DNA fingerprinting using multilocus probes MLPs

Roughly 3% of the human genome is comprised of tandem repeats (International Human Genome Sequencing Consortium, 2001) that - with the exclusion of satellite DNA - can be classified into two groups according to the size of the repeat unit and overall length of the repeat array. Human minisatellites or VNTR loci have repeat units ranging in length from 6 bp to more than 100 bp depending on the locus, with arrays usually kilobases in length. Human GC-rich minisatellites are preferentially found clustered in the recombination-proficient subtelomeric regions of chromosomes (Royle et al., 1988). Some minisatellite loci show very high levels of allele length variability.

Molecular Forensics. Edited by Ralph Rapley and David Whitehouse Copyright 2007 by John Wiley & Sons, Ltd.

The accidental discovery of hypervariable minisatellite loci detectable with MLPs in 1984 by Sir Alec Jeffreys launched a new age in forensic investigation (Jeffreys et al., 1985a). These minisatellites were detected by hybridization of probes to Southern blots of restriction-enzyme-digested genomic DNA, to reveal restriction fragment length polymorphisms (RFLPs). A common 10-15 bp 'core' GC-rich sequence shared between different minisatellite loci allowed MLPs to detect a wide array of minisatellites simultaneously, producing multi-band (barcode-like) patterns known as 'DNA fingerprints' (Figure 5.1).

Using only a single MLP designated 33.15, the match probability between unrelated individuals was estimated at <3 x 10-11, and in the case of two MLPs (33.15 and 33.6) that detect different sets of minisatellites the match probability is <5 x 10-19 (Jeffreys et al., 1985b). These probabilities are so low that the only individuals possessing identical DNA fingerprints are monozygotic twins. The

Figure 5.1 Examples of DNA fingerprint autoradiographs using multi-locus probes 33.6 and 33.15 obtained from a paternity test. The barcode-like patterns of mother (lane 1) and child (lane 2) are compared with two alleged fathers (lanes 3 and 4). One of the alleged fathers (lane 3) has not been excluded as the biological father because he possesses all the similar-sized bands inherent in the child that were not inherited from the mother. The remaining candidate (lane 4) was excluded from paternity due to a lack of common bands with the child

MLPs have proven their use in paternity testing (Jeffreys et al., 1991a) and immigration cases (Jeffreys et al., 1985c). However, a significant drawback associated with MLPs is the requirement for several micrograms of high-quality genomic DNA in order to obtain reliable DNA fingerprints. Because forensic specimens are often old and yield small quantities of degraded DNA, MLPs are not generally suitable for forensic analysis, although they were successfully implemented in a number of early criminal investigations (Gill and Werrett, 1987).

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