Development of a single-tube PCR-pyrosequencing method for the simultaneous and rapid detection of four variant...

CYP2C9 is a polymorphic enzyme that has been reported to metabolize several clinically useful drugs such as warfarin, phenytoin and non-steroidal anti-inflammatory drugs. We designed a rapid single-tube multiplex assay to detect four variant alleles of the CYP2C9 in a single polymerase chain reaction (PCR) and a single pyrosequencing reaction.

A multiplex PCR was designed to amplify two fragments simultaneously, one containing 430C>T (CYP2C9*2) polymorphism and other containing 1075A>C (CYP2C9*3), 1076T>C (CYP2C9*4) and 1080C>G (CYP2C9*5) polymorphisms.

Four variants of the CYP2C9 gene could be simultaneously detected using only two varieties of pyrosequencing primers in a single-tube. The success rate for the four SNPs (*2, *3,*4 and *5) was high. Genotypes obtained by the multiplex reaction were 100% concordant with genotypes obtained using direct DNA sequencing (n = 96). The analysis time was halved, compared with existing simplex pyrosequencing. The system allowed high-throughput analysis of over 384 samples per hour.

Our method reduces running cost and halves analysis time, compared to simplex pyrosequencing. Another advantage of this method is that it analyses and determines multiple bases around the polymorphic site thereby reducing the possibility of scoring a truncated PCR product.