PCR procedures to amplify GC-rich DNA sequences of Mycobacterium bovis

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 GCrich 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.

Automated summary

Amplifying high GC content genes using PCR poses a challenge in creating recombinant DNA constructs, and experiments comparing genes from different genomes with varying GC content have led to the development of an efficient PCR protocol for successful amplification of large GC-rich DNA templates.