"Everyone's different." We are all reminded of the veracity of this old adage just by looking around us in a crowd. With the exception of identical twins, it is not difficult to distinguish one individual from another. But recognizing differences in appearance between individuals is one thing; being able to genetically type them quickly and reliably to make the same distinctions is another.

An additional problem with information from blood groups was that blood samples had to be in good condition, and available in reasonable amounts. Although this was typically not an issue in paternity cases, where blood samples are usually drawn under ideal conditions, suitable samples are rarely if ever available in criminal cases.

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In this example, the top chromosome includes 12 motifs (a sequence of the bases A, G, C, and T) and the lower chromosome 17 motifs. The number of repeats varies by individual from as few as 7 to more than 40.

	Glossary
	Locus The site on a chromosome (or physical map) occupied by a gene. Homologous chromosomes generally have the same genes at the same loci. In everyday usage, locus is used synonymously with gene.

Visualizing the differences
Our ability to see the differences between individuals at the VNTR and STR loci depends on several key procedures and discoveries in the toolbox of molecular biology. DNA can be purified using reasonably simple and straightforward chemical procedures. However, the amount of biological material (e.g., blood, semen) that may be available is often quite limited and the amount of DNA that can be recovered may not be sufficient. To amplify the amount of DNA available, a procedure known as PCR (Polymerase Chain Reaction) can be used.

The lengths of DNA that can be amplified using PCR are limited, restricting its use for the most part to the amplification of STR loci. However, it is exceedingly sensitive. The follicle of a single hair can contain enough DNA to be amplified using PCR. The extreme sensitivity of the procedure requires that sterile laboratory procedures be stringently applied to avoid the possibility of contamination with foreign DNA. For example, laboratory workers must wear gloves when handling reaction tubes. DNA on the surface of the skin may be transferred to the tube with a single fingerprint, and confound the results.

Regents of the University of Michigan The phosphates in the DNA backbone give the molecules a negative charge. This makes DNA very soluble in water and gives us a way to attract them with a positive charge (like a magnet). To prevent all of the DNA in a sample from racing to the positive charge source immediately, it is forced to move through a Jello-like substance made from agarose (a polysaccharide extracted from red algae) and water. Smaller pieces of DNA can move more quickly through the thick gel, while larger pieces may hardly move at all. This has the effect of separating out the DNA sample by the size of their strands.

Courtesy, Pacific Northwest National Laboratory Since DNA dissolved in water is invisible, we need a way to be able to detect it. There are various DNA stains that can be used; the most common being Ethidium Bromide. Ethidium Bromide is a fluorescent compound that glows pink when excited by ultraviolet light. When a gel is finished, it is soaked in a staining solution (or the stain is previously mixed into the gel) and placed on a UV light box. Its picture is either acquired by a digital camera or a Polaroid photo is taken for documentation.

Regents of the University of Michigan The size of the strands is judged by running them next to a standard "ladder" of known lengths. This example shows part of a gel. The loading wells are the dark "holes" at the top. The position of the 2000bp ("base pairs" or nucleotides of double-stranded DNA) and 1000bp are marked. The first lane shows a piece of DNA about 1700 bp long; the second lane is just under 2000bp; and the third lane is the DNA "ladder". The fourth lane was read as a "bad" reaction; there was not much DNA and it is all smeared out. The last lane contains a ~1500bp fragment.

DNA fragments carry an electric charge, so small pieces of DNA can be separated by size if placed in a gel and an electric current is applied across the gel. This process is known as electrophoresis. Since for any one VNTR locus, a number of different alleles may exist, each with a different number of motif repeats, different alleles will have different lengths, determined by the number of motif repeats.

If the fragments of DNA containing the VNTR alleles are to be separated on the basis of size of the allele, we must create fragments of DNA that are cut at exactly the same position on either side of the locus. This task is already accomplished if the DNA is amplified by PCR, as amplification of a specific segment is intrinsic to the procedure. If PCR is not used, then we must cut the prepared DNA at specific points. For this task, enzymes that cleave the DNA at specific sequences are known. These are termed restriction enzymes or restriction endonucleases.

A curious feature of these enzymes is that they cleave the DNA at palindromic sequences that are from 4-8 bases in length.

Thus the enzyme EcoRI cleaves the DNA wherever the sequence,is found.

Now more than 60 of these enzymes are known, and by a suitable choice of restriction enzymes, the DNA can be cut at appropriate positions on either side of the VNTR locus.

If PCR is not used and the DNA from the sample is simply purified chemically, then the segments of DNA containing the VNTR alleles will be mixed with a myriad of other fragments of DNA. Separation of the DNA according to size by electrophoresis will result in a smear of hundred of thousands of different fragments with varying sizes on the gel. It is possible to specifically identify the VNTR containing fragments by making use of a property of DNA--namely that the molecule preferentially exists as a double helix, where each strand of the molecule is exactly complementary to the other strand.

Once all the fragments of DNA are separated, the double helices are denatured or separated into the component single helices, and radioactive purified VNTR DNA is added. This radioactive DNA re-forms a double helix only with the fragments of DNA containing the VNTR loci. Once the excess radioactive DNA is washed off, the gel is covered with a photographic film. The radioactive DNA exposes the emulsion and the resulting autoradiograph shows the position of the fragments of DNA containing the VNTR alleles.