Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples.
Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling. The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming, and labor intensive. We have previously described a method for quantitation of human DNA based on PCR amplification of a repetitive Alu sequence that uses a fluorescence plate reader. This manuscript describes and validates a variation of this assay using real-time PCR and SYBR Green I for quantitation. The advantages of the real-time assay over the plate reader assay are: reduced hands-on time, lower assay cost, and a greater dynamic range. The main disadvantage is the cost of the real-time instrument. However, for those forensic laboratories with access to a real-time instrument, this Alu-based assay has a dynamic range of 16 ng to 1 pg, is sensitive, specific, fast, quantitative, and uses only 2 microL of sample.
Nicklas
Janice A
JA
VT Forensic Laboratory, Department of Public Safety, 103 S. Main St., Waterbury, VT 05676, USA.
Buel
Eric
E
D020087:Alu Elements; D000818:Animals; D001419:Bacteria / Q000235:genetics; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D004330:Drosophila; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology; D005554:Forensic Medicine / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D020080:Tandem Repeat Sequences; D015003:Yeasts / Q000235:genetics
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA