Optimization of PCR conditions to amplify mitochondrial COI gene fragments of wildlife species in Kenya
Date
2012Author
Song'ore, E
Muigai, A
Kimwele, C
Gatebe, E
Mwaniki, M
Kinuthia, J
Osiemo, Z
Type
ArticleLanguage
enMetadata
Show full item recordAbstract
PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome c
oxidase I (COr) gene fragments of some of the targeted wildlife species in Kenya; butTalo,common zebra, grant's gazelle, warthog and common eland
and domestic samples purchased from the market as 'beef', 'goat'or 'mutton'. DNA from five wildlife species and one hundred of domestic samples were
extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixtures and annealing
temperatures to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature,
concentration of primer pair, amount of Dream Taq'" PCR Master Mix (2x) (Fermentas) and PCR cycle duration, were optimized by keeping the
amount of DNA template (2~L) and concentration ofPCR buffer, MgCl2 4mM and dNTP mixture constant (Fennentas). All genes were successfully
amplified, giving the correct fragment lengths of 700 base pair (bp), as assigned for both forward and reverse primer. The optimal conditions were
determined to be: O.SIlI(Spmoles) for each primer, 2SIlI of DreamTaq "1M PCR Master Mix (2x), 30 s of both denaturation and annealing cycles and
annealing temperature ofS6.S°C. PCR products obtained under these conditions produced excellent bands.
Publisher
University of Nairobi
Description
Journal Article