Potent Weapon in Forensic Medicine

Traditionally, one of the most accurate methods for placing an individual at the scene of a crime has been a fingerprint. With the advent of recombinant DNA technology, a more powerful tool is now available: DNA fingerprinting (also called DNA typing or DNA profiling).

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. These variations are restriction fragment length polymorphisms (RFLPs).

The detection of RFLPs relies on a specialized hybridization procedure called Southern blotting (Fig. 1). DNA fragments from digestion of genomic DNA by restriction endonucleases are separated by size electrophoretically, 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 labeled 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, as in Figure 9-9.

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 (see Fig. 24-8). 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. RFLP analysis sensitivity is augmented by using PCR (see Fig. 9-16a) 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 Figure 1, 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.

clones were more than 100,000 bp long, and modern sequencing techniques can resolve only 600 to 750 bp of sequence at a time, each clone had to be sequenced in pieces. The sequencing strategy used a shotgun approach, in which researchers used powerful new automated sequencers to sequence random segments of a given clone, then assembled the sequence of the entire clone by computerized identification of overlaps. The number of clone pieces sequenced was determined statistically so that the entire length of the clone was se-quenced four to six times on average. The sequenced DNA was then made available in a database covering the entire genome. Construction of the physical map was a time-consuming task, and its progress was followed in annual reports in major journals throughout the 1990s— by the end of which the map was largely in place. Completion of the entire sequencing project was initially projected for the year 2005, but circumstances and technology intervened to accelerate the process.

A competing commercial effort to sequence the human genome was initiated by the newly established Celera Corporation in 1997. Led by J. Craig Venter, the Celera group made use of a different strategy called "whole genome shotgun sequencing," which eliminates the step of assembling a physical map of the genome. Instead, teams sequenced DNA segments from through-

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.

FIGURE 1 The Southern blot procedure, as applied to DNA fingerprinting. This procedure was named after Jeremy Southern, who developed the technique.

Chromosomal DNA (e.g., Suspect 1)

Chromosomal DNA (e.g., Suspect 1)

Cleave with restriction endonucleases.

DNA fragments

Separate fragments by agarose gel electrophoresis (unlabeled).

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Radiolabeled ^lyi DNA probe

Incubate with probe, then wash.

Expose x-ray film to membrane.

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