title: Polymerase chain reaction optimization for amplification of Guanine-Cytosine rich templates using buccal cell DNA.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  Optimize a cost effective PCR assay to amplify the GC-rich DNA templates.
  
  
  METHODS
  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.
  
  
  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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, Sri Lanka.
authors.forename: C H W M R Chandrasekara
authors.initials: CH
authors.lastname: Bhagya
authors.forename: W S
authors.initials: WS
authors.lastname: Wijesundera Sulochana
authors.forename: N Perera
authors.initials: NP
authors.lastname: Hemamali
keywords: Enhancers; fragile X syndrome; guanine-cytosine-rich sequences; polymerase chain reaction; polymerase chain reaction additive
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