Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions.
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 (DeltaG). Null mutations were introduced to bring down these DeltaG 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.
Sahdev
Sudhir
S
Department of Biotechnology and Bioinformatics, New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon, India. sudhir.sahdev@ranbaxy.com
Saini
Shalini
S
Tiwari
Prabhakar
P
Saxena
Sanjeev
S
Singh Saini
Kulvinder
K
D017931:DNA Primers / Q000737:chemistry*; D018076:DNA, Complementary / Q000096:biosynthesis; D019281:Dimerization; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D020862:GC Rich Sequence / Q000235:genetics*; D019073:HT29 Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011972:Receptor, Insulin / Q000235:genetics; D013816:Thermodynamics; D019061:src-Family Kinases / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D011972:Receptor, Insulin; D019061:src-Family Kinases