Temporal temperature gradient electrophoresis for detection of single nucleotide polymorphisms.
The presence of single nucleotide polymorphisms (SNPs) in nuclear DNA and mitochondrial DNA (mtDNA) can be detected using a range of electrophoretic techniques, of which temporal temperature gradient electrophoresis (TTGE) is often the most user-friendly and reproducible. The technique operates on the same principle as denaturing gradient gel electrophoresis, but does not require a chemical gradient in the gel. Instead, TTGE relies on a steady and gradual increase in temperature during electrophoresis to denature and separate DNA sequences that differ by as little as one base pair. TTGE can be easily accomplished using DNA of high quality and it is a rapid-throughput method for SNP screening once conditions have been optimized. Detection of SNPs is, for example, important for the diagnosis of mitochondrial disorders such as heteroplasmy, the presence of more than one type of mitochondria within a cell or tissue. Here we describe the basic steps for TTGE and illustrate its utility for the detection of heteroplasmy in mtDNA control region sequences.
Jones
Bethan M
BM
National Oceanography Centre, School of Ocean and Earth Science, University of Southampton, Southampton, UK.
Knapp
Leslie A
LA
D020106:Acrylamide; D000818:Animals; D004272:DNA, Mitochondrial / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012984:Software; D012996:Solutions; D013696:Temperature; D013997:Time Factors
D004272:DNA, Mitochondrial; D012996:Solutions; D020106:Acrylamide