12 Apr


Detection of STR polymorphisms
After STR polymorphisms have been amplified using PCR, the length of the products must be measured precisely – some STR alleles differ by only one base pair. Gel electrophoresis of the PCR products through denaturing polyacrylamide gels can be used to separate DNA molecules between 20 and 500 nucleotides long with single base pair resolution [29]. Early systems detected the PCR products after electrophoresis on polyacrylamide slab-gels using silver staining [30, 31] but this limited the number of loci that could be incorporated into the multiplexes because the allelic size ranges of the different loci could not overlap. To overcome this limitation, fluorescence labelling of PCR products followed by multicolour detection has been adopted by the forensic community. A series of fluorescent dyes has been developed that can be covalently attached to the 5′ end of one of the PCR primers in each primer pair and detected real-time during electrophoresis. Up to five different dyes can be used in a single analysis which allows for considerable overlap of loci (Figure 6.3). The electrophoresis platforms have evolved from systems based on slab-gels to capillary electrophoresis (CE) that use a narrow glass tube filled with an entangled polymer solution to separate the DNA molecules [32-36]. Applied Biosystems provide the most commonly used

Figure 6.3 PCR multiplexes use up to five different dyes to label PCR products. The allelic ranges of three commonly used multiplexes, the AmpFlSTR® SGM Plus®, AmpFlSTR® Identifiler® and the PowerPlex® 16 are shown. The use of multiple dyes allows the detection of the internal-lane size standard (ROX in SGM Plus®, LIZ in Identifiler® and CXR in PowerPlex® 16) and three to four overlapping STR loci, where the use of different dyes allows the alleles to be assigned to the correct locus

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