Polymerase Chain Reaction
Basic mechanism and protocol
Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 22 September 2012, last major update July 2010
Copyright: (c) 2008-2010, PathologyOutlines.com, Inc.
Components and reagents of a typical PCR
● DNA Template: most often fractionated whole genomic DNA which likely contains the sequence to be amplified (Wikipedia-DNA replication); may be single or double stranded DNA
● Two oligonucleotide primers: single stranded DNA, often 20-30 base pairs (bp) which are complementary to the 3’ ends of the sense and anti-sense strands of the DNA sequence to be amplified
● DNA polymerase: usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70°C (see also below)
● Buffer solution and monovalent and divalent cations: includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity
● Deoxynucleoside triphosphates: provides the energy for polymerization and the building blocks for DNA synthesis
● PCR has many variations, but this protocol is typical
● Most protocols involve 15-40 repeated thermocycles, with reaction volumes of 5-100 microL
● Oligonucleotides, nucleoside triphosphates, DNA with the sequence to be amplified, buffer and a DNA polymerase are mixed together and subjected to different temperatures
● Often a “water only” control, with all required PCR reagents but water instead of DNA to be amplified, is simultaneously run to look for DNA contamination; this control should show no amplification; if it shows amplification, it indicates that a contaminating DNA is present
● The exact temperature and length of each PCR step in the thermocycling reaction varies according to the melting temperature of the oligonucleotides used and the length of the elongation step
1. Initialization: The reaction is heated to 94-96°C for 30 seconds to several minutes to completely denature the nucleic acids, which lowers non-specific nucleic acid interaction and inappropriate priming events. Additionally, some DNA polymerases require heat activation and require temperatures as high as 98° for several minutes to both denature the nucleic acids and activate the DNA polymerase. Typically, this step is done only once in a PCR reaction.
2. Denaturation: This is the first step in the thermocycling reactions that will be repeated. It causes nucleic acid denaturation or separation of the oligonucleotide primers from the template DNA to be amplified, resulting in all the DNA in the reaction to be single stranded. Usually this step involves heating the reaction to 94-98°C for 15-30 seconds.
3. Annealing: The reaction temperature is rapidly lowered to 50-64°C for 20-40 seconds. Since the template DNA and oligonucleotides are moving randomly in the reaction mix, the lower temperature will allow the template DNA and oligonucleotides to form Watson-Crick base pairs, resulting in double stranded DNA. The annealing temperature is relatively high, allowing only the most stable and therefore specific double stranded DNA structures to form. Double stranded DNA structures which have lower stability due to a few or many non-Watson-Crick base pairings are unlikely to form or initiate a non-specific DNA synthesis. Additionally, the DNA polymerase binds the double stranded DNA at this step and initiates DNA synthesis.
4. Elongation: Also known at extension, this step usually occurs at 72-80°C (most commonly 72°C). The length of the step depends on the length of the DNA being synthesized. Under optimal conditions, DNA polymerase will add about 1,000 bp/minute. The higher temperature used in elongation compared to annealing again lowers non-specific oligonucleotide-template interactions, increasing the specificity of the reaction. In this step, the DNA polymerase uses the nucleoside triphosphates to synthesize DNA in the 5’ to 3’ direction. After the first few rounds of synthesis, the length of the amplified DNA is limited by where each oligonucleotide binds, thus nearly all amplified DNA will be as long as the distance between two oligonucleotides on the template DNA. With each of the above temperature cycles (steps 2-4 above), the amount of DNA synthesized is doubled, resulting in exponential DNA amplification. Most PCR reactions do 15-40 cycles of steps 2-4.
5. Final elongation: This step in not always performed. The reaction is held at 70-74°C for several minutes after the last PCR to allow any remaining single stranded DNA to be fully extended.
6. Final hold: The reaction is complete and the resulting amplified nucleic acids are held at a low temperature (~4°C) until analysis.
7. Analysis: This step can involve many different methods. Often completed PCRs are analyzed by gel electrophoresis, where the amplified DNA is run into a gel and visualized via ethidium bromide staining seen under UV light. Usually a “water blank” reaction and a DNA ladder are also run into the gel as controls for possible DNA contamination and for molecular weight identification.
End of Molecular Pathology > Polymerase Chain Reaction > Basic mechanism and protocol
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