A system for the study of sequence polymorphisms in animal
mitochondrial DNA was developed. This system consists of two
candidate markers which are mitochondrial cytochrome b and D-loop.
Six novel primers were constructed for their amplifications by
polymerase chain reaction (PCR). The primers KY1 and KY2 amplified
a 359-bp port ion of the cytochrome b gene from chicken, water
buffalo, horse, goat and sheep. Primers UPM257 and UPM258 were used
to synthesize a 1.4 Kb-fragment containing the complete chicken
mi tochondrial D-loop. Primers LCH4-DO and LCH2-UP paired with
primers UPM257 and UPM258 respectively to amplify a 355-bp 5' region
and 395-bp 3' region of the D-loop. These primers were specific in
their amplifications. The mitochondrial DNA could be amplified from heterogeneous or degraded DNA preparations, as well as directly from
blood incubated in proteinase K thus bypassing lengthy DNA isolation
steps.Only 5ul of blood was required for a l00-ul PCR.
Fort he purification of PCR products, ethanol selective
precipitation was found to be both efficient and economical. Despite
its lower recovery compared to the Chroma Spin column, sufficient
quantity of DNA could be obtained for subsequent sequencing
