Chapter 6 - Temperature and Denaturing Gradient Gel Electrophoresis

Abstract

This chapter discusses the basic principles of temperature gradient gel electrophoresis (TGGE) and denaturing-gradient gel electrophoresis (DGGE). Both techniques are based on the principle that the electrophoretic mobility of double-stranded DNA fragments is significantly reduced by their partial denaturation. Because of the sequence dependence of the melting properties of DNA fragments, sequence variations are detected. TGGE/DGGE and related methods provide a very high sensitivity and are relatively easy and cheap to perform once the assays have been designed and optimized. The main advantages are the high detection rate and specificity and improved heterozygote detection. The methodology is simple, non-radioactive, and relatively non-toxic. Although the sensitivity of TGGE and DGGE in detecting point mutations in genetic disorders and other settings is reported to be close to 100 percent, these methods have never become popular, which is related to the perception that it is difficult to design adequate PCR primers and set up the assays. The disadvantages include the limitation of PCR fragment length to about 500 nucleotides, difficulties of analyzing GC-rich fragments, and the need for computer analysis of potential PCR fragments. However, once primers and conditions are chosen, TGGE/DGGE is a robust and easy to perform mutation screening method particularly well suited for the detection of known and unknown mutations in large genes, where high sensitivity is required and when large numbers of samples are to be tested.