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Polymerase Chain Reaction Protocol

  • Author: Erica Suchman1
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Colorado State University, Fort Collins, Colorado, 80523
  • Citation: Erica Suchman. 2011. Polymerase chain reaction protocol.
  • Publication Date : November 2011
  • Category: Protocol
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Figure 1

The sequence of events in the PCR is as follows. The temperature is raised to 92-98oC, causing the DNA strands to separate. Two primer sequences of approximately 20 nucleotides each are annealed to opposite strands of DNA. (RNA requires an initial reverse transcription step to create a double-stranded cDNA template.) The temperature is raised to the optimum for a polymerase from a thermophylic bacterium; usually Thermus aquaticus (Taq) is used at 72oC. Replication continues from the 3' OH of the primers, producing two copies of the DNA. The temperature is again raised to 92-98oC, causing the DNA strands to separate. Then the temperature is lowered to allow new primers to attach to each of the four strands created in the previous reaction. The temperature used during the annealing of primers must be optimized for each individual primer set. The Taq polymerase fortunately is stable during the DNA melting step and is able to begin a new cycle of synthesis. The process is repeated for 20 to 40 cycles so that additional copies arise exponentially, i.e., in a chain reaction. After amplification, the PCR product, sometimes called an amplicon, is analyzed on an agarose gel and is abundant enough to be detected with an ethidium bromide stain. The amplicon is compared to known sized molecular markers for production of bands of the correct size. (Erica Suchman, Colorado State University, Ft. Collins, CO)

Source: November 2011
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Figure 2

If quantification is desired one usually performs real-time quantitative PCR which requires the addition of a fluorescently labeled probe that hybridizes between the two primers (Taqman reverse transcription PCR) or the use of double-stranded DNA binding fluorescent dyes such as Syber green. PCR amplification is usually quantified using a fluorescence detector, and the number of cycles of amplification required to cross a threshold fluorescence value (cycle threshold or CT) is determined by the computer and manipulated by the user. The fewer the number of cycles required to cross the threshold, the more nucleic acids that are present in the sample. CT values of unknown samples can be compared to CT values of known concentration standards to quantify the amount of nucleic acids in samples. However, if one wants to quantify RNA, the RNA must first be reverse transcribed to cDNA and then used to perform real-time PCR. The probe is complementary to sequences between the two primers used to amplify the DNA and contains a 5' fluorescent reporter dye and a 3' quencher dye that disrupts (or quenches) the detectable signal from the fluorescent reporter dye when it is in close proximity via fluorescence resonance energy transfer. As Taq polymerase polymerizes the DNA, its 5' exonuclease activity will cleave the fluorescent dye from the primer liberating it. As it floats away from the quencher dye its fluorescence will be detected by the detector. (Erica Suchman, Colorado State University, Ft. Collins, CO)

Source: November 2011
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Figure 3

Ethidium bromide-stained agarose mini-gel visualizing the products of multiple PCR reactions amplifying a portion of the mecA gene that encodes for methicillin resistance in Staphylococcus aureus ("MRSA"). Distinct 533 base-pair bands are clearly visible in the samples containing the resistance gene. Less distinct bands, indicating smaller primer dimers and unincorporated primers, are also visible . (Labeled view) (Rebecca? Buxton, University of Utah, Salt Lake City, UT)



Source: November 2011
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Figure 4

Typical temperature program for a PCR reaction. (Rebecca? Buxton, University of Utah, Salt Lake City, UT)



Source: November 2011
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