Abstract
PCR amplification failure from cDNA libraries or RNA templates, under the optimal conditions is generally attributed to high GC content. Utilization of various additives without thorough analysis of secondary structures of the template as well as primers and subsequent PCR cycle conditions, generally leads to inadequate yields and/or truncated products. To address these concerns, we have examined two highly GC-rich human genes namely insulin receptor (IR) and
cSRC kinase.
In silico analysis of these genes revealed that their -5′ and -3′ sequences have >80% GC content. Primers designed through these GC-rich regions had high self-dimer free energy values (Δ
G). Null mutations were introduced to bring down these Δ
G levels below −5.0
kcal/mol. Oligo(dT)
18 primed cDNA was synthesized from HepG2 and HT29 total RNA to amplify IR and
cSRC kinase ORFs, respectively. A multi-prong strategy including primer modifications, various DMSO-betaine combinations and high denaturing temperature conditions was pursued during cDNA synthesis to achieve optimal PCR amplification. The reported approach can be utilized to improve the amplification of templates with high GC content, which are otherwise relatively difficult to resolve.