Single nucleotide polymorphism screening with denaturing gradient gel electrophoresis.
Denaturing gradient gel electrophoresis (DGGE) is a powerful technique for identifying DNA sequence-based differences. The method relies on the fact that double-stranded DNA molecules have unique denaturation rates that are based upon the specific nucleotide composition of the DNA sequence(s). While DGGE is typically used to screen for polymorphisms that vary by multiple nucleotides, it is equally useful for screening single nucleotide polymorphisms (SNPs). For most applications, it is possible to use computer software in advance to determine if SNPs can be differentiated using DGGE. The software can also model the effect of attaching a GC-rich clamp to the PCR primer to improve detection of SNPs. Once feasibility has been confirmed, a perpendicular DGGE can be used to identify the optimal denaturing gradient for the sequences of interest. Parallel gels can then be used to screen large numbers of samples at one time, eliminating the need for cloning and sequencing or direct sequencing of PCR products. This chapter provides step-by-step instructions on the use of DGGE and illustrates its application for detection of SNPs, as well as multiple nucleotide polymorphisms, in the major histocompatibility complex.
Knapp
Leslie A
LA
Primate Immunogenetics and Molecular Ecology Research Group, Department of Biological Anthropology, University of Cambridge, Cambridge, UK.
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012984:Software
D004247:DNA