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
Not only have PCR thermal cyclers become common in molecular biology labs, but they now have an increasing commercial importance. PCR can be used in a host of genetic tests and will only become more important as the function of specific genes is further elucidated from the sequencing revolution. There is significant effor to create lab-on-a-chip versions of PCR, that can perform massively-parallel replication of various genes. This problem is mainly one of microfluidic large-scale-integration, which has been revolutionized by Stephen Quake at Stanford.
Bartlett, J. M. S.; Stirling, D. (2003). "A Short History of the Polymerase Chain Reaction". PCR Protocols. 226. pp. 3–6.
Saiki, R.; Gelfand, D.; Stoffel, S.; Scharf, S.; Higuchi, R.; Horn, G.; Mullis, K.; Erlich, H. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science 239 (4839): 487–491.
White, R. A., Blainey, P. C., Fan, H. C., & Quake, S. R. (2008). Digital PCR provides sensitive and absolute calibration for high throughput sequencing. BMC Genomics, 10(1), 116.