Sequencing And Typing Strategies

Standard methods for genetic testing employ what is called the "polymerase chain reaction" or PCR. In order to test for the presence of a particular gene or allele's of a gene, a sample of DNA is isolated by means of two "probes"~each a 10 to 20 base long strand of DNA complimentary in sequence to DNA sequences at each end of the target gene. As the sample DNA is heated up, the two complimentary strands of DNA that form the double helix separate. When cooled again, the short probes are able to "hybridize" or bond to the target strands of DNA (one probe for each complimentary strand at opposite ends). Then as the sample is incubated at a moderate temperature, polymerase enzyme initiates the formation of a complimentary strand of DNA starting at the probe. The cycle is repeated twenty to forty times and the result is that the target DNA has been amplified exponentially. The sample is then placed on an electrophoretic gel and even single base pair differences in length of the target gene can be resolved visually on the gel.

Sequencing of genes employes the same electrophoretic. gel technique but the amplified DNA is first divided into four separate solutions and "digested" by enzymes that specifically cleave the DNA at one of the four base pairs that make up the molecule and a flourescent tag is then added. The resulting solutions are then passed through an electrophoretic gel, and the sequence of the DNA is reconstructed by summation of the differing lengths of DNA. All standard sequencing machines employ computer assisted reading and analysis to reconstruct the sequence.

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