Amplified fragment length polymorphisms AFLPs

Some minisatellite loci with relatively short (-1000 bp) alleles can be amplified via polymerase chain reaction (PCR) to yield AFLPs. Before PCR-based typing of microsatellite loci was established, one such minisatellite locus called D1S80 (Nakamura et al., 1988; Kasai et al., 1990) was used extensively within the realm of forensic DNA analysis. (Figure 5.3a). In the D1S80 system, fragments between the range of 14-42 repeat units (16 bp per repeat) are amplified to yield alleles considerably smaller than the fragments normally analysed in DNA fingerprinting and profiling.

In contrast to RFLP analysis, D1S80 alleles fall into discrete size classes and thus can be compared directly to a standard composite of most alleles (an allelic ladder) on the same gel (see Figure 5.3a). This was a significant improvement that was also employed in subsequent STR systems. In D1S80, the amplified DNA fragments were commonly detected using a silver stain, with the final profile usually showing two bands from which the numbers of repeat units can be easily estimated. Although the D1S80 locus in particular contains four common alleles with frequencies of >10% in Japanese people, the likelihood of discrimination between two unrelated individuals is still high (0.977 in Japanese people; Member of TWGFDM, 1995).

This has been the preferred locus for analysing forensic specimens worldwide. In addition to this locus, PCR-based methods utilizing commercial kits that

Figure 5.3 Detection of MCT118 (D1S80). (a) Agarose gel electrophoresis followed by silver-staining. Top figures indicate bloodstain (lane 1), victim (lane 2) and suspect (lane 3). The allelic ladder marker (AL) is displayed with the number of repeats indicated to the left. Positive and negative controls are expressed in lanes P and N, respectively. (b) Fluorescent labeled primer and capillary electrophoresis. Figures to the left indicate sample numbers, and AL, PC and NC represent the allelic ladder, positive control and negative control, respectively. The upper numbers below the peaks in each sample indicate the number of repeats and the lower numbers indicate the peak intensity exploit single nucleotide polymorphisms (SNPs), including HLA-DQa and the five PolyMarker loci (LDLR, GYPA, HBGG, D7S8 and Gc), were popularly used in the 1990s. Recently, the National Institute of Police Science (Japan) developed a novel genotyping method that involves PCR and fluorescent-labelled primers detected by capillary electrophoresis (Figure 5.3b). This method has allowed for higher accuracy since the results are not prone to human error.

However, after the detailed evaluation of allele frequency data in the world population at D1S80 (Peterson et al., 2000) and increasing practical usage of multiplex STR systems, D1S80 has been gradually abandoned in favour of STRs.

For Forensic and Research Use Only

Figure 5.3 (b) Continued

For Forensic and Research Use Only

Figure 5.3 (b) Continued

Was this article helpful?

0 0

Post a comment