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Polymerase chain reaction

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Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a technique for replicating a specific section of DNA and amplifying it exponentially.[1] It was first conceived by Kary Mullis in 1983,[2] who thought of the idea while thinking of a way to analyze mutations in DNA. Later in 1993, he was awarded the Nobel Prize in Chemistry for his work.[2][3] He later said in a scientific journal, "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."[1]

Requirements

In order for PCR to work, there are a few requirements that are needed. First, it needs a DNA template with the desired region of DNA that will be amplified. Second, there have to be one or more primers, which designate the area of DNA that will be amplified by PCR. Third, it needs a DNA polymerase with an optimum temperature around 70oC, which will synthesize a copy of the DNA fragment. Fourth, it needs the four deoxyribonucleotide triphosphates dATP, dGTP, dCTP, and dTTP, which are the building blocks of the new DNA.[4] Fifth, it needs a buffer solution, which allows for a suitable environment without contamination. Finally, it needs divalent cation magnesium ions and monovalent cation potassium ions.[2]

Primers

Without primers, PCR would be unable to amplify a specific section of DNA. Primers are usually very short, usually no more than 25 base pairs long, but they are necessary for PCR to work. The primers are made up of nucleotides that are complimentary to the nucleotides on each end of the DNA fragment that will be amplified. This means that an Adenine (A) nucleotide will pair with a Thymine (T) nucleotide, and a Cytosine (C) nucleotide will pair with a Guanine (G) nucleotide.

Taq Polymerase

Taq Polymerase is a DNA polymerase that was isolated from the temperature resistant bacteria Thermus aquaticus, and is used during PCR to amplify the DNA fragment.[1] Taq is commonly used in PCR because it is able to withstand temperatures of above 70oC, which is extremely important in order for the reaction to work. Taq catalyzes the polymerization of DNA in the 5' to 3' direction. This means that there is no proofreading technique available to it, and the error rate of Taq is 2.2 x 10-5 per nt per cycle.[3] This is about 1 error for every 10,000 nucleotides.[4]

Uses

The Polymerase Chain Reaction is used commonly for many things. It is extremely easy to do, and requires very little equipment, but it is vitally important for a lot of the work involving DNA. These are just a few of the many techniques that PCR is used in.

DNA Sequencing

DNA shown using gel electrophoresis.
Main Article: DNA sequencing

DNA sequencing is used to find the nucleotide order of a DNA fragment. Knowing the sequence of the nucleotides is very important to find any hereditary diseases, or just to understand DNA better. PCR is used to amplify the section of DNA that needs to be sequenced so that enough samples are available. The DNA is then divided into four separate single-stranded samples. Each of the samples has its own separate dideoxynucleotide (ddNTP), which is radioactively labeled so that it can be seen later. Once the samples are separated by using gel electrophoresis (see image), they are compared side by side. The first nucleotide is either A, T, C, or G, depending on which ddNTP was used in that sample. For example, if the first strip in the gel was in the sample that used ddATP, then that nucleotide will be an ATP. If the next strip in the gel was in the ddGTP sample, then that will be the GTP nucleotide. This nucleotide reading is continued down the strip until the complete nucleotide sequence of that section of DNA is known.[5]

Genetic Fingerprinting

Main Article: DNA fingerprinting

This is the technique of identifying someone by using any organic part of them. For example, a single drop of blood or a strand of hair could be used if they are in a good enough condition. PCR is used in this process to amplify different sections of the DNA, which are then analyzed by using gel electrophoresis. Forensics teams often use this technique at crime scenes to find suspects.[6]

Cloning Genes

Main Article: Cloning

PCR is used when cloning genes to amplify that gene, which is then inserted into a vector. Gene cloning is very different from cloning a living organism, like a sheep. Instead, it is the process of isolating a gene from one organism and transfering it into another. This is very useful to study the effects of a single gene in another organism.[7]

Procedure

The polymerase chain reaction step by step

PCR has three major steps, which are repeated anywhere from 20 to 40 times. The three steps are:

1. Denaturation - The DNA strand is heated to approximately 94oC, and it splits apart to become a single strand of DNA. The heat breaks the hydrogen bonds that are keeping the DNA strands together.

2. Annealing - The primers and their complimentary nucleotides on the single strand of DNA form and break ionic bonds until the bonds become strong enough to not break.

3. Extension - During this step, the temperature is heated back up to around 72oC, and the Taq polymerase polymerizes the bases that have formed on the DNA fragment. Polymerization adds nucleotides to the 3' end of each primer that is attached to the DNA strand. This forms new strands of DNA that contain the section of DNA that was originally desired.

These steps are repeated many times so that enough fragments are obtained to be used in DNA sequencing and DNA replication.[8]

References

  1. 1.0 1.1 Hartl, Daniel L (2008) (in Portuguese). Princípios de Genética de População [A Primer of Population Genetics] (3rd ed.). São Paulo: Funpec Editora. p. 10. ISBN 978-85-7747-022-8. 
  2. 2.0 2.1 Bartlett, J. M. S.; Stirling, D. (2003). "A Short History of the Polymerase Chain Reaction". In Bartlett, J. M. S.; Stirling, D. PCR Protocols 226 (2nd ed.). Totowa, New Jersey: Humana Press. p. 3-6. ISBN 1-59259-384-4. 
  3. "The Nobel Prize in Chemistry 1993". Nobel Foundation. http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/. Retrieved August 20, 2013. 
  4. Griffiths, Anthony J. F.; Wessler, Susan R.; Lewontin, Richard C.; Carroll, Sean B (2008). Introduction to Genetic Analysis (9th ed.). New York: W. H. Freeman. p. 731-732. ISBN 978-0-7167-6887-6. 

External links