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How a scrimillion copies of DNA translate into a test result (Christine Funk, Minnesota Board of Public Defense Trial Team)The DNA tests currently being used in forensic laboratories all make use of a procedure known as polymerase chain reaction, or PCR. PCR is a procedure that allows a small amount of DNA (which by itself would not be enough to type) to be ampli.ed into an amount large enough for typing. It does this by making millions of copies of DNA fragments from a polymorphic area (or areas) of the genome. PCR is not a genetic test itself, but merely a tool to increase the amount of genetic material to be tested. The "amplification" of DNA takes place in a test tube. The DNA that is extracted from each sample is placed in a separate tube, along with a mixture of primers, enzymes, and other reagents. The tubes are then placed in a machine known as a thermal cycler, which can control their temperature precisely while going through a series of heating and cooling cycles. Each cycle has three steps. First, the tubes are heated to approximately 94 degrees Celsius. At this temperature the DNA denatures--that is, the double-stranded molecule "unzips" to form two complementary single strands. In the second step, the tubes are cooled to about 60 degrees Celsius. At this temperature the primers anneal (bond) to the single strands of DNA. The primers are similar to genetic probes. They are single strands of organic bases (nucleotides), synthesized in a laboratory, that are complementary to speci.c target areas on the single stands of human DNA. The primers are designed to anneal at positions that ank the polymorphic areas to be amplified, thereby marking those areas.In the third step, the tubes are heated to about 72 degrees Celsius. At this temperature, an enzyme known as Taq DNA polymerase acts as a catalyst, causing single DNA strands in the areas marked by the primers to attract and bond with complementary bases that are oating in the solution. Each single strand of DNA from the marked areas thus becomes one side of a new double strand. When this process is completed, the number of identical double strands of DNA from the polymorphic areas is twice what it was at the beginning of the cycle. This three step cycle is repeated 25-35 times, doubling the number of copies of the target DNA each time, and producing literally billions of copies. The target DNA (from a polymorphic area, or areas), which was initially like a needle in a haystack of other DNA, is ampli.ed to the point that there are far more needles than hay, at which point the needles can be typed using a variety of methods. As a result of its ability to generate usable amounts of material from as little DNA as that which comes from a single cell, PCR-based approaches are amazingly sensitive and have the additional advantage of being much faster than RFLP analysis and better able to generate interpretable results even when evidentiary samples are degraded by exposure to the environment. It is important to remember that PCR is simply a procedure for replicating DNA. It is not a method for typing DNA, although some courts have used the term incorrectly as a description for the DQ-alpha and Polymarker tests. In fact, PCR is a component of every current DNA typing method. MaterialsPresentation 1: Evidence collectionPresentation 2: DNA extractionPresentation 3: How they make a scrimillion copies of the DNA |
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