title: Different real time PCR approaches for the fine quantification of SNP's alleles in DNA pools: assays development, characterization and pre-validation.
abstract: Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5 % to 95 % and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1 % for both the approaches, allowing to extend the range of quantifications from 1 % to 99 %. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.
authors.affiliation: Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy.
authors.forename: Elia
authors.initials: E
authors.lastname: Mattarucchi
authors.forename: Milena
authors.initials: M
authors.lastname: Marsoni
authors.forename: Giorgio
authors.initials: G
authors.lastname: Binelli
authors.forename: Alberto
authors.initials: A
authors.lastname: Passi
authors.forename: Francesco
authors.initials: F
authors.lastname: Lo Curto
authors.forename: Francesco
authors.initials: F
authors.lastname: Pasquali
authors.forename: Giovanni
authors.initials: G
authors.lastname: Porta
mesh_terms: D000483:Alleles; D004247:DNA; D006801:Humans; D025202:Molecular Diagnostic Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA