Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA |
| |
Authors: | C H W M R Chandrasekara Bhagya W S Wijesundera Sulochana N Perera Hemamali |
| |
Institution: | Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, Sri Lanka;1.Department of Psychological Medicine, Faculty of Medicine, University of Colombo, Sri Lanka |
| |
Abstract: | CONTEXT:Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters.AIM:Optimize a cost effective PCR assay to amplify the GC-rich DNA templates.SETTINGS AND DESIGN:Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5’ untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells.MATERIALS AND METHODS:A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase.RESULTS:Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used.CONCLUSIONS:It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases. |
| |
Keywords: | Enhancers fragile X syndrome guanine-cytosine-rich sequences polymerase chain reaction additive polymerase chain reaction |
|
|