Amplification of high GC content genes by PCR is a major challenge during the creation of recombinant GC-rich DNA constructs. This may be due to the difficulty in DNA denaturation or the possibility of forming secondary structures from DNA templates. Tools have been described to address the technical problems associated with the amplification of shorter sequences (<1000 bp). However, obstacles of synthesizing larger-sized GC-rich sequences by PCR continue to exist. This study aims to investigate the amplification of long and high GC content genes by PCR from the Mycobacterium bovis, a genome with GC content >60%, in comparison to amplifying a gene from the Listeria monocytogenes genome, a genome with a 37.8% GC content. Three PCR protocols were designed and experimented at various conditions with two M. bovis genes, Mb0129, a large gene of 1794 bp with 77.5% GC content, mpb83, a smaller gene of 663 bp in length with moderate GC content of 63%, together with LMHCC_RS00060, a large L. monocytogenes gene of 1617 bp with a lower GC content of 41.5%. The result demonstrated the superiority of the 2-step PCR protocol over other protocols in PCR amplification of Mb0129 when specific high fidelity DNA polymerases were used in the presence of an enhancer. The study highlighted the importance of manipulating the cycling conditions to perform the annealing and extension steps at higher temperatures while adjusting the ramp speed at a lower speed for a successful PCR amplification of a large GC-rich DNA template. A final PCR protocol was developed and enabled the amplification of 51 GC-rich targets. This can be a valuable tool for the amplification of long GC-rich DNA sequences for various downstream applications.

通过 PCR 扩增高 GC 含量的基因是创建富含 GC 的重组 DNA 构建体的主要挑战。这可能是由于 DNA 变性困难或从 DNA 模板形成二级结构的可能性。已经描述了用于解决与较短序列（<1000 bp）扩增相关的技术问题的工具。然而，通过 PCR 合成更大尺寸的富含 GC 的序列仍然存在障碍。本研究旨在研究通过 PCR 扩增牛分枝杆菌（GC 含量 >60% 的基因组）与扩增单核细胞增生李斯特菌的基因相比基因组，具有 37.8% GC 含量的基因组。设计了三种 PCR 方案并在不同条件下对两个牛支原体基因进行实验，Mb0129，一个 1794 bp 的大基因，GC 含量为 77.5%，mpb83，一个长度为 663 bp 的较小基因，GC 含量为 63%，一起与LMHCC_RS00060，大L.单核细胞1617个碱基，41.5％的较低GC含量的基因。结果证明了两步 PCR 方案在Mb0129 的PCR 扩增中优于其他方案当在增强子存在的情况下使用特定的高保真 DNA 聚合酶时。该研究强调了操纵循环条件以在较高温度下执行退火和延伸步骤的重要性，同时以较低速度调整斜坡速度以成功 PCR 扩增富含 GC 的大型 DNA 模板。制定了最终的 PCR 协​​议，并能够扩增 51 个富含 GC 的目标。这可以成为为各种下游应用扩增富含 GC 的长 DNA 序列的宝贵工具。