Studies aiming to overcome these problems gave special consideration to the primers, enhancer solutions and cyclic conditions. In addition, cloning genes of high GC content by PCR using long primers can pose an extra challenge to the amplification process because of the high melting temperature ( Tm) due to the extra hydrogen bonding between cytosine and guanine base pairs (Borah, 2011). Such structures can block the DNA polymerase and/or prevent primers from annealing to the templates thus terminating the synthesis of the new DNA strand during PCR. This problem is complicated by the high probability of forming secondary structures such as hairpin within DNA templates, self and cross primer dimer formation. The amplification of target DNA sequences of high GC content (>60%) can be an obstacle to a successful PCR. This can be a valuable tool for the amplification of long GC-rich DNA sequences for various downstream applications. A final PCR protocol was developed and enabled the amplification of 51 GC-rich targets. 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. 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. monocytogenes gene of 1617 bp with a lower GC content of 41.5%. 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. Three PCR protocols were designed and experimented at various conditions with two M. Tools have been described to address the technical problems associated with the amplification of shorter sequences (60%, in comparison to amplifying a gene from the Listeria monocytogenes genome, a genome with a 37.8% GC content. This may be due to the difficulty in DNA denaturation or the possibility of forming secondary structures from DNA templates. Amplification of high GC content genes by PCR is a major challenge during the creation of recombinant GC-rich DNA constructs.
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