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Real-Time Multiplex SNP Melting Assay to Discriminate Individuals

NCJ Number
Journal of Forensic Sciences Volume: 53 Issue: 6 Dated: November 2008 Pages: 1316-1324
Date Published
November 2008
9 pages

This article describes the development of multiplex, single nucleotide polymorphism (SNP), fluorescence resonance energy transfer-based (FRET) real-time polymerase chain reaction (PCR) assays as a method of quickly and inexpensively differentiating crime-scene samples from multiple donors, so as to expedite casework analysis by allowing the selection of probative items that require comprehensive testing.


These multiplex FRET assays, using quenching probes, can determine the genotype of an individual for five to seven SNPs plus gender determination in a fast (2 hours), accurate manner, using a real-time PCR instrument. The 6-plex A assay was sensitive, giving correct typings at comparable template amounts to that required for STR analysis using standard typing kits. The assay did fail to give the correct genotype for heterozygotes at low template amounts, detecting only one or no alleles. One allele may still be helpful as a screening procedure when the data are meant only to sort between a limited number of individuals. This SNP assay is the first report of testing that used six SNPs in one multiplex assay in a real-time instrument. Other real-time methodologies such as TaqMan, can perform three assays at most in a multiplex, because of the limit of using two colors (dye channels) per SNP--one color for each allele. The DNA samples used in this study were mostly in-house controls used for STR testing in the laboratory (lab personnel) or DNA samples from convicted offenders. In addition to the DNA samples, the descriptions of methods and materials address the FRET hybridization probe quenching melting assay to detect SNPs; SNP selection and primer design; the real-time multiplex SNP assay; the real-time quantization assay; and sequencing. 5 tables, 5 figures, 35 references, and appended SNP identity percentage calculation, the sharing of SNP genotypes by the same-gender siblings, and the sharing of SNP genotypes by the same-gender parent and child

Date Published: November 1, 2008