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

• Published in: Clinical biochemistry 2011-06, Vol.44 (8), p.692-698
• Main Authors: Li, Li-Yan, Li, Qiang, Yu, Yan-Hong, Zhong, Mei, Yang, Lei, Wu, Qing-Hong, Qiu, Yu-Rong, Luo, Shen-Qiu
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; DNA Primers - genetics; Electrophoresis; Exons - genetics; GC Rich Sequence - genetics; GC-rich sequences; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Polymerase chain reaction; Polymerase Chain Reaction - methods; Primer design
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2011.02.001

To establish a primer design method for amplification of GC-rich DNA sequences. A group of 15 pairs of primers with higher T m (> 79.7 °C) and lower level Δ T m (< 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%). All the DNA sequences in this study were successfully amplified. Statistical analyses show that the T m and Δ T m were the main factors influencing amplifications. This 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.