Fluorescence energy transfer‐labeled primers for high‐performance forensic DNA profiling

Abstract

A fluorescence energy transfer (ET) dye-labeled STR typing system (ET 16-plex) is developed for the markers used in the commercial STR typing kit PowerPlex 16, and its performance assessed using a 96-lane microfabricated capillary array electrophoresis (muCAE) system. The ET 16-plex amplicons displayed 1.6-9-fold higher fluorescence intensities compared to those produced using the single-dye (SD)-labeled multiplex kits. The ET multiplex delivered full STR profiles from 62.5 pg of DNA; half the input required for the SD kits while maintaining a similar heterozygote allele balance. This increased sensitivity should improve typing of poor-quality DNA samples by making minor or imbalanced alleles more readily detectable at the low copy number (LCN) threshold. The ET 16-plex also generated complete profiles with only 28 PCR cycles; this capability should improve LCN typing by reducing the amplification time and drop-in allele incidence. To confirm the practical advantages of ET-labeled primers, six previously problematic casework samples were tested and only the ET 16-plex kit was able to capture additional allele data. The successful development and demonstration of ET primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The superior spectral properties and universal compatibility with any primer sequence provided by ET cassettes will make future multiplex construction more facile and straightforward. The pairing of ET cassette technology with the muCAE system illustrates not only an enhanced STR typing platform, but a significant step toward a higher-efficiency forensic laboratory enabled by better chemistry and microfluidics.

Forensic trace DNA: a review

“…Newer dyes, such as Molecular Probes' BODIPY or Alexa Fluor Series, have become commercially available, with the latter being the brightest and most photostable oligonucleotide conjugates available, with average fluorescence greatly exceeding that of even 6-FAM [174]. Energy transfer (ET) cassettes, which contain both donor and acceptor dyes separated by a sugar-phosphate spacer, give substantially higher sensitivities than single dye fluorophores [175,176]. The incorporation of these more sensitive dyes into the commercial STR amplification kits may decrease the amount of template/PCR product by lowering the limit of detection.…”

Microchip‐based forensic short tandem repeat genotyping

“…To address this issue, Yeung et al. used the fluorescence energy transfer (ET) dye labeled primers for STR loci amplification . The ET dye consists of a pair of fluorescent dyes.…”

Recent advances in microfluidic technologies for biochemistry and molecular biology

“…The high analytical efficiencies, fast processing times, reduced consumption of expensive biological reagents and portability make it suitable for a range of applications including diagnostics (46,52,(62)(63)(64)(65)(66)(67)(68)(69)(70), protein and cell-based high throughput drug screening (26,27,71,72) and biomolecule production through directed evolution (20). For example, microfluidic point-of-care diagnostic systems have gained much attention in recent years and proof of concept microfluidic diagnostic devices based on PCR (64,65), DNA/microRNA profiling (66,67), immunoassay (52,70), and protein profiling (69) have all been demonstrated. Soh and Colleagues developed a microfluidic device for genetic analysis of the H1N1 influenza virus from throat swab samples (64).…”