Long sections of mitochondrial DNA amplified from fourteen orders of insects using conserved polymerase chain reaction primers

The combination of highly conserved or universal polymerase chain reaction (PCR) primers with techniques that allow for the amplification of PCR produ cts greater than a few thousand base pairs (long PCR) makes it possible to amplify the complete mitochondrial genome of virtually any insect as a smal l number of overlapping segments. Twelve conserved primers from 7 mitochond rial genes were used in 17 pair combinations. The size of the amplified seg ments ranged from 3.3 to 14.1 kb. Total genomic DNA from 33 insect species representing 14 orders served as the template. In most instances, 2 fragmen ts sufficed to include the whole mitochondrial DNA (mtDNA). Less frequently 3 or more fragments were required to cover the complete mtDNA Fragments th at combined contain all of the mtDNA were amplified from 26 of 33 species. For the remaining species, >67% of the mtDNA was amplified. Anp of the larg e amplicons are convenient for restriction fragment comparisons and they ar e also suitable as a template for nucleotide sequencing of either small mtD NA regions or the complete mtDNA of diverse taxa in conjunction with popula tion or phylogenetic investigations. The procedures described here can be u sed to amplify the A+T or control region as part of a larger fragment and p rovides an opportunity for a more detailed analysis of this region.