October 1995: DNA Goes to Court

RFLP Technology

Currently, the most powerful DNA profiling technique is the RFLP (restriction fragment length polymorphism) method illustrated in Figure 2. At many crime scenes, sufficient quantities of DNA can be recovered from dried blood spots or semen samples to make this analysis possible. DNA is extracted from the evidence and digested with a restriction enzyme. Fragments are separated by gel electrophoresis, denatured, and transferred onto a nitrocellulose membrane by a process called Southern blotting. The membrane is bathed in a solution containing a radioactively labeled "probe" or single-stranded DNA molecule containing VNTR locus sequences. Probe molecules hybridize with complementary sequences in the immobilized fragment, which can be visualized on X-ray film. Forensic investigations commonly use probes for four or five different loci on a single sample to improve the validity of the test. However, one probe must be completely stripped from the membrane before the next one is applied. The entire process may take two months to complete; a nerve-wracking time for defendant, prosecutors and defense attorneys.

The final DNA fingerprint is a pattern on X-ray film of light and dark bands similar to the bar codes found on retail goods. Homozygotes will show one dark band rather than two lighter ones since they have received the same DNA sequence from each parent. The two-band pattern for a heterozygote is illustrated in Figure 1.

Although these techniques are standard practice in many laboratories, great care must be taken in carrying out DNA typing tests. Forensic samples of DNA are rarely pure. DNA from bacteria or fungi may show up in the fingerprint; dyes from denim can interfere with restriction enzymes; and proteins in the evidence sample can retard the migration of DNA fragments in gels, a problem known as band shift.

PCR Technology

If the forensic sample is too minuscule for RFLP testing, or if the DNA is degraded, the polymerase chain reaction (PCR) can be used to obtain a DNA profile. PCR is a molecular copying process used to amplify specific DNA sequences. With this technique it is possible to obtain enough DNA from a single hair follicle or a single sperm cell to determine an individual's DNA profile. The remarkable sensitivity of PCR is the procedure's main advantage. Beginning with a single molecule of DNA, thirty cycles of PCR can generate 100 billion molecules in one afternoon.

PCR can be used to selectively amplify DNA fragments containing either length or sequence polymorphisms. Sequence polymorphisms, such as occur within the genes of the highly polymorphic HLA complex, are the result of single nucleotide base changes. Length polymorphisms are exemplified by the variable number of tandem repeat loci (VNTR). Length variation at a given VNTR locus is detected by size-fractionation of PCR products in gels. PCR-amplified DNA products can be directly visualized by staining after electrophoresis, eliminating the need for radioactive probes.

Calculating the Odds

If the DNA profile of crime scene evidence does not match the profile of the suspect, the suspect is completely exonerated. DNA techniques have proven extremely useful in excluding suspects. The FBI finds exclusions in about 30 percent of the comparisons it carries out, making DNA-typing a major source of protection for the innocent.

Figure 2 RFLP method: steps to a genetic signature.

However, the converse is not true with absolute certainty. Thus, if DNA profiles from the evidence and a suspect are judged to match, the strength of this evidence is measured by a "match probability," the likelihood that an individual chosen randomly from an appropriate population will match the crime profile. A good deal of controversy has centered on the methods for calculating this match probability.

Our DNA comprises about 3 billion base pairs and, with the exception of identical twins, the DNA of any two individuals differs greatly, probably by over a million base differences. There is, therefore, sufficient information in the DNA to identify the culprit unequivocally from a DNA sample recovered from a crime scene. However, DNA typing methods analyze only a tiny fraction of all the potentially variable sequences within the genome. Thus, DNA typing does not identify an individual, but rather results in a high probability of identification.

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