Diagnostic applications of the polymerase chain reaction (PCR). A

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Research Techniques Made Simple: Polymerase Chain Reaction

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Diagnostic applications of the polymerase chain reaction (PCR). A Diagnostic applications of the polymerase chain reaction (PCR). A. A clinical specimen (eg, pus, tissue) contains DNA from many sources as well as the chromosome of the organism of interest. If the DNA strands are separated (denatured), the PCR primers can bind to their target sequences in the specimen itself. B. Amplification of the target sequence by PCR. (1) The target sequence is shown in its native state. (2) The DNA is denatured, allowing the primers to bind where they find the homologous sequence. (3) In the presence of the special DNA polymerase, new DNA is synthesized from both strands in the region between the primers. (4-6) Additional cycles are added by temperature control of the polymerase with each new sequence acting as the template for another. The DNA doubles with each cycle. After 25 to 30 cycles, enough DNA is present to analyze diagnostically. C. Internal probe. The amplified target sequence is shown. A probe can be designed to bind to a sequence located between (internal to) the primers. D. Analysis of PCR amplified DNA. (1) The amplified sequence can be cloned into a plasmid vector. In this form, a variety of molecular manipulations or sequencing may be carried out. (2) Direct hybridizations usually make use of an internal probe. The example shows three specimens, each of which went through steps A and B. After amplification, each was bound to a separate spot on a filter (dot blot). The filter is then reacted with the internal probe to detect the PCR-amplified DNA. The result shows that only the middle specimen contained the target sequence. (3) The amplified DNA may be detected directly by agarose gel electrophoresis. The example shows detection of amplified fragments in two of three lanes on the gel. (4) The sensitivity of detection may be increased by use of the internal probe after Southern transfer. The example shows detection of a third fragment of the same size that was not seen on the original gel because the amount of DNA was too small. Source: Principles of Laboratory Diagnosis of Infectious Diseases, Sherris Medical Microbiology, 6e Citation: Ryan KJ, Ray C. Sherris Medical Microbiology, 6e; 2014 Available at: https://accessmedicine.mhmedical.com/DownloadImage.aspx?image=/data/Books/1020/rya_ch4_f018.png&sec=56986872&BookID=1020&ChapterSecID=56968646&imagename= Accessed: November 12, 2017 Copyright © 2017 McGraw-Hill Education. All rights reserved