Primer Design and Optimisation of Pongo pygmaeus CFTR Gene: Eight High Mutation Exon (Range No. 1-11)
Abstract
Orangutans are known to have respiratory diseases similar to cystic fibrosis
symptoms in humans. Cystic fibrosis is a respiratory disease caused by mutations
in the cystic fibrosis transmembrane regulator gene (CFTR). Research on the CFTR
gene in Bornean orangutans is still rarely done. Therefore, this study will detect the
CFTR gene mutation of the Bornean orangutan samples from the BOSF
rehabilitation centre. This study aimed to design specific primers for the CFTR gene
in several predefined gene exons with the densest mutation and to obtain the optimal
annealing temperature for each primer. We used UGENE version 40.1 for manual
primers design. Optimisation of PCR amplification program and primer annealing
temperature was determined with pre-denaturation at 94 °C, denaturation at 95 °C,
different annealing temperatures for each primer, and final extension at 72 °C. The
optimum condition for annealing temperatures of designed specific primers for
exons 3, 4, 6, 8, and 11 of the orangutan CFTR gene was at 51-58 °C. Primer can
amplify the exon and intron regions flanking the target exon.
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