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(Polymerase Chain Reaction)

What is PCR (polymerase chain reaction)?

Polymerase chain reaction (PCR) is a technique that is used to amplify trace amounts of DNA (and in some instances, RNA) located in or on almost any liquid or surface where DNA strands may be deposited. The key to understanding PCR is to know that every human, animal, plant, parasite, bacterium, or virus contains genetic material such as DNA (or RNA) sequences (nucleotide sequences or pieces of DNA or RNA) that are unique to their species, and to the individual member of that species. Consequently, if a sample contains segments of DNA or RNA, PCR is a method used to amplify (make many more identical copies) of these unique sequences so they can then be used to determine with a very high probability the identity of the source (a specific person, animal, or pathogenic organism) of the trace DNA or RNA found in or on almost any sample of material.

PCR amplification is only part of the identifying test, however. Once the amplification is done (see below), the amplified segments need to be compared to other nucleotide segments from a known source (for example, a specific person, animal, or pathogenic organism). This comparison of unique segments is often done by placing PCR-generated nucleotide sequences next to known nucleotide sequences from humans, pathogens, or other sources in a separating gel. Electrical current is run through the gel and the various nucleotide sequences form bands that resemble a "ladder" according to their electrical charge and molecular size. This is termed gel electrophoresis. Bands or "ladder" like steps that migrate to the same levels in the gel show identity of nucleotide sequences. This method is one of the most popular ways PCR tests are completed (See Fig 1).

Figure 1, Bands or "ladder" like steps of PCR produced DNA of Mycobacterium (courtesy of the CDC)

Bands or ladder like steps of PCR produced DNA of microbacterium

Figure. Repetitive element (Rep)–PCR (A) and pulsed-field gel electrophoresis (PFGE) (B) patterns of Mycobacterium cosmeticum isolates from 2 patients in Ohio and 1 patient in Venezuela. Rep-PCR was performed by using BOXA1R primer (3), and PFGE was performed with restriction enzyme AseI. Lanes 1, 2, Ohio isolates OH1 and OH2; lanes 3, 4, control strains ATCC BAA-878T and ATCC BAA-879; lane 5, Venezuelan isolate VZ1. DNA size standards are 100-bp (S1) and 48.5-kb marker (S2).

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PCR (Polymerase Chain Reaction) Definition

PCR (polymerase chain reaction): PCR (polymerase chain reaction) is a technique in molecular genetics that permits the analysis of any short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA for analysis. Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient so that untold numbers of copies can be made of the DNA. What is more, PCR uses the same molecules that nature uses for copying DNA:

  • Two "primers", short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;
  • An enzyme called polymerase that moves along the segment of DNA, reading its code and assembling a copy; and
  • A pile of DNA building blocks that the polymerase needs to make that copy.

SOURCE: PCR (polymerase chain reaction).

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