BIOLOGY Volume 3 - A Guide to General Biology - 2004

25. APPLIED GENETICS

25.7. Human Genetics

25.7.12. Genetic Fingerprinting and Genotyping

The technique of genetic fingerprinting was developed by Alec Jeffreys and his colleagues in 1984 at the University of Leicester. A later and more sensitive variation of this method is known as genotyping. The method gained widespread public recognition through its use in high-profile criminal cases, such as the O. J. Simpson trial in the United States in 1995.

The Human Genome contains about 100,000 genes (according to recent data, significantly fewer). Genes encode Proteins, yet about 95% of DNA is non-coding. The function of such DNA is not yet fully understood, but it may be involved in establishing the proper Structure of Chromosomes within The Cell. About 30–40% of this DNA consists of short base sequences that are repeated many times. Some of these repeated sequences are scattered throughout the DNA, while others are clustered together, or in other words, arranged in tandem. Such "tandem repeats" are called satellite DNA (they were first isolated as a distinct DNA fraction following centrifugation). Each cluster of repeated sequences is referred to as a satellite. The number of repeats in satellites varies. In some of them (known as minisatellites), the sequences are repeated only a few times. Individuals exhibit enormous diversity in the number of repeats of these short sequences (making them "hypervariable"). Therefore, minisatellites are sometimes designated as VNTR (variable number tandem repeats). Each individual possesses two allelic minisatellites at a given locus: one inherited from the mother and one from the father. Genetic fingerprinting is the Analysis of the lengths of these minisatellite sequences in a given individual.

Procedure

DNA is extracted from Cells and treated with a restriction enzyme. Agarose gel Electrophoresis is used to separate the resulting fragments; the fragments are thereby sorted by size, as already explained in Section 25.1 and shown in Fig. 25.4. The subsequent procedure is described in Section 25.7.9 and illustrated in Fig. 25.34. Southern blotting is used to transfer the DNA onto a nitrocellulose or nylon filter. The DNA is then hybridized with a radioactive DNA probe, which is a sequence complementary to the repetitive minisatellite sequence, and the fragments bound to the probe are identified by autoradiography.

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Fig. 25.34. Genetic fingerprinting.

As already noted, minisatellite sequences vary in length among different individuals. The probe binds to minisatellites, which appear in different positions on the filter for different individuals because their lengths differ. For each individual, the resulting pattern (the distribution of minisatellites) is characteristic and is therefore called a fingerprint (Fig. 25.34). If a probe recognizes and binds to several types of minisatellites across The Genome, it is called a multilocus probe, and it will produce numerous bands on the autoradiograph. The more bands there are, the more unique the pattern. The probability of a random match for four bands between two people is approximately 1 in 250, whereas for 20 bands it is much lower—1 in 10¹² (there are about 4.5 · 10⁹ people on Earth).

Multilocus probes yield the best results when using pure, uncontaminated DNA. However, forensic scientists rarely work with fresh, high-quality material. As a rule, samples are contaminated with substances such as soil or Bacteria. A more "powerful" method is often required. This is achieved using a single-locus probe. Such a probe can be used with small pieces of DNA (for example, partially degraded DNA) as well as with minute quantities of material. The probe recognizes only a single short repetitive sequence that is unique to one minisatellite and is therefore found only on a specific pair of homologous chromosomes. Restriction Digestion produces two characteristic fragments for each individual, and consequently, two bands will appear on the autoradiograph, one of maternal and one of paternal origin (Fig. 25.35). If two single-locus probes are used, four bands will appear; if three, six bands, and so on. If a very high degree of certainty is required in distinguishing between two individuals, A large number of probes must be used (see below). This method is called genotyping, and the resulting profile is the one most frequently used in forensic science.

The method can be made even more sensitive by amplifying The amount of DNA using the Polymerase Chain Reaction (PCR). This means that a DNA profile can be obtained from an extremely small sample of DNA (for example, a piece of a postage stamp licked by a suspect and punched out with a hole punch). Anyone who has left a flake of dandruff or sneezed at a crime scene can become a suspect!

Fig. 25.35. Inheritance of minisatellites and the resulting diversity of DNA profiles. Selection/30.html">The population as a whole is characterized by enormous diversity in minisatellite lengths, so the probability that two individuals will have identical DNA profiles is extremely small (see text).

Applications

Genetic fingerprinting plays the same revolutionary role in modern legal proceedings that standard fingerprinting played in the early 1900s. DNA is usually extracted from small amounts of biological material found at a crime scene, such as bloodstains, Hair, or saliva. Semen is used in cases of rape (Fig. 25.36). Paternity testing has become a routine procedure. Fig. 25.37 shows an example of using DNA profiles to resolve a disputed paternity case.

Fig. 25.36. Genetic fingerprinting of the victim's Blood, semen (sample), and the suspects' blood.

Fig. 25.37. DNA profiles of individuals involved in a paternity dispute (M — mother, C — child, A — alleged father).

25.8. Look at Fig. 25.37. Which of the mother's children is the son or daughter of the alleged father?

Genetic fingerprinting was first used in forensic science in the UK in 1986. In 1983, a schoolgirl was found raped and murdered in a village near Leicester; a second body was found in 1986. The suspect confessed to the second crime, but the police suspected that he was also responsible for the 1983 crime. The police asked Dr Jeffreys of the University of Leicester to perform DNA fingerprinting on semen samples collected from the two crime scenes and a blood sample from the suspect. The analysis showed that the man was innocent of both crimes! All local men (about 1500 individuals) were tested, but again without a positive match. Eventually, the murderer was caught As a result of an overheard conversation in a pub. DNA fingerprinting confirmed his guilt. However, in approximately 30% of cases, genetic fingerprinting fails to yield a match between the tested DNA and the DNA profile found at the crime scene.

Reliability and Validity

Reliability refers to the consistency of obtaining the same result upon repeated testing. For example, would two different laboratories produce the same result using the same sample? To improve reliability, measures are being implemented across Europe to standardize the procedure. This is crucial for international criminal investigations, such as those involving terrorism and drug trafficking.

By validity, it is meant that a given test satisfies all required standards. In judicial proceedings, the greatest concern arises from the fact that two individuals may share the exact same profile or DNA fingerprint. Court cases concerning this issue have occasionally become headline news. Jeffreys calculated that two people would exhibit the same band using a multilocus probe in approximately one out of four cases. Consequently, the probability is one chance in 4ⁿ, where n is the number of bands. Thus, There is a 1 in 256 chance that two people will share 4 identical bands, and less than a 1 in 10¹² chance that they share 20 to 30 identical bands (the typical number of bands in a standard DNA fingerprint). However, it should be kept in mind that forensic experts often have to work with poor-quality material and degraded DNA, which makes it impossible to obtain a definitive answer. Another difficulty is that the probability of a match is calculated only for the population as a whole. A higher degree of similarity will be found among relatives.

It is considerably more difficult to perform statistical calculations for single-locus probes, since each individual band occurs in the population with a different frequency. On average, when using a single such probe, bands will match in at most 1 out of 100 cases; when using two probes, in 1 out of 10,000 cases; and when using three probes, in 1 out of 1,000,000 cases. Variations exist among ethnic groups, and therefore distinct Databases must be used for different population groups.

Here are several Examples of the applications of DNA profiling:

1) for paternity testing;

2) for confirming purebred status in animals;

3) for monitoring genetic diversity in the breeding of endangered animal species;

4) in civil inheritance disputes;

5) in forensic science for identifying criminal suspects.