An Improved Technique for Genotyping the ABCB1 Gene Variant of Exon 21

ZUCCOLI, JOHANNA ROMINA - CORRESPONDING AUTHOR; PAGNOTTA, PRISCILA AYELÉN; MELITO, VIVIANA ALICIA; LAVANDERA, JIMENA; PARERA, VICTORIA; BUZALEH, ANA MARÍA

Methods and Protocols

MDPI

Lugar: Basel; Año: 2023 vol. 6

The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobioticsand antiretroviral drugs. Some variants of the ABCB1 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 ahigh 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 severalenzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay andHigh-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype thethree variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primersand the digestion of the PCR product with two restriction enzymes: BrsI to identify A allele andBseYI 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.