Agarose

Load Load known unknown DNA samples

Load Load known unknown DNA samples

Sample 2 is positive

Sample 1 is negative

Sample 2 is positive to determine whether the unknown sample of blood is from a human, than a unique sequence from humans must be determined. Since many different genomes are currently being sequenced, this data is easily obtained. Next, two primers must be designed and synthesized using the sequence information. Small samples of DNA of unknown origin can then be tested by PCR using these primers. After the PCR reaction the DNA generated is run on an agarose gel to separate it according to size. If the DNA sample tested contains the target or known sequence, the PCR product will be of the predicted length (e.g., Fig. 23.20; unknown sample No. 2). If the test DNA is not from the same organism, no band will be generated (e.g., Fig. 23.20; unknown sample No. 1). The key to this experiment is the primers and how well they anneal to the target sequence. The primers may bind to closely related sequences, but the DNA made by PCR can be sequenced to determine whether or not this occurred. Increasing the temperature in which the primers anneal to the template can increase the stringency for this reaction also.

Clearly, PCR can be used in a variety of diagnostic tests. For example, visible symptoms of AIDS only appear a long time after infection, often several years. However, using PCR primers specific for sequences found only in the HIV genome, scientists can test for HIV DNA in blood samples, even when no symptoms are apparent. Another example is tuberculosis. Unlike many bacteria, Mycobacterium, which causes this disease, grows very slowly. Originally, to test for tuberculosis, the bacteria were cultured on nutrient plates, but this test took nearly a month. In contrast, PCR identification of mycobacterial DNA can be done in a day. Faster medical diagnoses are critical to help prevent the spread and progression of these diseases.

PCR is a powerful tool for amplifying small amounts of DNA. The DNA from 1/100th of a milliliter of human blood contains about 100,000 copies of each chromosome. If the target sequence for PCR is 500 base pairs, then there is about one-tenth of a picogram (10-12 gram) by weight of a target sequence. A good PCR run will amplify the target sequence and yield a microgram (10-6 gram) or more of DNA. A microgram may not seem much but is plenty for complete sequencing or cloning. Obviously, it is possible to identify an organism from an extremely small trace of DNA-containing material. In fact, the DNA from a single cell can be used to amplify a specific target sequence. This technique has revolutionized the criminal justice system by allowing highly accurate identification of individuals from very small samples.

Was this article helpful?

0 0

Post a comment