A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the Identification of 9 New Mutations Previously Unidentified by DGGE

Cliff J Meldrum,Lesley Thomas,Rodney J Scott,Ted Edkins,Rohanna Creegan,Michael Agrez,Renee Crooks,Mary Mcphillips,Ewan Miller

doi:10.1186/1897-4287-1-1-39

Abstract

Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC) has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE) with denaturing high performance liquid chromatography (DHPLC) in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE.From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.

Highlights

hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited disorder that predisposes mutation carriers to an increased risk of early onset colorectal cancer in the absence of any premalignant marker [1]
All 130 samples that had been subjected to denaturing gradient gel electrophoresis (DGGE) analysis were tested by denaturing high performance liquid chromatography (DHPLC) to determine if this methodology was at least as reliable as DGGE
The analysis of genes associated with the colorectal cancer predisposition HNPCC remains problematic since there are a number of different genes, which predispose to this disorder

Summary

Introduction

HNPCC is an autosomal dominantly inherited disorder that predisposes mutation carriers to an increased risk of early onset colorectal cancer in the absence of any premalignant marker [1]. DNA mismatch repair is a complex process that involves many genes, four of which have been directly associated with the disease. These genes include hMSH2 on chromosome 2p [5, 6], hMLH1 on chromosome 3q [7], hPMS2 on chromosome 2 [8], and hMHS6 on chromosome 7 [9], for a detailed review see Papadopoulos and Lindblom

Methods

Results

Conclusion