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3 Forensic Biology (continued) + ward ' s science DNA Separation and Detection Separation and detection of the amplified products is required following PCR. There are many different methods to achieve typing. These include agarose gel electrophoresis (Fig. 2); polyacrylamide gel electrophoresis (PAGE) followed by silver staining or, if the primers are fluorescently tagged, detection by fluorescent gel scanners; and capillary electrophoresis (CE) with laser-induced fluorescence. The latter method has become the most commonly used method of detection, as it is highly auto- mated (there is no gel to pour and load), samples can be easily be reinjected (robotically), and since the DNA traverses the entire length of the capillary, the resolution of the higher-mo- lecular-weight loci is usually better than in the PAGE methods. Forensic Genetic Markers The genetic markers currently being typed in most forensic biology laboratories include autosomal short tandem repeats (STRs), mitochondrial DNA, and Y chromosome STRs. Short Tandem Repeats Short tandem repeats consist of regions of 2–7 base pairs that are repeated consecutively. Individuals may vary in the number of repeats and/or the content of the repeats. The variation in the content of the repeats occurs in either a change in the base within a repeat unit or as a deletion in the repeat unit. STRs used in forensics are either tetranucleotide or pentanucleo- tide repeats. STRs are highly abundant and well-studied in the human genome, and their small size and the alleles' small size range facilitate typing from highly degraded, small quantities of starting material. Twenty CODIS core loci have been upload- ed into the national DNA database. Mitochondrial DNA Mitochondrial DNA (mtDNA) is useful for forensic DNA in that it exists in high copy numbers in each cell, and therefore has a better chance of being detected in small samples. Moreover, mtDNA is maternally inherited, so any individual within the maternal lineage may provide an mtDNA reference sample. Finally, since the size of the amplicons (the DNA segments to be generated by PCR) is small, mtDNA can be typed from degraded DNA. Mitochondrial DNA hypervariable regions I and II are the most commonly sequenced targets in forensic DNA laboratories. The most commonly used method of mtDNA typ- ing is fluorescent Sanger's dideoxy sequencing. Y Chromosome Markers Y chromosome markers, including Y STRs, have been recently used in many forensic DNA crime laboratories. The interest in Y chromosome markers is well supported for the following reasons: 1. The total number of male cells that are present at a rape scene may be very small in the case of rapists who are azoospermic (having no sperm) or oligospermic (having a low sperm count). 2. The total number of male cells may be low due to loss of sample or degradation. 3. Multiple semen donors may need to be identified in a multiple rape case. Fig. 2: Agarose gel electrophoresis apparatus. When an electrical field is applied across trough containing agarose gel, DNA migrates toward the positively charged (anode) terminal (red wire). (Credit: iStock.)