An Improved Technique for Genotyping the Gene Variant of Exon 21.

The Multidrug Resistance protein (, ) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have a high incidence in Caucasians. Several protocols have been used for genotyping the exon 21 variants, such as allele-specific PCR-RFLP using adapted primer to generate a digestion site for several enzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay and High-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype the three variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primers and the digestion of the PCR product with two restriction enzymes: to identify A allele and to differentiate between G or T. An improvement of this methodology was also described. The proposal technique here described is demonstrated to be very efficient, easy, fast, reproducible, and cost-effective.