Amplification of Filia, a Bovine GC Rich Gene, by Optimization of Primer Design

Filia is a maternal effect gene, disorder in which causes decreased fertility in mouse and abortion in human. Bovine Filia transcript is predicted in NCBI database. Nucleotide analysis shows that it has a higher GC content in comparison with its mouse or human orthologs, and this can make problems in PCR amplification. This study was performed to investigate the possibility of amplification of bovine Filia transcript with high GC content by optimization of primer design. Two pairs of primers were designed to amplify 443 and 230 bp fragments. High Tm of primers and low ΔTm conditions were considered to design only the first pair of primers. Bioinformatic analysis was performed on the two fragments to analyze the GC contents and possible secondary structures. RNA was extracted from bovine oocytes. After reverse transcription, PCR amplification was performed with different annealing temperatures in two or three steps. Results showed that the 443 bp fragment has a CpG island and a higher GC content and more possibility of secondary structure formation compared with the 230 bp fragment. Nevertheless only the 443 bp fragment with the primers of interest was amplified with a sharp band. In conclusion we showed that low ΔTm, high Tm of primers and annealing temperatures and also a two-step PCR help specific amplification of GC-rich sequences.