Department of Cytobiology, Southern Medical University, Guangzhou 510515, PR China.
Clin Biochem. 2011 Jun;44(8-9):692-8. doi: 10.1016/j.clinbiochem.2011.02.001. Epub 2011 Feb 18.
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 ΔT(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 ΔT(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.
建立一种用于扩增富含 GC 序列的引物设计方法。
设计了 15 对 Tm 值较高（>79.7°C）且 ΔTm 值较低（<1°C）的引物，用于扩增 GC 含量较高（66.0%-84.0%）的序列。对引物参数和 PCR 产物的 GC 含量进行了统计分析，并与文献进行了比较。本研究还通过缩短富含 GC 的 PCR 扩增引物进行了其他控制实验，并对缩短的引物参数和 PCR 产物的 GC 含量进行了统计分析，与未缩短的引物进行了比较。设计了 26 对引物，以测试该引物设计策略在扩增 GC 含量较低（35.2%-53.5%）序列中的适用性。
本研究中的所有 DNA 序列均成功扩增。统计分析表明，Tm 和 ΔTm 是影响扩增的主要因素。
该引物设计策略为扩增富含 GC 的序列提供了一种完美的工具。这证明在较高的退火温度条件下（>65°C），无法形成二级结构，我们可以通过设计引物和使用较高的退火温度来轻松克服这一困难。
