title: Assessment of DHPLC usefulness in the genotyping of GSTP1 exon 5 SNP: comparison to the PCR-RFLP method.
abstract: Single-nucleotide polymorphism (SNP) analysis can be performed by several methods such as PCR-RFLP, real time PCR and mass spectrometry. Denaturating High Pressure Liquid Chromatography (DHPLC) analysis allows the detection of DNA mutations in heteroduplex samples. GSTP1 exon 5 gene presents a single-nucleotide polymorphism (a to g) that results into an amino-acid substitution (Ile to Val). Ile and Val variants are identified respectively by a and b alleles. This polymorphism affects enzyme activity and is highly frequent within Caucasian populations and therefore widely studied in the context of SNP related to cancer susceptibility. Our goal was to evaluate DHPLC usefulness in detecting a well-known SNP in comparison to PCR-RFLP, in the field of molecular epidemiological studies. Fifty Caucasian people were genotyped by both methods. Heterozygous samples were identified easily at two temperatures using the DHPLC method. Discrimination between a/a and b/b homozygous genotypes was done by pooling every homozygous sample with a known a/a sample. Our genotyping using both methods resulted in the characterisation of 32 (64%) a/a homozygous, 18 (36%) a/b heterozygous and 5 (10%) b/b homozygous. All samples were also identically genotyped by the two methods. Our results show that DHPLC is a good alternative to classical PCR-RFLP method in genotyping SNPs. Advantages of this chromatographic method were no restriction site needed and a reduced technical time thanks to an automated injection. Moreover, unlike classical RFLP gel analysis, DHPLC chromatograms provided objective criteria for sample classification.
authors.affiliation: GRECAN-EA1772, UFR des Sciences Pharmaceutiques, Université de Caen et Laboratoire de Cancérologie Expérimentale, Centre François Baclesse, Route de Lion-sur-Mer, 14076 Caen cedex 05, France.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Abbas
authors.forename: Maud
authors.initials: M
authors.lastname: Lepelley
authors.forename: Mathilde
authors.initials: M
authors.lastname: Lechevrel
authors.forename: François
authors.initials: F
authors.lastname: Sichel
mesh_terms: D000217:Acyltransferases / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005838:Genotype; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013696:Temperature
chemical_list: D004247:DNA; D000217:Acyltransferases; C043403:fatty acyl ethyl ester synthase