Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application Androniki Psifidi 1 , Chrysostomos Dovas 2 *, Georgios Banos 1 1 Department of Animal Production, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece, 2 Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece 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. Significance: 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. Citation: Psifidi A, Dovas C, Banos G (2011) Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application. PLoS ONE 6(1): e14560. doi:10.1371/journal.pone.0014560 Editor: Katy C. Kao, Texas A & M University, United States of America Received July 16, 2010; Accepted December 20, 2010; Published January 19, 2011 Copyright: ß 2011 Psifidi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Sixth Framework Program of the European Commission, project RISKSCRA, grant number: 030278, FPC-2004-SMW- COLL. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dovas@vet.auth.gr Introduction Single Nucleotide Polymorphisms (SNP) are the most common type of genetic markers and refer to a position where two alternative bases occur at appreciable frequency (.1%) in a population [1]. The identification of SNP is fundamental to basic life science, human hereditary disease diagnosis and pharmacoge- nomics [2,3]. SNP detection is also of great importance to agriculture and animal science, addressing quantitative trait loci (QTL) identification, marker assisted selection, genome mapping, food traceability, parental tests, detection of genetically modified organisms in food and feed, diagnosis of animal genetic diseases, and disease susceptibility. Typically, in genetic diseases caused by mutations in high proportion of DNA, sensitivity of diagnostic assays is not a critical factor for SNP detection. However, in cases where mutated DNA molecules correspond to a small fraction of the wild-type DNA, for example oncogenes or tumour suppressor genes and microbial or viral mutations associated with drug resistance, the sensitivity of SNP detection becomes a critical factor [4]. Estimation of allele frequency is required in a number of applications of great interest that use pooled DNA samples, such as monitoring minimal residual disease in the course of malignant haematopathies and the quantitative assessment of post transplant chimerism. Association studies of non-Mendelian complex diseases have emphasized cost benefits of allele frequency estimation in DNA pools [5] and raised the need for reliable, highly sensitive, fast throughput screening assays. In addition, DNA pooling can reduce the genotyping effort and cost. For example, in milking ruminants, bulk milk (meaning the total amount of milk produced and collected on a given day in a certain farm) has already been used as a source of DNA pooled across all animals raised on the PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e14560