Primer Design and Optimisation of Pongo pygmaeus CFTR Gene: Five High Mutation Exon (Range No. 12−27)

Abstract

Respiratory disease is one of the leading causes of captive orangutan mortality. Some symptoms resemble cystic fibrosis caused by mutations in the CFTR gene. One study identified mutations that may cause cystic fibrosis-like disease in orangutans. Only several pieces of research focused on respiratory diseases in orangutans involve genetic analysis. Therefore, this study aims to design an effective primer for the Bornean orangutan CFTR gene amplification. This study tested reference primers for six exons with the densest mutation using UCSC PCR in-silico. Primers with no result in PCR in silico were re-designed manually using UGENE and measured via OligoAnalyzer™ Tool. Primer optimisation was conducted using Bornean orangutan DNA blood samples. The sequence result was further analysed using the BLAST-N program to confirm the effectiveness of the primers. Seven sets of primers for exons 12, 14, 17, 20, and 23 of the Bornean orangutan CFTR gene were designed with decent parameter quality. The primers amplified the intended exon along with several bases of the flanking intron.