Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA.

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dc.contributor.authorBhagya, C H W M R Chandrasekara
dc.contributor.authorSulochana, W S Wijesundera
dc.contributor.authorHemamali, N Perera
dc.date.accessioned2013-08-01T07:59:23Z
dc.date.available2013-08-01T07:59:23Z
dc.date.issued2013-01
dc.description.abstractCONTEXT: 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.en_US
dc.identifier.citationBhagya 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.en_US
dc.identifier.urihttps://imsear.searo.who.int/handle/123456789/147640
dc.language.isoenen_US
dc.source.urihttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3722634/?report=classicen_US
dc.subjectEnhancersen_US
dc.subjectfragile X syndromeen_US
dc.subjectguanine-cytosine-rich sequencesen_US
dc.subjectpolymerase chain reaction additiveen_US
dc.subjectpolymerase chain reactionen_US
dc.subject.meshCheek --cytology
dc.subject.meshCytosine --analogs & derivatives
dc.subject.meshDNA --genetics
dc.subject.meshEnhancer Elements, Genetic --genetics
dc.subject.meshFragile X Syndrome --genetics
dc.subject.meshGuanine --analogs & derivatives
dc.subject.meshNucleic Acid Amplification Techniques
dc.subject.meshPolymerase Chain Reaction --methods
dc.titlePolymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA.en_US
dc.typeArticleen_US
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