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Multiplex PCR–single‐base extension genotyping of multiple glutathione S‐transferase polymorphisms
Author(s) -
Faber Ana,
Ferreira Guilherme N. M.,
Silva Susa.,
Rueff José,
Gaspar Jorge
Publication year - 2005
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20040038
Subject(s) - genotyping , biology , genetics , genotype , multiplex polymerase chain reaction , primer extension , polymerase chain reaction , multiplex , primer (cosmetics) , single nucleotide polymorphism , genomic dna , gene , molecular inversion probe , microbiology and biotechnology , computational biology , nucleotide , chemistry , organic chemistry
Identification of genetic polymorphisms has recently gained increased interest, since they can be used as markers to identify the genes that predispose to disease. This emerging role of genetic polymorphism in clinical association has created the need for high‐throughput genotyping methodologies. The present study describes the development of an SBE (single‐base extension) methodology for the parallel identification of genetic polymorphisms in glutathione S‐transferase genes, a superfamily of phase II drug‐metabolizing enzymes. Oligonucleotide PCR primers were designed for simultaneous amplification of GSTM1 , GSTP1 and GSTT1 gene loci SBE primers were also designed to be specific for each loci and to stop one nucleotide 5′‐upstream of the polymorphic location. A specific tag was associated with each SBE primer to guarantee further discrimination by length. After simultaneous amplification of the target gene loci from genomic DNA extracted from human blood samples, SBE reactions were performed with fluorescently labelled dideoxynucleotide triphosphates. Individual genotypes were identified after separation of each tag‐SBE probe by PAGE. The multiplex/SBE methodology was validated with previously genotyped DNA samples extracted from 21 individuals and it was used in a blind assay to genotype additional 64 individuals. The results show that SBE leads to the same results as the current ‘gold standard’ restriction‐fragment‐length‐polymorphism‐based genotyping methodologies, since SBE is a robust and accurate genotyping methodology that enables the parallel identification of multiple polymorphisms in the same reaction.