Different Real Time PCR Approaches for the Fine Quantification of SNP's Alleles in DNA Pools: Assays Development, Characterization and Pre-validation
2005
Mattarucchi, Elia (University of Insubria, Varese, Italy) | Marsoni, Milena (University of Insubria, Varese, Italy) | Binelli, Giorgio (University of Insubria, Varese, Italy) | Passi, Alberto (University of Insubria, Varese, Italy) | Curto, Francesco Lo (University of Insubria, Varese, Italy) | Pasquali, Francesco (University of Insubria, Varese, Italy) | Porta, Giovanni (University of Insubria, Varese, Italy), E-mail: [email protected]
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.
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