PCR

Polymerase Chain Reaction
PCR is a technique that replicates DNA molecules without using living organisms, but by using enzymes in vitro (in an artificial environment). The technique allows a small amount of DNA to be amplified exponentially and thus analyzed easier- making genetic fingerprinting and the diagnosis of diseases far easier.In practice, PCR is used to amplify a specific region of the DNA strand, be it a single gene, part of a gene, or even non-coding sequences.Further more by using PCR, it can amplify a specific sequence of DNA by as many as one billion times and it is important in biotechnology, forensics, medicine, and genetic research.
 * __1. What is PCR?__**

The purpose of PCR (Polymerase Chain Reaction) is to duplicate huge numbers of gene. This process is necessary to have enough starting template for sequencing.
 * __2. What is the purpsose of PCR?__**

The PCR starts with a denaturing step. Samples of DNA are separated by subjecting them to temperatures of 94-96 degrees Celsius. The temperature is then lowered to 50-65 degrees for one to several minutes. This allows primers that are created by scientists to attach to their specifically assigned regions and “bracket out” the DNA region to be amplified. The temperature is then raised to 72 degrees for several minutes to allow the //Taq// Polymerase enzyme to attach at each primer site, synthesizing a new DNA strand. This cycle can be repeated over and over until many replicated strands of the DNA are made.
 * __3. How does PCR work?__**

Three major steps are required during PCR (Polymerase Chain Reaction), which are repeated 30 ~ 40 times over. This is done on an automated cycler ( - cycling reaction) which can heat and cool the tubes with the reaction mixture in a very short time thus allowing polymerase to break and stick to synthesis a new DNA strand.
 * **The cycling reactions**

The first step of PCR is the denaturing step. The samples are heated to 94 ~ 96 °C for one minute approximately to separate the single strands of the target DNA. While the denaturation process is occuring, the double stranded DNA melts open cause of the heat to a single stranded DNA, which makes all enzymatic reactions to stop.
 * **Denaturation (Step 1)**

Next the temperature is lowered to 50-65°C for around one minute. This lowering of temperature allows the left and right polymers to anneal to their complementary base sequence. The Primers are designed to signify the specific part of a DNA to be amplified. Polymerase that wanders around can attach to the Double DNA strand and start copying the template.
 * **Annealing (Step 2)**

The temperature is increased to 72°C for several minutes. Since in this stage it is the ideal condition (temperature) for polymerase to attach, Polymerase is attached at each priming site (template) and synthesizes or extends a new DNA strand.
 * **Extension (Step 3)**

Since both strands of the original DNA is amplified the numbers from now on there is an exponential increase happening, which means the number of gene increases by X2 (X meaning total number of genes). See the figure below.



http://www.dnalc.org/ddnalc/resources/pcr.html The reaction of enzymes to replicate a DNA strand happens in a heat controlled environment- specifically, small reaction tubes that are inserted into a thermal cycler. This “thermal cycler” is a machine that heats and cools the reaction tubes within it so each step of the PCR is undergone at optimal temperatures. "Polymerase Chain Reaction." __Gene Almanac__. Dolan DNA learning center (Cold Spring Harbor Labs). 15 Feb. 2007 <[|http://www.dnalc.org/ddnalc/resources/pcr.html>.] PCR (Polymerase Chain Reaction) Also see the website below to see another type of PCR process animation. [|http://users.ugent.be/~avierstr/principles/pcrani.html]
 * __4. To view an interesting PCR process animation, go to Cold Spring Harbor Laboratory’s website:__**

Although Polymerase Chain Reaction is an effective and strong technique, it is quite hard to perform PCR. Firstly, polymerase reaction depends on the size of the DNAs to be amplified. This means that the reaction could be limited by distance between the primers. Secondly, taq polymerases make mistakes. It is reported that polymerases make frequent mistakes during the process of PCR. Thirdly, primer design is extremely important during the amplification. The primers for the reaction must be well-organized and specific for the DNA sample to be amplified. Additionally, PCR is really sensitive to temperatures, nucleotides, and other factors.
 * __5. Difficulties of Polymerase Chain Reaction__**

__**6. Applications of Polymerase Chain Reaction**__ Polymerase chain reaction can be used in various cases. PCR can be used for identifying diseases caused by the infectious agent’s DNA. In forensic use, the test can be used to compare two samples of DNA. PCR can be also used in taxonomic classification to help showing evolutionary relationships between organisms on the molecular level. PCR method can be used when only very small samples of DNA are available. Therefore even tiny pieces of preserved tissue from extinct animals can be tested by PCR.

__**7. Links:**__ http://www.accessexcellence.org/RC/VL/GG/polymerase.html http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html http://www.dnalc.org/ddnalc/resources/pcr.html http://users.ugent.be/~avierstr/principles/pcr.html http://faculty.plattsburgh.edu/donald.slish/PCR.html "polymerase chain reaction." __The American Heritage® Dictionary of the English Language, Fourth Edition__. Houghton Mifflin Company, 2004. //Answers.com// 15 Feb. 2007. http://www.answers.com/topic/polymerase-chain-reaction "polymerase chain reaction: Development and Applications." //The Columbia Electronic Encyclopedia.// © 1994, 2000-2006, on Infoplease. © 2000–2007 Pearson Education, publishing as Infoplease. 01 Mar. 2007 http://www.infoplease.com/ce6/sci/A0860463.html.