Polymerase chain reaction
Prepared by Max Darnell - AP225 Fall 2011
Polymerase chain reaction (PCR) is a technique to massively replicate short sequences of DNA. Developed in 1983, it has become extremely valuable in molecular biology, especially in terms of being able to detect small amount of a gene of interest by amplifying it to detectable quantities. This technique is based on thermal cycling and takes advantage of DNA replication machinery already found in the cell. This work resulted in the 1993 Nobel Prize in Chemistry.
The figure below is a schematic of how PCR works.
There are six main steps in PCR:
1) Initializing - This step may or may not be included, depending on the types of enzymes, but it involves heating the DNA of interest to just under 100 C.
2) Denaturation - This step heats the DNA to the extent that the two complementary strands dissociate, leaving only single-stranded DNA.
3) Annealing - This step attaches the primers to the single-stranded DNA. Primers are the short DNA sequences that will act as the building blocks for the new complementary strand of DNA. These are pre-made and are heated to just under their melting temperature.
4) Elongation - In this step, the temperature is set to the optimum for the activity of DNA polymerase, which builds off of the primer and attaches free nucleotides to the growing complementary DNA strand.
5) Secondary elongation - This step is similar to the previous and just ensures that all of the complementary strands have been completed.
6) Repeat - Now that the amount of DNA has been doubled, the process can be repeated, breaking apart the strands and again producing complementary strands.
Applications/Connections to Soft Matter
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Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J. "Integrating Cells Into Tissues". Molecular Cell Biology (5th ed.). New York: WH Freeman and Company. pp. 197–234.
Sluijter, J. P. G., Smeets, M. B., Velema, E., Pasterkamp, G., & De Kleijn, D. P. V. (2004). Increase in collagen turnover but not in collagen fiber content is associated with flow-induced arterial remodeling. Journal of Vascular Research, 41(6), 546-555.
Lee, H.-jin, Ahn, S.-hyun, & Kim, G. H. (2011). Three-Dimensional Collagen / Alginate Hybrid Scaffolds Functionalized with a Drug Delivery System ( DDS ) for Bone Tissue Regeneration. Chemistry of Materials.