Polymerase Chain Reaction (PCR): Difference between revisions
No edit summary |
No edit summary |
||
| Line 11: | Line 11: | ||
Typically these steps are repeated in a cycle about 30 times generating a large amount of identical DNA copies. Therefore, PCR is often used just before doing an [[Electrophoresis|electrophoresis]]. | Typically these steps are repeated in a cycle about 30 times generating a large amount of identical DNA copies. Therefore, PCR is often used just before doing an [[Electrophoresis|electrophoresis]]. | ||
The most important part of the PCR reaction is the initial design of the [[Primers|primers]]. The [[Primers|primers]] are normally between 18 to 20 [[Base pairs|base pairs]] in length and must be completely complimentary to the ends of the [[DNA|DNA]] region of interest. Included in the experiment must be both the forward and reverse [[Primer|primers]]. <br> | The most important part of the PCR reaction is the initial design of the [[Primers|primers]]. The [[Primers|primers]] are normally between 18 to 20 [[Base pairs|base pairs]] in length and must be completely complimentary to the ends of the [[DNA|DNA]] region of interest. Included in the experiment must be both the forward and reverse [[Primer|primers]]. <br> | ||
PCR is carried out in a [[Thermal cycler|thermal cycler]] and the [[Enzyme|enzyme]] '[[Taq Polymerase|Taq Polymerase]]' (isolated from''Thermus aquaticus'') is used as it is [[Thermostable|thermostable]] (is not denatured by high temperatures), therefore is not denatured at the high temperatures. [[Pfu|Pfu]] (''[[Pyrococcus furiosus|Pyrococcus furiosus]]'') [[DNA Polymerase|DNA Polymerase]] can also be used as it has better thermostability than [[Taq Polymerase|Taq Polymerase]] and it possesses 3' to 5' [[Proof reading|proof reading]] activity. | PCR is carried out in a [[Thermal cycler|thermal cycler]] and the [[Enzyme|enzyme]] '[[Taq Polymerase|Taq Polymerase]]' (isolated from ''[http://en.wikipedia.org/wiki/Thermus_aquaticus Thermus aquaticus]'') is used as it is [[Thermostable|thermostable]] (is not denatured by high temperatures), therefore is not denatured at the high temperatures. [[Pfu|Pfu]] (''[[Pyrococcus furiosus|Pyrococcus furiosus]]'') [[DNA Polymerase|DNA Polymerase]] can also be used as it has better thermostability than [[Taq Polymerase|Taq Polymerase]] and it possesses 3' to 5' [[Proof reading|proof reading]] activity. | ||
The technique was developed by Kary Mulis in 1983 for which he was awarded the [[Nobel Prize|Nobel Prize]] in Chemistry in 1993. <ref>http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html</ref> | The technique was developed by Kary Mulis in 1983 for which he was awarded the [[Nobel Prize|Nobel Prize]] in Chemistry in 1993. <ref>http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html</ref> | ||
PCR can be done using water baths at varying temperatures. | PCR can be done using water baths at varying temperatures. | ||
<references /> | <references /> | ||
Revision as of 11:02, 11 November 2010
Polymerase Chain Reaction (PCR) is a technique used for the amplification and identification of DNA or RNA. Also see mRNA (including transcriptase).
PCR has three main stages:
1. Heat DNA to 95°C to melt the strands
2. Cool to 50 - 65°C to allow primers to anneal
3. Heat to 72°C to allow elongation
Typically these steps are repeated in a cycle about 30 times generating a large amount of identical DNA copies. Therefore, PCR is often used just before doing an electrophoresis.
The most important part of the PCR reaction is the initial design of the primers. The primers are normally between 18 to 20 base pairs in length and must be completely complimentary to the ends of the DNA region of interest. Included in the experiment must be both the forward and reverse primers.
PCR is carried out in a thermal cycler and the enzyme 'Taq Polymerase' (isolated from Thermus aquaticus) is used as it is thermostable (is not denatured by high temperatures), therefore is not denatured at the high temperatures. Pfu (Pyrococcus furiosus) DNA Polymerase can also be used as it has better thermostability than Taq Polymerase and it possesses 3' to 5' proof reading activity.
The technique was developed by Kary Mulis in 1983 for which he was awarded the Nobel Prize in Chemistry in 1993. [1]
PCR can be done using water baths at varying temperatures.