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DNA fingerprinting is based on sequence polymorphisms, slight sequence differences (usually single base-pair changes) between individuals, 1 bp in every 1,000 bp, on average.
Each difference from the prototype human genome, sequence (the first one obtained) occurs in some fraction of the human population; every individual has some differences.
Some of the sequence changes affect recognition sites for restriction enzymes, resulting in variation in the size of DNA fragments produced by digestion with a particular restriction enzyme.
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These variations are restriction fragment length polymorphisms (RFLPs). The detection of RFLPs relies on a specialized hybridization procedure called Southern blotting. DNA fragments from digestion of genomic DNA by restriction endonucleases are separated by size electrophoretic-ally, denatured by soaking the agarose gel in alkali, and then blotted onto a nylon membrane to reproduce the distribution of fragments in the gel.
The membrane is immersed in a solution containing a radioactively labelled DNA probe. A probe for a sequence that is repeated several times in the human genome generally identifies a few of the thousands of DNA fragments generated when the human genome is digested with a restriction endonuclease. Autoradiography reveals the fragments to which the probe hybridizes.
The genomic DNA sequences used in these tests are generally regions containing repetitive DNA (short sequences repeated thousands of times in tandem), which are common in the genomes of higher eukaryotes. The number of repeated units in these DNA regions varies among individuals (except between identical twins).
With a suitable probe, the pattern of bands produced by DNA fingerprinting is distinctive for each individual. Combining the use of several probes makes the test so selective that it can positively identify a single individual in the entire human population. However, the Southern blot procedure requires relatively fresh DNA samples and larger amounts of DNA than are generally present at a crime scene.
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RFLP analysis sensitivity is augmented by using PCR to amplify vanishingly small amounts of DNA. This allows investigators to obtain DNA fingerprints from a single hair follicle, a drop of blood, a small semen sample from a rape victim, or samples that might be months or even many years old.
These methods are proving decisive in court cases worldwide. In the example in Fig. 4.21, the DNA from a semen sample obtained from a rape and murder victim was compared with DNA samples from the victim and two suspects. Each sample was cleaved into fragments and separated by gel electrophoresis. Radioactive DNA probes were used to identify a small subset of fragments that contained sequences complementary to the probe.
The sizes of the identified fragments varied from one individual to the next, as seen here in the different patterns for the three individuals (victim and two suspects) tested. One suspect’s DNA exhibits a banding pattern identical to that of a semen sample taken from the victim. This test used a single probe, but three or four different probes would be used (in separate experiments) to achieve an unambiguous positive identification.
Such results have been used to both convict and acquit suspects and, in other cases, to establish paternity with an extraordinary degree of certainty. The impact of these procedures on court cases will continue to grow as societies agree on the standards and as formal methods become widely established in forensic laboratories. Even decades-old murder mysteries can be solved: in 1996, DNA fingerprinting helped to confirm the identification of the bones of the last Russian czar and his family, who were assassinated in 1918.
