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A Real‐Time Multiplex SNP Melting Assay to Discriminate Individuals
Author(s) -
Nicklas Janice A.,
Buel Eric
Publication year - 2008
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/j.1556-4029.2008.00877.x
Subject(s) - multiplex , snp , computational biology , förster resonance energy transfer , polymerase chain reaction , single nucleotide polymorphism , genetics , biology , microbiology and biotechnology , computer science , fluorescence , genotype , gene , physics , quantum mechanics
A method that quickly and inexpensively differentiates crime scene samples from multiple donors would expedite casework analysis by allowing the selection of probative items requiring comprehensive testing. This new method need not be perfectly definitive nor give a complete 13 locus short tandem repeat (STR) profile; it simply must be able to differentiate between most victim and suspect samples. We describe the development of multiplex, single nucleotide polymorphism (SNP), fluorescence resonance energy transfer‐based real‐time polymerase chain reaction (PCR) assays to fulfill this need. Dual probes, one fluorescently labeled and the other labeled with a quencher, are monitored during a melt analysis to reveal an increase in fluorescence, which allows the assessment of the two SNP alleles. Two alternate 6‐plex assays (with and without gender determination) have been developed for the six‐color RG6000 real‐time instrument (Corbett Robotics, Inc.) and one seven SNP plus gender assay (performed as two 4‐plex assays, one with gender the other without) have been developed for use in four/five color real‐time instruments. This technique can discriminate between 95% and 99% of samples from different individuals. This assay is fast (∼2 h), much less expensive than STR analysis, and uses a real‐time PCR instrument which is found in most forensic and molecular biology labs.