title: A rapid screening method using DNA binding dyes to determine whether hair follicles have sufficient DNA for successful profiling.
abstract: We report a simple screening method to assess the viability of successful DNA profiling from single hair follicles. A total of 48 hair samples (shed and plucked) were collected from male and female donors and the root tips (0.5cm) were stained using one of three DNA binding dyes (EvaGreen™, Diamond™ Nucleic Acid Dye and RedSafe™) at 20× concentration. The hairs were subsequently viewed under a Nikon Optiphot fluorescent microscope to count the approximate number of nuclei in one plane of view. The hairs were then processed using either (1) a DNA extraction kit (QIAmp(®) Mini Kit) and then amplified using the AmpFLSTR(®) NGM™ kit, which amplifies 15 short tandem repeat (STR) loci plus the gender marker amelogenin, or (2) by direct PCR amplification using the same DNA profiling kit. Diamond™ dye had the lowest background signal and plucked hairs treated with this dye produced full DNA profiles when amplified directly and was chosen to screen a further 150 mixed hair samples. These hairs were separated into one of five categories (1, >100 nuclei; 1.5, 50-99 nuclei; 2, 1-49 nuclei; 2.5, no nuclei but high fluorescent signal; 3, no nuclei and very low fluorescent signal) from which 60 of the hairs were chosen to undergo direct amplification using the NGM™ kit. It was found that there was a direct correlation to the category designation and the ability to obtain a DNA profile up-loadable to the Australian DNA Database. Approximately 91% of category 1 hairs resulted in either a full or high partial (12-29 alleles) profile by direct PCR whereas about 78% of category 3 hairs exhibited no amplification. The results show that this method can be used to predict successful STR amplification from single hair follicles. It is a rapid, sensitive, cheap, non-destructive and easy to perform methodology applicable for screening multiple hairs in order to aid forensic investigators in predicting hairs that will yield DNA results.
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, Australia. Electronic address: alicia.haines@flinders.edu.au.
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Haines
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, Australia.
authors.forename: Adrian
authors.initials: A
authors.lastname: Linacre
mesh_terms: D053523:Amelogenin / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D016172:DNA Fingerprinting / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry*; D018859:Hair Follicle / Q000737:chemistry*; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats; D008853:Microscopy; D016133:Polymerase Chain Reaction
chemical_list: D053523:Amelogenin; D005456:Fluorescent Dyes; D004247:DNA
keywords: Diamond™ Nucleic Acid Dye; Fluorescence microscopy; Forensic evidence recovery; Hair follicles; STR analysis