Denaturing high pressure liquid chromatography (dHPLC) is an efficient method 
for discovery of unknown mutations by heteroduplex analysis of PCR fragments. 
For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial 
genome, we developed a set of 67 primer pairs defining overlapping PCR fragments 
that are well suited for heteroduplex analysis. The aim of our optimization 
efforts was to ensure that point mutations are detectable at every nucleotide 
position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) 
were found to have unfavourable melting profiles in all possible amplicons, 
therefore requiring GC-clamps at the end of one or both oligonucleotide PCR 
primers. Following detection of a heteroduplex pattern by dHPLC, our primers can 
also be employed for DNA sequencing to identify the underlying mutation. In case 
of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment 
collector is useful to recover the heteroduplex peak, which contains mutant and 
wildtype DNA molecules in a 1:1 ratio.