The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA

Abstract

A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples.

Mutant DNA Quantification by Digital PCR Can Be Confounded by Heating During DNA Fragmentation

“…Current DNA fragmentation methods include double-strand breakage by heating (9), site-directed restriction enzyme digestion (4), digestion with an enzyme that generates random double-strand breaks (e.g., Fragmentase) (10), acoustic shearing based on focused-ultrasonication (e.g., using the Covaris focused-ultrasonicator), fragmentation with a biopolymer-based shredding system (QIAshredder, Qiagen) (11), and fragmenting by electromagnetic energy from microwave irradiation (12). Of these methods, high temperature is the simplest and least expensive approach.…”

Optimization of STR locus enrichment for STR profiling of fragmented DNA

“…Magnetic bead hybridization was shown to have potential to be applied to the recovery and STR amplification of degraded forensic DNA . Target STR DNA sequence was enriched from fragmented genomic DNA by hybridization of an allele‐specific biotinylated probe and streptavidin‐coated magnetic particles.…”

“…Another strategy to increase the success of STR typing of degraded samples is the enrichment of specific STR loci before DNA profiling analysis . Approximately 330 copies of each STR locus are present in 1 ng of genomic DNA.…”

“…Approximately 330 copies of each STR locus are present in 1 ng of genomic DNA. If some of the STR loci are intact in degraded samples, the intact STR loci could be enriched via hybridization of degraded genomic DNA with locus‐specific biotinylated oligonucleotide, which can be collected with streptavidin‐coated magnetic particles . This paper will present the details of experimental conditions to improve the efficiency of recovery of alleles from artificially degraded DNA and degraded forensic samples, and discuss considerations on the use and limitations of allele enrichment for increasing success in STR typing.…”

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics

“…These techniques can be generally divided into two categories: (1) novel DNA extraction procedures to purify the target DNA prior to PCR amplification; and (2) modification of the DNA amplification process to minimize the effect of inhibitors that are not removed. The pre-PCR removal methods produce PCR-compatible DNA samples using silica-based extraction [9][10][11][12][13], magnetic separation [14][15][16], column chromatography [17][18][19], and other new protocols [20,21]. Mitigation of inhibition during PCR can also be achieved by extensive dilution of DNA extracts [22,23], utilization of genetically engineered DNA polymerases with better resistance to inhibitors (e.g.…”