DNA Fingerprinting Depends on Differences in Length of Simple-Sequence DNAs
Within a species, the nucleotide sequences of the repeat units composing a simple-sequence DNA tandem array are highly conserved among individuals. In contrast, the number of repeats, and thus the length of simple-sequence tandem arrays containing the same repeat unit, is quite variable among individuals. These differences in length are thought to result from unequal crossing over within regions of simple-sequence DNA during meiosis. As a consequence of this unequal crossing over, the lengths of some tandem arrays are unique in each individual.
In humans and other mammals, some simple-sequence DNA exists in relatively short 1–5-kb regions made up of 20–50 repeat units, each containing 14–100 bp. These regions are called minisatellites, in contrast to microsatellites made up of tandem repeats of 1–13 bp. Even slight differences in the total lengths of various minisatellites from different individuals can be detected by Southern blotting (see Figure 6-24). This technique was exploited in the first application of DNA fingerprinting, which was developed to detect DNA polymorphisms (i.e., differences in sequence between individuals of the same species) (Figure 8-7). Today the far more sensitive polymerase chain reaction (PCR) technique (see Figure 6-18) is generally used in forensic genetic testing. Microsatellites consisting of tandem repeats of four bases in 30–50 copies are usually analyzed today. The exact number of repeats at a specific location in the genome generally varies between the two homologous chromosomes of an individual (one inherited from the mother and one from the father) and between the Y chromosomes of different males. A mixture of pairs of PCR primers that hybridize to unique sequences flanking 13 of these short tandem repeats and a Y-chromosome short tandem repeat are used to amplify DNA in a sample from one individual. The resulting mixture of PCR product lengths is unique in the human population, except for identical twins. The use of PCR allows analysis of minute amounts of DNA, and individuals can be distinguished more precisely and reliably than by conventional fingerprinting.
FIGURE 8-7 Distinguishing individuals by DNA fingerprinting. (a) In this analysis of paternity, several minisatellite repeat lengths were determined by Southern blot analysis of restriction enzyme–digested genomic DNA and hybridization with a probe for a sequence shared by several minisatellite sequences. This method generated hypervariable multiband patterns for each individual called “DNA fingerprints.” Lane M shows the pattern of restriction fragment bands using the mother’s DNA; C, using the child’s DNA; and F1 and F2 using DNA from two potential fathers. The child has minisatellite repeat lengths inherited from either the mother or F1, demonstrating that F1 is the father. Arrows indicate restriction fragments from F1, but not F2, found in the child’s DNA. (b) In these “DNA fingerprints” of a specimen isolated from a rape victim and three men suspected of the crime, it is clear that minisatellite repeat lengths in the specimen match those of suspect 1. The victim’s DNA was included in the analysis to ensure that the specimen DNA was not contaminated with DNA from the victim.