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Detection of p53 Gene in Breast Cancer by Denaturing Gradient Gel Electrophoresis and the DCode System

Akira Ichikawa and Seiji Igarashi
Tochigi Cancer Center, Tochigi, Japan


Introduction

Denaturing gradient gel electrophoresis (DGGE) is one of the most consistently used mutation scanning methods. It has evolved enormously over the last decade to be widely used particularly in gene diagnostic laboratories. A practical method on this topic has been reported recently.1 The more frequently used mutation screening methods are DGGE and Single-Strand Conformational Polymorphism (SSCP). However, the sensitivity of DGGE is far higher (~95%) than that of SSCP, making it an attractive technique for screening unknown mutations.

The principle of DGGE is based on the fact that DNA duplexes melt at high temperature in the presence of a gradient of chemical denaturant. DNA melting is sequence specific and occurs in discrete segments called melting domains. Since melting involves breaking the hydrogen bonds that hold the base pair together, a G-C rich region melts at a higher temperature than an A-T rich region.

A PCR* product begins to melt from the region which has the lowest melting point (lowest Tm) first when it is exposed to chemical denaturant and high temperature. If there are point mutations in the PCR product, the melting point will change, and thus its mobility on a polyacrylamide gel will be different from the mobility of the wild type DNA. In other words, the mobility of the DNA fragment-containing mutation in the gel will be different from that of the wild type. In this paper we describe the use of DGGE to screen p53 mutations in breast cancer.


Method

One hundred and two genomic DNA sam
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