Sequencing: Not Always the “Gold Standard”

Abstract

For the detection of point mutations (or variant alleles), PCR followed by restriction fragment length polymorphism (RFLP) detection is widely used and is probably one of the simplest methods for detection of known mutations. This method depends on the fact that the mutation of interest disrupts a recognition sequence for a restriction enzyme. When a restriction site is not present around the mutation, mismatch primers need to be designed that do create a restriction site. Such a mismatch primer is thus usually located directly adjacent to the mutated nucleotide. We validate our PCR-RFLP assays by direct sequencing of the PCR product. In two of our assays, however, we found that none of the three samples found to be heterozygous by PCR-RFLP was confirmed as heterozygous by direct sequencing. Detection of the CYP3A53 polymorphism (A6986G) is based on amplification of a 293-bp DNA fragment digested with Ssp 1 (1). For amplification, we used 40 pmol of primers P1 (5′-CATGACTTAGTAGACAGATGAC-3′) and P2 (5′-GGTCCAAACAGGGAAGAAATA-3′) in a final volume of 50 μL. The reverse primer P2 [which contains a mismatch (underlined nucleotide) to create a restriction site for the Ssp 1 restriction enzyme] is located directly next to the mutated nucleotide. Fig. 1A⇓ shows the …