Short Tandem Repeat DNA Profiling Using Perylene-Oligonucleotide Fluorescence Assay

Wereport an amplification-free genotyping method to determinethe number of human short tandem repeats (STRs). DNA-based STR profilingis a robust method for genetic identification purposes such as forensicsand biobanking and for identifying specific molecular subtypes ofcancer. STR detection requires polymerase amplification, which introduceserrors that obscure the correct genotype. We developed a new methodthat requires no polymerase. First, we synthesized perylene-nucleosidereagents and incorporated them into oligonucleotide probes that recognizefive common human STRs. Using these probes and a bead-based hybridizationapproach, accurate STR detection was achieved in only 1.5 h, includingDNA preparation steps, with up to a 1000-fold target DNA enrichment.This method was comparable to PCR-based assays. Using standard fluorometry,the limit of detection was 2.00 +/- 0.07 pM for a given target.We used this assay to accurately identify STRs from 50 human subjects,achieving >98% consensus with sequencing data for STR genotyping.