Fluorescence Imaging in Human Identity Testing

We have investigated the use of fluorescence detection and the FluorImager S1 System (Molecular Dynamics) for analyzing a comprehensive set of human DNA typing tests. We used an alkaline phosphatase-conjugated YNH24 oligonucleotide probe to the repeat-containing D2S44 locus to detect both alleles in 50 ng of human genomic DNA (0.025 amol) by Southern hybridization using a chemifluorescent substrate. We used a similar approach to quantify human DNA using an enzyme-conjugated oligonucleotide probe to the D17Z1 locus. Both fluorescent nucleic acid gel staining and direct fluorescent labeling methods were tested to detect PCR-based D1S80 and short tandem repeat (STR) multiplex allele profiles. The fluorescent staining method sensitively detected these allelic profiles in both denaturing and non-denaturing acrylamide gels using a simple, 10-min procedure. Fluorescent primers eliminate the doublet band patterns often seen with staining methods, which label both strands of the amplified products. This complicates interpretation of STR typing tests. Only one primer for each locus is labeled, so only one strand of the DNA product is detected. Fluorescein end-labeled primers were used in multiplex PCR to amplify, detect and type STRs.