Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies
1 Ludwig Institute for Cancer Research, São Paulo, Brazil
2 Hospital Alemão Oswaldo Cruz. R. João Julião, 245, 1° andar, Cep. 01323-903, São Paulo (SP), Brazil
3 Instituto de Química, Universidade de São Paulo, Brazil
4 Av. Prof. Lineu Prestes, 748, Butantã, Cep. 05508-900, São Paulo (SP), Brazil
5 Departments of Oncology and Epidemiology, McGill University.546 Pine Ave W, Montreal, Quebec H2W1S6, Canada
6 Department of Social and Preventive Medicine and Ste-Justine Hospital, University of Montreal, 3175 Côte Ste-Catherine, Montreal, Quebec H3T 1C5, Canada
BMC Genetics 2010, 11:44 doi:10.1186/1471-2156-11-44Published: 26 May 2010
Mutations in TP53 are common events during carcinogenesis. In addition to gene mutations, several reports have focused on TP53 polymorphisms as risk factors for malignant disease. Many studies have highlighted that the status of the TP53 codon 72 polymorphism could influence cancer susceptibility. However, the results have been inconsistent and various methodological features can contribute to departures from Hardy-Weinberg equilibrium, a condition that may influence the disease risk estimates. The most widely accepted method of detecting genotyping error is to confirm genotypes by sequencing and/or via a separate method.
We developed two new genotyping methods for TP53 codon 72 polymorphism detection: Denaturing High Performance Liquid Chromatography (DHPLC) and Dot Blot hybridization. These methods were compared with Restriction Fragment Length Polymorphism (RFLP) using two different restriction enzymes. We observed high agreement among all methodologies assayed. Dot-blot hybridization and DHPLC results were more highly concordant with each other than when either of these methods was compared with RFLP.
Although variations may occur, our results indicate that DHPLC and Dot Blot hybridization can be used as reliable screening methods for TP53 codon 72 polymorphism detection, especially in molecular epidemiologic studies, where high throughput methodologies are required.