| BRIEF REPORT |
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| Year : 2013 | Volume
: 19
| Issue : 1 | Page : 78-83 |
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Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA
C. H. W. M. R. Chandrasekara Bhagya1, WS Wijesundera Sulochana1, N Perera Hemamali2
1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, Sri Lanka
2 Department of Psychological Medicine, Faculty of Medicine, University of Colombo, Sri Lanka
Correspondence Address:
C. H. W. M. R. Chandrasekara Bhagya
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, P. O. Box 271, Kynsey Road, Colombo
Sri Lanka
 Source of Support: National Science Foundation, Conflict of Interest: None
DOI: 10.4103/0971-6866.112898
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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. |
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