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

作者：Li, Li-Yan; Li, Qiang; Yu, Yan-Hong; Zhong, Mei; Yang, Lei; Wu, Qing-Hong; Qiu, Yu-Rong; Luo, Shen-Qiu^*

来源：Clinical Biochemistry, 2011, 44(8-9): 692-698.

DOI：10.1016/j.clinbiochem.2011.02.001

• Polymerase chain reaction
• GC-rich sequences
• Primer design

Objectives: To establish a primer design method for amplification of GC-rich DNA sequences. @@@ Design and methods: A group of 15 pairs of primers with higher T(m) (>79.7 degrees C) and lower level Delta T(m) (<1 degrees 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%). @@@ Results: All the DNA sequences in this study were successfully amplified. Statistical analyses show that the T(m) and AT, were the main factors influencing amplifications. @@@ Conclusions: 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 degrees C), and we can overcome this difficulty easily by designing primers and using higher annealing temperature.

• 出版日期2011-6
• 单位南方医科大学