DNA Sex Test: A New Rapid and Quantitative Forensic Approach Using Amelogenin Gene Based Fluorescent PCR

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Abstract

For forensic PCR applications, it is recommended that the investigation of X and Y sequences should be carried out in parallel or simultaneously, and the distinction of male and female DNA cannot be made based solely on the absence of a band [1]. Several PCR-based tests have been developed for gender identification, among them multicopy repeat sequences on the X and Y chromosomes can be amplified together and provide a highly sensitive assay but quantitation of the relative X/Y product is not possible because of significant differences in repeat copy number [2–4]. Alternatively, single copy X-Y homologous regions such as amelogenin offer the advantage of requiring only one pair of primers and both X and Y sequences are of equal copy number [5]. Primers described by Sullivan and coll. flank a 6bp deletion within intron 1 of the X homologue resulting in 106bp and 112bp PCF, products from the X and Y chromosomes respectively [6]. Dye labelled PCR products were generated using one primer coupled with fluorescent dye ‘FAM’ via a 5’ aminolinker. These primers allowed DNA samples ranging from 10pg to 100ng to be amplified through 35 cycles comprising 1 minute at 94°C, 1 minute at 60°C and 1 minute at 72°C in a Perkin Elmer 480 thermal cycler. PCR products (20 μl) were visualised simply after electrophoresis in a 4% agarose gel for 90 mins at 100V and by ethidium bromide staining.