Hot start PCR

Hot start PCR is a modified form of polymerase chain reaction (PCR) which avoids a non-specific amplification of DNA by inactivating the DNA polymerase at lower temperatures.[1]

Background

The polymerase chain reaction (PCR) is a method in molecular biology used to amplify a single copy or a few copies of specific pieces of DNA across several orders of magnitude, generating thousands to millions of copies of a target DNA sequence. In conventional PCR, the DNA polymerase is slightly active at room temperature, and to a lesser degree, even on ice.[2] In some instances, when all the reaction components are put together, nonspecific primer annealing can occur at these low temperatures. This nonspecific annealed primer can then be extended by the DNA polymerase, generating nonspecific products and lowering product yields.

The fundamental idea of hot start PCR is to reduce misprimed products during setup of the experiment. Early methods of hot start PCR involved excluding or limiting the concentration of one of the PCR reagents until the denaturation stage begins.[3] This method later evolved into separating the reagents by covering one of the reagents in a wax which is solid at room temperature and dissolves during the initial stage in PCR.[4]

Technique

In hot start PCR, antibodies, affibodies, chemical modifications, or aptamers specific to DNA Polymerase (usually modifications of the lysine with organic acid anhydride) are used to block the activity of the enzyme during reaction setup. An initial activation step at roughly 95℃ is required for activation of the protein. This step both denatures antibodies linked to the active center of the DNA polymerase, and also removes any lysine modifications made with acid anhydride. The anti-DNA polymerase antibodies reduce the DNA polymerase activity below 72℃, the optimal temperature at which the enzyme extends the primers. When the specific antibodies detach from DNA polymerase, the amplification proceeds with greater specificity.

The hot start PCR method is being further improved with research utilizing carbon nanotubes, nanoparticles, and quantum dots to enhance activity of the DNA polymerase.[5]

Advantages

Hot start PCR significantly reduces nonspecific priming, the formation of primer dimers, and often, increases product yields.[6] Classic methods, while effective, involve additional handling and increased risk of contamination.[7]

Disadvantages

If the DNA polymerase is chemically modified, the re-activation time during the initual denaturation stage of the PCR cycle is increased because you need to wait for the enzyme to activate. This increased heating time could damage the DNA. Studies have also shown that some modifications by chemicals can cause issues for amplifying long strands of DNA (larger than 3kb).[8]

A disadvantage of using antibodies for hot start PCR is that you need a unique antibody per enzyme, which can be expensive if you are running a large number of PCRs.[9] The antibodies are also derived from an animal cell, which could contaminate the results.[8]

References

  1. ↑ Paul, N. (2010). Hot start PCR. Methods in Molecular Biology. 630. pp. 301–318. doi:10.1007/978-1-60761-629-0_19. ISBN 978-1-60761-628-3. PMID 20301005.
  2. ↑ "DNA Polymerase—Four Key Characteristics for PCR | Thermo Fisher Scientific - US". www.thermofisher.com. Retrieved 2018-10-02.
  3. ↑ Coleman, William; Tsongalis, Gregory (2016). Diagnostic Molecular Pathology. Academic Press. pp. 15–23. ISBN 9780128008867.
  4. ↑ Kaijalainen, S. (1993). "An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose". Nucleic Acids Research. 21 (12): 2959–2960. PMC 309708. PMID 8332517.
  5. ↑ Sang, Fuming (2012). "Quantum dots induce hot-start effects for Taq-based polymerase chain reaction". Journal of Biomedical Science and Engineering. 5 (6): 295–301. doi:10.4236/jbise.2012.56038.
  6. ↑ Birch, David E.; Kolmodin, L.; Wong, J.; Zangenberg, G. A.; Zoccoli, M. A.; McKinney, N.; Young, K. K. Y. (1996). "Simplified hot start PCR". Nature. 381 (6581): 445–446. doi:10.1038/381445a0. PMID 8632804.
  7. ↑ Primrose, S. B.; Richard M. Twyman; R. W. Old (2001). Principles of gene manipulation. Wiley-Blackwell. p. 23. ISBN 978-0-632-05954-6.
  8. 1 2 "How is Hot-Start Technology Beneficial For Your PCR | Thermo Fisher Scientific - US". www.thermofisher.com. Retrieved 2018-10-02.
  9. ↑ "Hot Start PCR". www.biogene.com. Retrieved 2018-10-02.


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