An automated DNA sequencer utilizing high sensitivity infrared (IR) fluorescence technology together with Polymerase Chain Reaction (PCR) methodology was used to detect several sex differentiating loci on the X and Y chromosomes from various samples often encountered in forensic case work. Amplifications of the X-Y homologous amelogenin gene, the alpha-satellite (alphoid) repeat sequences and the X and Y chromosome zinc finger protein genes ZFX and ZFY (ZFX/ZFY) were performed. DNA extracted from various forensic specimens was amplified using either Taq, Tth or ThermoSequenase. Multiplexing using primers for all three loci in one reaction tube was achieved using Tth and ThermoSequenase.
Two IR labeling strategies for detection of PCR products were utilized. In the first strategy, one of the PCr primers contained a 19-base extension at its 5′ end identical to an IR-labeled universal M13 Forward (−29) primer which was included in the amplification reactions. During PCR the tailed primer generates sequence complementary to the M13 primer which subsequently primes the initial amplification products, thereby generating IR-labeled PCR products. In the second strategy, dATP labeled with an IR dye (IR-dATP) was included in the amplification reaction. During amplification IR-dATP was utilized by the polymerase and incorporated into the synthesized DNA, thus resulting in IR-labeled PCR products. X and Y specific bands were readily detected using both labeling methodologies.
Amplified products were electrophoretically resolved using denaturing Long-Ranger gels and detected with an automated detection system using IR laser irradiation. A separation distance of 15 cm allowed run times of less than 2 h from sample loading to detection. Because the gels could be run more than once, at least 120 samples (2 loads × 60 samples/load) can be typed using a single gel.
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