Rapid, long-range molecular haplotyping of thiopurine S-methyltransferase (TPMT) *3A, *3B, and *3C

Clinical Chemistry
Nicolas von AhsenMichael Oellerich

Abstract

Haplotyping is an important technique in molecular diagnostics because haplotypes are often more predictive for individual phenotypes than are the underlying single-nucleotide polymorphisms (SNPs). Until recently, methods for haplotyping SNPs separated by kilobase distances were laborious and not applicable to high-throughput screening. In the case of thiopurine S-methyltransferase (TPMT*), differentiating among TPMT*3A, *3B, and *3C alleles is sometimes necessary for predictive genotyping. The genomic region including the two SNPs that define TPMT*3A, *3B, and *3C alleles was amplified by long-range PCR. The resulting PCR product was circularized by ligation and haplotyped by allele-specific amplification PCR followed by product identification with hybridization probes. Critical points were the long-range PCR conditions, including choice of buffer and primers, optimization of the ligation reaction, and selection of primers that allowed for strict allele-specific amplification in the second-round PCR. Different underlying TPMT haplotypes could then be differentiated. Results from the haplotyping method were in full agreement with those from our standard real-time PCR method: TPMT*1/*3A (n = 20); TPMT*1/*3C (n = 4); TPMT*1/*1 (n...Continue Reading

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Citations

Oct 31, 2007·Genetic Epidemiology·B KulleL Wojnowski
Aug 22, 2013·British Journal of Clinical Pharmacology·Lynne Lennard
Feb 1, 2006·Personalized Medicine·Monica ArenasJeremy Sanderson
Jan 13, 2006·Journal of the American Chemical Society·Akimitsu OkamotoIsao Saito

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