PCR

= Polymerase Chain Reaction =

General description
toc

The **Polymerase Chain Reaction** (PCR) is a technique, developed by [|Kary Mullis], used to amplify a chosen region of a DNA molecule. Different regions of many types of DNA can be used, so long as the sequences at the borders of the region are known. The amplification is due to //Taq polymerase// action, which results in multiple copies of the region of interest belonging to DNA molecule.

**Figure - 1:** PCR machine.

History
PCR is a technique that was developed by Kary Mullis in 1983. Kary Mullis and a team of researchers at Cetus Corporation conceived of a way to start and stop a polymerase's action at specific points along a single strand of DNA. Mullis also realized that by harnessing this component of molecular reproduction technology, the target DNA could be exponentially amplified. This DNA amplification procedure was based on an in vitro rather than an in vivo process. Previous techniques for isolating a specific piece of DNA relied on gene cloning – a tedious and slow procedure. PCR, on the other hand Kerry Mullis stated “lets you pick the piece of DNA you’re interested in and have as much of it as you want”. When other Cetus scientists eventually succeeded in making the polymerase chain reaction perform as desired in a reliable fashion, they had an immensely powerful technique for providing essentially unlimited quantities of the precise genetic //material// molecular biologists and others required for their work. Since the first report in1985, many scientific papers were published. Furthermore, the large number of publications of course makes it impossible to review all the important contributions to the development and application of PCR technology. Karry Mullis was awarded the Nobel Prize for Chemistry in 1993 for his discovery of PCR. However, this discovery is contested amongst many scientists, all of which may have contributed to unlocking this puzzle. It has also been said that PCR did not exist until it was made to work in an experimental system. With this in mind, merely the thought of a concept is not sufficient; a concept must have been successfully been put into practice. Although there is doubt as to the ultimate creator of PCR, and doubt as to the possibility that PCR may somehow or sometime be replaced, there is little doubt the impact that PCR has created over a short time span on the study of molecular biology and life. Since March 28, 2005, researchers in the U.S. no longer need a license to practice the basic PCR amplification process, which was covered by U.S. Patents 4,683,195, 4,683,202 and 4,965,188. This much-anticipated opportunity has opened the door for an influx of suppliers hoping to provide Taq DNA polymerase to scientists for this specific application without the associated royalty burden. Once Taq DNA polymerase has been incorporated into a scientists toolbox, it likely remains there for many years, if not for their entire research career.

Products and Companies
The following table presents a list of some companies that produce various PCR applications.


 * = **Logo** ||= **Companies Name** ||= **Products** ||
 * = [[image:http://www.scubeindia.com/premier_biosoft/images/PremierBiosoft_logo.jpg]] ||= Premier Biosoft International ||= This companie have authored software for technologies as varied as PCR, cloning and qPCR to tissue microarrays. ||
 * = [[image:http://www.illumina.com/images/logos/sabiosciences.gif]] ||= SABiosciences ||= This companie have RT² qPCR products for gene expression analysis, RT² qPCR reagent kits and data analysis software. ||
 * = [[image:http://media.biocompare.com/Images/Layout/MainLogo.gif width="171" height="71" caption="Biocompare - Home" link="http://www.biocompare.com/Default.aspx"]] ||= biocompare ||= This companie have PCR software. ||
 * = [[image:http://www.primerdesign.co.uk/PrimerDesign%20images/small%20logo%20no%20slogan.jpg link="index.html"]] ||= Primer Design ||= This companie have many products for Real Time PCR. ||
 * = [[image:http://www.geneworks.com.au/library/Kapa_logo.gif width="174" height="105"]] ||= KAPABIOSYSTEMS ||= This companie have a portfolio of next-generation reagents that contain novel DNA polymerases engineered specifically for PCR application. ||
 * = [[image:http://proger-cdd.crihan.fr/Images/LogoBIORAD.jpeg width="242" height="70"]] ||= BIO-RAD ||= This companie have many aplications of PCR related devices. ||

**Table 1:** Companies and their products.

This technique is used to amplify a chosen region of a DNA molecule, and in order to do so we need to know the borders and synthesized two short oligonuceotides, which act as primers for the DNA synthesis and delimit the region to be amplified. These primers are the key to the success or failure of a PCR experiment. They must be complementary to the borders of the amplified region. Also the DNA fragment to be amplified should have 3-10kb and the primers a length between 17-28 bp. If they are too short they might hybridize to non-target sites giving undesired amplification products and if they are too long they hybridize at a slower rate.

The Polymerase Chain Reaction includes many steps. In first place we have to denature the double helix of DNA by using high temperatures (94 ºC), add oligonucleotides primers that will hybridize the sequences, flanking the target regions on the template molecule (50-65ºC), and afterwards we add //Taq polymerase//, some dNTPs to the solution and the amplification carries on (72 º C). We repeat this cycle approximately 30 times. To analyze the results it’s normally used gel electrophoresis. Another important thing that can determine the efficiency of PCR is the temperature, so we have to use specific temperatures in the different steps.





**Figure - 2:** PCR steps.

In this technique, like we can deduce, it’s the //Taq Polymerase// or DNA polymerase I from //Thermus aquaticus// that’s responsible to amplify the interest region. This enzyme is thermostable, giving it resistance to denaturation by heat treatment. Other types of polymerases like //Pyrococcus furiosus//, //Pyrococcus woesi, Pyrococcus sp., thermotoga maritime,// are also used. One problem implicit to the amplification carried by //Taq Polymerase// is the impossibility of error correction. If the fragments are for cloning purposes, the product may have any differences compared to the original sequence.


 * Figure - 3:** Differences in temperature during the PCR cycle.




 * Table 2:** Some properties of PCR reagents.

PCR is very used nowadays because it has many applications, some of them are:

>
 * cloning genes;
 * diagnostic of pathogens;
 * forensic medicine;
 * gene expression;
 * molecular typing;
 * sequence determination;
 * site-directed mutagenesis;
 * taxonomy.

Since then new procedures were developed that have in basis the PCR technique discovered by Kary Mullis, for example: AFLP PCR, Allele-Specific PCR, Alu PCR, Assembly PCR, Assymetric PCR, Colony PCR, HDA, Hot Start PCR, Inverse PCR, In Situ PCR, ISSR PCR, Late-PCR, Long PCR, Nested PCR, RT-PCR, Real Time PCR, RT-PCR, Single Cell PCR and Standard PCR.

> > **IRE-PCR (Interspersed Repeat Element PCR):** a cloning strategy where by insertion of a new piece of DNA into a vector inactivates a gene carried by the vector. > > **Multiplex PCR:** a PCR carried out with more than one pair of primers and hence targeting two or more sites in DNA being studied. > > **RACE (Rapid Amplification of cDNA ends):** a PCR-based technique for mapping the end of an RNA molecule. > > **Repetitive DNA PCR:** a clone fingerprinting technique that uses PCR to detect the relative positions of repeated sequences in cloned DNA fragments. > > **RT-PCR (Reverse Transcription PCR)**: a PCR technique in wich the starting material is RNA. The first step in the procedure is conversion of the RNA to cDNA with reverse transcriptase. > > > > Joana Vieira, MEBiom, nº 55738 >