OUTGROUP HETERODUPLEX ANALYSIS USING TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS - HIGH-RESOLUTION, LARGE-SCALE, SCREENING OF DNA VARIATION IN THE MITOCHONDRIAL CONTROL REGION

The ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol Outgroup Heteroduplex Analysis (OHA). We assess this technique's sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433-base-pair fragment of Control Region mitochondrial DNA from Melomys cervinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient. When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.