Polymerase Chain Reaction (PCR): Difference between revisions
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Polymerase Chain Reaction<ref>Hartl D. L., Ruvolo M. (2012), Genetics: Analysis of genes and genomes, Eight Edition, Jones and Bartlett learning (Chapter 2 DNA Structure and Genetic Variation)</ref> (PCR) is a technique used for the [[Amplification|amplification]] and identification of [[DNA|DNA]] or [[RNA|RNA]] of known sequence to give exponential products or copies. Also see [[MRNA|mRNA]] (including [[Transcriptase|transcriptase]]). It allows scientists to produce many millions of copies of a certain DNA sequence in a couple of hours. This technique was developed by an american biochemist [[Kary Mullis|Kary Mullis]] in 1984 <ref>Berg, J.M., Tymoczko, J.L. and Stryer, L. (2012). Biochemistry, 7 th Edition, New York , W.H.Freeman and Co Ltd. pg 151</ref> for which he was awarded the Nobel Prize in Chemistry in 1993 . <br> | Polymerase Chain Reaction<ref>Hartl D. L., Ruvolo M. (2012), Genetics: Analysis of genes and genomes, Eight Edition, Jones and Bartlett learning (Chapter 2 DNA Structure and Genetic Variation)</ref> (PCR) is a technique used for the [[Amplification|amplification]] and identification of [[DNA|DNA]] or [[RNA|RNA]] of known sequence to give exponential products or copies. Also see [[MRNA|mRNA]] (including [[Transcriptase|transcriptase]]). It allows scientists to produce many millions of copies of a certain DNA sequence in a couple of hours. This technique was developed by an american biochemist [[Kary Mullis|Kary Mullis]] in 1984 <ref>Berg, J.M., Tymoczko, J.L. and Stryer, L. (2012). Biochemistry, 7 th Edition, New York , W.H.Freeman and Co Ltd. pg 151</ref> for which he was awarded the Nobel Prize in Chemistry in 1993 . <br> | ||
[[PCR|PCR has]] three main stages: | [[PCR|PCR has]] three main stages: | ||
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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. 18 to 20 base pairs for a primer are ideal because a 18-2 base sequence is quite unique and is therefore unlikely to be present in any other section other than the target DNA. This ensures that the primers bind only to the flanking sequences associated with the target DNA sequence. For shorter genomes a smaller primer can be used. Along with this included in the PCR reaction must be both the [[Forward primers|forward]] and [[Reverse primer|reverse]] [[Primer|primers]], in addition to [[Taq polymerase|Taq Polymerase]] (which requires MgCl<sub>2 </sub> for its effective activity) and the DNA template. Alongside these substances the following must also be added; [[Magnesium Chloride|Magnesium Chloride]], free [[Nucleotides|nucleotides]] ([[DATP|dATP]], [[DCTP|dCTP]], [[DTTP|dTTP]] and [[DGTP|dGTP]]) and a [[Tris-HCl|Tris-HCl]] (pH 8.0) [[Buffer|buffer]].<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. 18 to 20 base pairs for a primer are ideal because a 18-2 base sequence is quite unique and is therefore unlikely to be present in any other section other than the target DNA. This ensures that the primers bind only to the flanking sequences associated with the target DNA sequence. For shorter genomes a smaller primer can be used. Along with this included in the PCR reaction must be both the [[Forward primers|forward]] and [[Reverse primer|reverse]] [[Primer|primers]], in addition to [[Taq polymerase|Taq Polymerase]] (which requires MgCl<sub>2 </sub> for its effective activity) and the DNA template. Alongside these substances the following must also be added; [[Magnesium Chloride|Magnesium Chloride]], free [[Nucleotides|nucleotides]] ([[DATP|dATP]], [[DCTP|dCTP]], [[DTTP|dTTP]] and [[DGTP|dGTP]]) and a [[Tris-HCl|Tris-HCl]] (pH 8.0) [[Buffer|buffer]].<br> | ||
PCR is carried out in a [[Thermal cycler|thermal cycler]], (a machine that is capable of varying temperature) when this is unavailable water baths can be used instead, and the [[Enzyme|enzyme]] '[[Taq Polymerase|Taq Polymerase]]' (isolated from ''[[Thermus aquaticus|Thermus aquaticus]]'') is used as it is [[Thermostable|thermostable]], which forms the core of PCR.Originally, DNA polymerase was added to the PCR reaction but it was denatured by the high temperatures, so had to be added at the end of every cycle. However, because Taq polymerase is thermostable, it isn't denatured so only needs to be added at the beginning of the reaction. [[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]], (a machine that is capable of varying temperature) when this is unavailable water baths can be used instead, and the [[Enzyme|enzyme]] '[[Taq Polymerase|Taq Polymerase]]' (isolated from ''[[Thermus aquaticus|Thermus aquaticus]]'') is used as it is [[Thermostable|thermostable]], which forms the core of PCR.Originally, DNA polymerase was added to the PCR reaction but it was denatured by the high temperatures, so had to be added at the end of every cycle. However, because Taq polymerase is thermostable, it isn't denatured so only needs to be added at the beginning of the reaction. [[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 technique has many importance amongst which is it's use in identification of the orientation of cloned inserts, also, it is used in other fields such as forensic science- at crime scenes, science in general- to diagnosize diseases.<br> | PCR technique has many importance amongst which is it's use in identification of the orientation of cloned inserts, also, it is used in other fields such as forensic science- at crime scenes, science in general- to diagnosize diseases.<br> | ||
PCR is a very useful tool for scientists for several reasons. | PCR is a very useful tool for scientists for several reasons. It can provide valuable diagnostic information in medicine as bacteria and viruses can easily be detected by the use of specific primers. For example, PCR can reveal the presence of a small amount of DNA from the human immunodeficiency virus (HIV) in persons who have not yet mounted an immune response to the pathogen. In these patients, assays designed to detect antibodies against the virus would yield a false negative test result. This is useful in the early diagnosis of HIV as well as helping to reduce the likelihood of further transmission. | ||
PCR shows high sensitivity and specificity to even small pieces of DNA, the results of PCR can be made available in a short amount of time (a few hours usually) and it is a relatively cheap process at £1-2 per reaction. | |||
=== References === | === References === | ||
<references /> | <references /> | ||
<br> | <br> | ||
Revision as of 02:01, 26 October 2012
Polymerase Chain Reaction[1] (PCR) is a technique used for the amplification and identification of DNA or RNA of known sequence to give exponential products or copies. Also see mRNA (including transcriptase). It allows scientists to produce many millions of copies of a certain DNA sequence in a couple of hours. This technique was developed by an american biochemist Kary Mullis in 1984 [2] for which he was awarded the Nobel Prize in Chemistry in 1993 .
PCR has three main stages:
- Strand Seperation : Heat dsDNA to 95°C for 15s to melt and seperate the strands.
- Hybridisation of Primer : Cool to 50 - 65°C to allow primers to anneal to the DNA strands.
- DNA Synthesis : 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. 18 to 20 base pairs for a primer are ideal because a 18-2 base sequence is quite unique and is therefore unlikely to be present in any other section other than the target DNA. This ensures that the primers bind only to the flanking sequences associated with the target DNA sequence. For shorter genomes a smaller primer can be used. Along with this included in the PCR reaction must be both the forward and reverse primers, in addition to Taq Polymerase (which requires MgCl2 for its effective activity) and the DNA template. Alongside these substances the following must also be added; Magnesium Chloride, free nucleotides (dATP, dCTP, dTTP and dGTP) and a Tris-HCl (pH 8.0) buffer.
PCR is carried out in a thermal cycler, (a machine that is capable of varying temperature) when this is unavailable water baths can be used instead, and the enzyme 'Taq Polymerase' (isolated from Thermus aquaticus) is used as it is thermostable, which forms the core of PCR.Originally, DNA polymerase was added to the PCR reaction but it was denatured by the high temperatures, so had to be added at the end of every cycle. However, because Taq polymerase is thermostable, it isn't denatured so only needs to be added at the beginning of the reaction. 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.
PCR technique has many importance amongst which is it's use in identification of the orientation of cloned inserts, also, it is used in other fields such as forensic science- at crime scenes, science in general- to diagnosize diseases.
PCR is a very useful tool for scientists for several reasons. It can provide valuable diagnostic information in medicine as bacteria and viruses can easily be detected by the use of specific primers. For example, PCR can reveal the presence of a small amount of DNA from the human immunodeficiency virus (HIV) in persons who have not yet mounted an immune response to the pathogen. In these patients, assays designed to detect antibodies against the virus would yield a false negative test result. This is useful in the early diagnosis of HIV as well as helping to reduce the likelihood of further transmission.
PCR shows high sensitivity and specificity to even small pieces of DNA, the results of PCR can be made available in a short amount of time (a few hours usually) and it is a relatively cheap process at £1-2 per reaction.
References