title: Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application.
abstract: BACKGROUND
  Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples.
  
  
  METHODS
  The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep.
  
  
  CONCLUSIONS
  The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively.
  
  
  CONCLUSIONS
  The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.
authors.affiliation: Department of Animal Production, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece.
authors.forename: Androniki
authors.initials: A
authors.lastname: Psifidi
authors.forename: Chrysostomos
authors.initials: C
authors.lastname: Dovas
authors.forename: Georgios
authors.initials: G
authors.lastname: Banos
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D005787:Gene Frequency; D022521:Ligase Chain Reaction / Q000379:methods* / Q000592:standards; D057230:Limit of Detection; D008722:Methods; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep
chemical_list: D005456:Fluorescent Dyes; D004247:DNA