A primer design strategy for PCR amplification of GC-rich DNA sequences

ObjectivesTo establish a primer design method for amplification of GC-rich DNA sequences.Design and methodsA group of 15 pairs of primers with higher Tm (> 79.7 °C) and lower level ΔTm (< 1 °C) were designed to amplify GC-rich sequences (66.0%–84.0%). The statistical analysis of primer parameters and GC content of PCR products was performed and compared with literatures. Other control experiments were conducted using shortened primers for GC-rich PCR amplifications in this study, and the statistical analysis of shortened primer parameters and GC content of PCR products was performed compared with primers not shortened. A group of 26 pairs of primers were designed to test the applicability of this primer designing strategy in amplifications of non-GC-rich sequences (35.2%–53.5%).ResultsAll the DNA sequences in this study were successfully amplified. Statistical analyses show that the Tm and ΔTm were the main factors influencing amplifications.ConclusionsThis primer designing strategy offered a perfect tool for amplification of GC-rich sequences. It proves that the secondary structures cannot be formed at higher annealing temperature conditions (> 65 °C), and we can overcome this difficulty easily by designing primers and using higher annealing temperature.