Creating a DNA Profile
If they're all supposed to arrive at a similar result -- a unique DNA profile -- then why are there so many different techniques for analysis? Which technique to use depends on a couple of factors, including cost, time available for analysis and the quality and amount of the DNA sample available.
The first method for creating a DNA profile was RFLP, or restriction fragment length polymorphism. RFLP is not used as often today because it requires a large sample of DNA -- as much as 25 hairs or a nickel-sized spot of bodily fluid -- and can take as long as a month to complete [source: Baden]. It also requires examining multiple sections of the DNA strand to find variations, which is time-consuming and leaves more room for human error. Some of the steps for RFLP analysis are also used in other types of DNA profiling. For RFLP, the steps are:
- Separate white and red blood cells with a centrifuge.
- Extract DNA nuclei from the white blood cells. This is done by bathing the cells in hot water, then adding salt, and putting the mixture back into the centrifuge [source: University of Arizona].
- Cut DNA strand into fragments using a restriction enzyme.
- Place fragments into one end of a bed of agarose gel with electrodes in it. Agarose gel is made from agar-agar, a type of seaweed that turns into gelatin when dissolved in boiling water.
- Use an electric current to sort the DNA segments by length. This process is called agarose gel electrophoresis. Electrophoresis refers to the process of moving the negatively-charged molecules through the gel with electricity. Shorter segments move farther away from their original location, while longer ones stay closer. The segments align in parallel rows.
- Use a sheet of nitrocellulose or nylon to blot the DNA. The sheet is stained so the different lengths of DNA bands are visible to the naked eye. By treating the sheet with radiation, an autoradiograph is created. This is an image on x-ray film left by the decay pattern of the radiation. The autoradiograph, with its distinctive dark-colored parallel bands, is the DNA profile.
PCR (polymerase chain reaction) analysis is usually the first step in the creation of a DNA profile today. PCR can replicate a small amount of DNA to create a larger sample for analysis. It does this using a repeating process that takes about five minutes. First, a heat-stable DNA polymerase -- a special enzyme that binds to the DNA and allows it to replicate -- is added. Next, the DNA sample is heated it to 200 degrees F (93 degrees C) to separate the threads. Then the sample is cooled and reheated. Reheating doubles the number of copies. After this process is repeated about 30 times, there is enough DNA for further analysis.
PCR is the first step in analyzing STRs (Short Tandem Repeats), which are very small, specific alleles in a variable number tandem repeat (VNTR). Alleles are pairs of genes that occur alternately at a specific point, or loci, on a chromosome. STRs are explained further in How DNA Evidence Works. Analyzing STRs is more accurate than the RFLP technique because their small size makes them easier to separate and to tell apart.
A variation on STR analysis is Y-STR. Only STRs found on the Y-chromosome (which only males have) is analyzed. STR analysis is useful if the sample has mixed DNA (from both men and women) or in sexual assault cases with a male assailant. Y-STR is otherwise processed just like a regular STR.
AmpFLP, amplified fragment length polymorphism, is another technique that uses PCR to replicate DNA. Like RFLP, it first uses a restriction enzyme. Then, the fragments are amplified using PCR and sorted using gel electrophoresis. AmpFLP's advantage over other techniques is that it can be automated and doesn't cost very much. However, the DNA sample must be high quality or errors may result, which is the case with most DNA analysis techniques. Analysts can have a time telling the longer strands apart because they bunch up tightly.
In this article, we'll look at how DNA profiles are used and why it's creating controversy.