers in the BRCA1 gene were investigated, and the programs had no nonspecific signals that were made of theoretic sequences. Moreover, the genotyping of the SNPs could also be distinguished easily. The results indicated that this method can be used to prepare high quality single-stranded DNA templates for pyrosequencing and allows pyrosequencing be lower in cost, simpler in operation, and easier in automation, and the cross-contamination from sample preparation was also reduced.