Introduction

DNA fingerprinting (also known as DNA profile analysis and DNA typing), is a method of distinguishing between individuals by analyzing patterns in their DNA. This project focuses on the first method of DNA fingerprinting to be developed, by Sir Alec Jeffreys at the University of Leicester in 1985 (Wikipedia contributors, 2006).

Jeffreys discovered regions of DNA—that he named microsatellites—that are highly variable between individuals. Microsatellites occur in "non-coding" DNA, that is, DNA that is not transcribed into messenger RNA to code for proteins. Random variations in non-coding DNA accumulate more rapidly than in coding DNA. This is because variations in coding DNA are more likely to have negative effects, since they can cause changes in the amino acid sequence of proteins, leading to changes in protein function. While some regions of non-coding DNA are involved in regulation of gene expression, many non-coding regions have no known function, and are sometimes referred to as "junk DNA." Microsatellites are one example of such "junk DNA."

Microsatellites contain variable numbers of short, repeated sequences of DNA at a specific, identifiable locus (place) in the DNA sequence. The number of repeats is highly variable between individuals.

So how is microsatellite DNA used to distinguish between individuals? The DNA sample is first cut into smaller pieces using one or more restriction enzymes. Restriction enzymes recognize specific DNA sequences and cut the DNA molecule at or near this recognition site. Because microsatellite length varies between individuals, the resulting DNA fragments will have different lengths (the technical terms is restriction fragment length polymorphisms, or RFLPs).

In order to visualize the different fragment lengths (RFLPs), the cut DNA is loaded into a slab of agarose gel in a salt solution. An electric current is applied, which causes the DNA (which is negatively charged at neutral pH) to migrate through the gel. The gel acts to separate the DNA fragments by size, since shorter fragments move faster than larger fragments through the cross-linked structure of the gel. Figure 1 shows a simple example of cut DNA fragments separated by gel electrophoresis. An actual DNA fingerprint would contain many more bands (see Figure 2, below).

restriction enzyme digestion and DNA fragments on gel
Figure 1. Simplified example of DNA fragments cut by a restriction enzyme (Eco R1) and separated by gel electrophoresis. Sample A has a shorter repeated sequence (4 repeats, 40 base pairs total), while sample B has a repeated sequence nearly twice as long (7 repeasts, 70 base pairs total). At right, the resulting fragments are visualized after being run on a gel. The shorter fragment in column A has run further down the gel than the longer fragment in column B. Column M is a set of DNA fragment size standards, with fragments of known length (Rosner and Smith, 2004).

Next, the pattern of the DNA bands containing the microsatellites is visualized. This is done by denaturing the DNA in the gel (separating the double strands into single strands) and transferring it to a nitrocellulose or nylon membrane. In this transfer process, the DNA retains the banding pattern generated in the gel, with the fast-moving, smaller fragments toward the bottom and the slow-moving, larger fragments toward the top. To detect the microsatellite-containing fragments, radioactively labeled "probes" (short sequences of DNA complementary to the microsatellite sequence) are loaded over the membrane, and then washed off. Under the proper conditions, the probes will only bind to the matching (complementary) DNA sequence, so the probes specifically label the microsatellite-containing fragments.

The membrane is then placed next to a piece of x-ray film. The emissions from the radioactive probes react with silver grains in the film, so that when it is developed, the location of the microsatellite-containing bands from the gel can be seen on the film (see Figure 2). The NOVA webpage in the Bibliography (Groleau, 2000) has a "virtual" DNA fingerprinting experiment that you can run in your web browser, which is a fun way to get an overview of the procedure.

DNA 'fingerprints' from six individuals
Figure 2. DNA "fingerprints" from six individuals (Rosner and Smith, 2004).

The experimental procedure in the recommended kit is similar to the one just described. You will be digesting DNA samples from two "suspects" with restriction enzymes (non-human DNA is actually used in the kit). The DNA fragments for each "suspect" are then separated by gel electrophoresis and directly stained and visualized in the gel itself (no radioactive probes necessary!) You will use the banding pattern of the fragments generated by the restriction enzymes to match "suspect" DNA to "crime scene" DNA.