Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA.
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Date
2013-01
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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.
Description
Keywords
Enhancers, fragile X syndrome, guanine-cytosine-rich sequences, polymerase chain reaction additive, polymerase chain reaction
Citation
Bhagya C H W M R Chandrasekara, Sulochana W S Wijesundera, Hemamali N Perera. Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA. Indian Journal of Human Genetics. 2013 Jan; 19(1): 78-83.