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Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA.
Indian J Hum Genet ; 2013 Jan; 19(1): 78-83
Artigo em Inglês | IMSEAR | ID: sea-147640
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.
Assuntos

Texto completo: DisponíveL Índice: IMSEAR (Sudeste Asiático) Assunto principal: DNA / Bochecha / Reação em Cadeia da Polimerase / Elementos Facilitadores Genéticos / Técnicas de Amplificação de Ácido Nucleico / Citosina / Síndrome do Cromossomo X Frágil / Guanina Tipo de estudo: Guia de Prática Clínica Idioma: Inglês Revista: Indian J Hum Genet Ano de publicação: 2013 Tipo de documento: Artigo

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Texto completo: DisponíveL Índice: IMSEAR (Sudeste Asiático) Assunto principal: DNA / Bochecha / Reação em Cadeia da Polimerase / Elementos Facilitadores Genéticos / Técnicas de Amplificação de Ácido Nucleico / Citosina / Síndrome do Cromossomo X Frágil / Guanina Tipo de estudo: Guia de Prática Clínica Idioma: Inglês Revista: Indian J Hum Genet Ano de publicação: 2013 Tipo de documento: Artigo