■ Figure 11-8 Short tandem repeat TH01 (repeat unit TCAT) linked to the human tyrosine hydroxylase gene on chromo-some11p15.5. Primers are designed to amplify short regions containing the tandem repeats. Allelic ladders consisting of all alleles in the human population (flanking lanes in the gel shown at bottom right) are used to determine the number of repeats in the locus by the size of the amplicon. The two alleles shown contain 7 and 8 repeats. If these alleles were found in a single individual, that person would be heterozygous for TH01 with a genotype of 7/8.
amplification, each sample PCR product is combined with allelic ladders (sets of fragments representing all possible alleles of a repeat locus) and internal size standards (molecular weight markers) in formamide for elec-trophoresis. After electrophoresis, detection and analysis software will size and identify the alleles. In contrast to RFLPs and VNTRs, STRs are discrete allele systems in which a finite number of alleles are defined by the number of repeat units in the tandem repeat (see Fig. 11-8). Several commercial systems are available consisting of labeled primers for one locus to more than 16 loci. The allelic ladders in these reagent kits allow accurate identification of the sample alleles (Fig. 11-9).
Advances in fluorescence technology have increased the ease and sensitivity of STR allele identification (Fig. 11-10). Although capillary electrophoresis is faster and more automated than gel electrophoresis, a single run through a capillary can resolve only loci whose allele ranges do not overlap. The number of loci that can be resolved on a single run was increased by the use of multicolor dye labels. Primer sets labeled with dyes that can be distinguished by their emission wavelength generate products that are resolved according to fluorescence color as well as size (Fig. 11-11). Test DNA amplicons, allelic ladders, and size standards for multiple loci are thus run simultaneously through each capillary. Genotyping software such as GeneMapper (Applied Biosystems), STaR Call, and FMBIO Analysis Software (Hitachi Software Engineering) provide automated resolution of fluorescent dye colors and genotyping by comparison with the size standards and the allelic ladder.
As in RFLP testing, an STR "match" is made by comparing profiles followed by probability calculations. The AmpliType HLA DQa Forensic DNA Amplification and Typing Kit (Promega) has been used in conjunction with the PM system to generate highly discriminatory allele frequencies. For example, the chance of a set of alleles occurring in two unrelated individuals at random is 1 in 106—7 X 108 Caucasians or 1 in 3 X 106-3 X 108 African Americans.
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