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
Article
in English
| 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 ANDDESIGN:
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 ANDMETHODS:
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.
Full text:
Available
Index:
IMSEAR (South-East Asia)
Main subject:
DNA
/
Cheek
/
Polymerase Chain Reaction
/
Enhancer Elements, Genetic
/
Nucleic Acid Amplification Techniques
/
Cytosine
/
Fragile X Syndrome
/
Guanine
Type of study:
Practice guideline
Language:
English
Journal:
Indian J Hum Genet
Year:
2013
Type:
Article
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