NCJ Number
172051
Journal
Journal of Forensic Sciences Volume: 42 Issue: 3 Dated: (May 1997) Pages: 452-460
Date Published
1997
Length
9 pages
Annotation
An automated DNA sequencer using high-sensitivity infrared (IR) fluorescence technology together with Polymerase Chain Reaction (PCR) methodology was used to detect several gender differentiating loci on the X and Y chromosomes from various samples often encountered in forensic casework.
Abstract
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 used. In the first strategy, one of the PCR primers contained a 19-base extension at its 5-foot end identical to an IR-labeled universal M13Forward (- 29) primer that was included in the amplification reactions. During PCR the tailed primer generates sequence complementary to the M13 primer that 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 used by the polymerase and incorporated into the synthesized DNA, thus resulting in IR-labeled PCR products. X and Y specific bands were readily detected by using both labeling methodologies. Amplified products were electrophoretically resolved by 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 hours from sample loading to detection. Because the gels could be run more than once, at least 120 samples can be typed using a single gel. 5 figures and 31 references