Development of a tetraplex PCR assay for CYP2D6 genotyping in degraded DNA samples

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty-two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.