DNA Fingerprinting—Electrophoresis at Work

Introduction

“DNA Fingerprinting—Electrophoresis at Work” is an introductory activity for students about to begin the study of biotechnology and electrophoresis. Students become medical forensic experts as they read a case in which a woman has been attacked or a case where an elderly man has been murdered. The entire case rests on their expert ability to interpret the results from DNA tests.

Concepts

• DNA fingerprinting
• Restriction enzymes

Background

The world of forensic science was revolutionized recently with the discovery of a scientific technique for identifying humans and animals by using their DNA. This technology, DNA fingerprinting, can be used to identify the source of DNA in not only forensic medicine, but has been utilized in paternity cases, identification of disaster victims, and in the diagnosis of genetic diseases.

DNA fingerprinting was developed and the term first used in 1984 by the British researcher Alec Jeffreys at the University of Leicester. Jeffreys was also the first to use the technique in paternity, immigration and murder cases.

DNA (deoxyribonucleic acid) is found in the chromosomes of every cell in the body. It contains the chemical instructions for all life processes. It also serves as a sort of “biological template,” regulating development of the organism and ultimately determining its outward morphology. Each individual, as evidenced by his or her outward appearance, carries a unique set of DNA patterns with the notable exception of identical twins. It is this variation in genetic material, from one individual to another, that provides the potential for virtually unmistakable identification.

Of further significance to the process of DNA fingerprinting is the fact that up to 30% of human DNA consists of relatively short, (4–8 base pairs) highly repetitive sequences. From one individual to another the size and number of these sequences varies significantly. The key to capitalizing on these variations is an array of naturally occuring bacterial proteins—restriction enzymes.

Restriction enzymes are present in bacterial cells and are “used” by the bacteria to combat (or “restrict”) invasions by foreign genetic material. For example, DNA from an invading bacteriophage (virus) is attacked and cut apart (fragmented) to render it harmless and prevent the phage from taking over the bacterial cell. Each of these restriction enzymes has evolved to recognize very specific sites on the foreign DNA. These are termed restriction sites. An increasing number of these restriction enzymes have been isolated and analyzed so that a unique nomenclature has been developed to identify them. They are named according to the name of the bacterial species from which they were isolated, the particular strain of that bacterial species, and their order of discovery. For example, the restriction enzyme EcoR1 is derived from Escherichia coli, Strain RY13, and was the first such enzyme isolated from this particular strain. Table 1 lists a few of the most widely used restriction enzymes, their source, and the specific recognition site of each.

The arrows over the restriction sites show the precise location where the restriction enzyme cuts the DNA strand. To further illustrate, look at the following segment of double stranded DNA:


Note: Restriction enzymes “read” DNA from the 5′ end to the 3′ end.

The segment is “digested” with the Hae111 restriction enzyme giving the following result:


The DNA is cut in two places yielding three fragments of varying lengths. If you can imagine extrapolating this process over an individual’s entire genetic complement, you can also imagine the result—a large number of fragments of different lengths. The number of fragments and their relative sizes make up the DNA fingerprint for that individual.

Once the DNA sample has been fragmented, the investigator has to have some means of separating the fragments and of visualizing the fingerprint that the separation process produces. The separation technique used is electrophoresis.

Electrophoresis is a separation technique similar in principle to various forms of chromatography (i.e., particles moving through a medium at varying rates depending on properties of those particles). For DNA samples the medium generally used is an agarose gel (agarose is derived from agar, the gelling agent used in bacteriological culture media). In electrophoresis an electrical current is passed through the gel and this current serves to “drive” the migration of the DNA sample through the gel.

The forensic investigator introduces the fragmented DNA sample onto one end of the gel to initate the separation. Smaller sized fragments will migrate more quickly through the gel than the larger fragments and similar sized fragments move together in a “band.” Due to the size of the human genome, a very large number of bands is produced and each individual band is somewhat difficult to distinguish from those adjacent to it. Simply staining the gel at this point would result in a nearly continuous smear. The final step in the procedure is Southern Blot analysis—a technique by which a discreet number of the resulting bands are labeled and photographed.

In a Southern Blot the DNA bands are transferred (blotted) from the gel to a nitrocellulose membrane. The membrane is then treated with a radioactive DNA probe. The probe is a short DNA segment that recognizes and binds to a particular repetitive sequence present in several of the bands produced by the electrophoretic separation. A piece of X-ray film is applied to the membrane and the radioactively labelled bands expose the film to produce the final result—the DNA fingerprint. The banding patterns included with this exercise were produced in this precise fashion.

DNA fingerprinting, when properly performed, provides positive evidence of an individual’s identity and is a much more powerful tool than more familiar, classic forensic techniques such as fingerprinting and blood typing. These classic techniques are means of phenotypic (or exclusionary) testing. Phenotypic testing can only be used to prove that the crime scene evidence does not match the suspect.

DNA fingerprinting requires only a relatively small tissue sample and can identify any person from a crime scene who may have left only the tiniest trace of evidence. Potential sources of DNA include: blood samples (even though mature red blood cells carry no nuclear material, sufficient white blood cells are usually present), sperm samples, dried blood or semen from a fabric, a few skin cells from under a victim’s fingernails, or several hairs with roots attached.

A brief summary of the procedure follows:

• DNA is extracted from a tissue sample.
• DNA samples are digested with restriction enzymes which produce a series of different sized fragments of DNA.
• Selected bands are separated into bands by agarose gel electrophoresis, resulting in a banding pattern unique to that individual.
• The fragments are visualized by performing a Southern Blot analysis resulting in an X-ray photograph of the DNA fingerprint.
• The DNA fingerprint is compared to similarly prepared evidence and a determination is made.

Currently forensic scientists are limited by the quantity of DNA available and the degree of degradation of the sample. A more recent technique being adapted for forensic testing is known as the Polymerase Chain Reaction (PCR). This technology can be used to amplify (make numerous exact duplicates of) as little as a single molecule of DNA. The amplified sample may then be digested by restriction enzymes, electrophoresed, stained, and analyzed. PCR technology will make any detectable DNA sample, no matter how small, available to the forensic investigator.

In forensic testing, DNA samples from the victim, suspect, and the evidence from the crime scene are DNA fingerprinted and compared. Matches between the banding patterns provide evidence that can be used in a court of law. See the diagram of fingerprints below. Victim bands are included to demonstrate that the evidence must indeed have come from the suspect. An overhead transparency is provided so students can see and learn how to match the banding patterns.

Materials

Procedure

1. Some background information is provided. You will want to incorporate this information with other facts you already have on biotechnology and electrophoresis. An overhead transparency is provided on how to match banding patterns. Instruct students on how to match banding patterns before passing out the case materials.
2. Two different case studies are provided. Within each case, you can select one of four outcomes. You will want to switch cases and outcomes each class period.
3. Distribute to each group of students the DNA case you have selected, one Suspect DNA Analysis Card, one Evidence Card (B, C or D) and one Evidence Card (A, E, F or G). Students now have all the materials needed to make the comparisons and decide who is the guilty party.
4. Evidence Cards B, C and D are bands of the victim’s DNA. They will not match any of the bands found on the suspect DNA Analysis Card. Each group of students should get only one of these cards.
5. Evidence Cards A, E, F and G are evidence (suspect’s DNA) taken from the victim. These bands will match the bands found on the suspect DNA Analysis Card. Each group of students should get only one of these cards.

6. If you want to have some fun with the students, give them two Evidence Cards (B, C or D) and have them match up the bands with those found on the Suspect DNA Analysis Card. Once the students have discovered that none of the cards given to them match, tell them “the lab has just called to say they made a mistake. They sent the wrong cards in error.” Pass out one new Evidence Card (B, C or D) and one Evidence Card (A, E, F or G) and have them try again.
7. Have students keep their answers to themselves until all groups have completed their comparisons.

References

Molecular Cell Biology. James Darnell, Harvey Lodish, David Baltimore. 2nd Edition. 1990 Scientific American Books, Inc., New York.
A Sourcebook of Biotechnology Activities, 1990. Published by the National Association of Biology Teachers and the North Carolina Biotechnology Center.