Polymerase chain reaction
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Introduction
Kary Mullis developed the technique of the polymerase chain reaction (PCR) in 1983. He won a Nobel Prize for the procedure in 1993 as it became quite clear PCR had revolutionized the worlds of biochemistry and genetics. PCR enables researchers to amplify extremely small samples of DNA, even one molecule, into virtually unlimited quantities. PCR is now a common laboratory procedure, and because of its broad range of applications, it is rapidly reaching the nonscientific community as well. For example, PCR is used in paternity testing. The genes of the father can be amplified and compared to the child's DNA for similarities. Courts are also using PCR to compare genetic evidence found at a crime scene to that of a suspect's DNA. This usage enables even the smallest amount of DNA to act as a witness to the crime allowing a greater resolution between guilt and innocence. PCR is also helping in the isolation of genes and the decoding of the human genome itself.

 


How it works
The process of PCR occurs in three steps: denaturing, annealing and extension. Each repetition of these steps constitutes one cycle and doubles the amount of DNA present in the sample. Each of these steps is conveniently triggered by temperature. Temperature regulation enables the ingredients for each step to be mixed in the same container. All the researchers need do is mix up the recipe. The raising and lowering of the temperature does the rest. In denaturing, the temperature is raised to approximately 94'F. The heat breaks the hydrogen bonds between the amino acids of each strand of DNA's double helix. The helix thus unwinds and separates into two strands. In the next step, annealing, the temperature is lowered to about 58'F and allows the primers to attach. Primers are small segments of DNA that bind in a complementary fashion to the strand intended for copy. Primers can be made synthetically (de novo) or by breaking up an original sample of the initial strand. The primers attach to the singular strands of the sample by reforming hydrogen bonds. The primers enable the attachment of the DNA polymerase during extension. The polymerase must have a segment of intact DNA on the complementary strand to begin the process of attaching base pairs and reforming or copying the strand. Since the DNA polymerase can only copy in one direction along the strand, the primers can be selectively chosen to amplify a specific segment of DNA or the entire strand. For extension the mixture is warmed to activate the polymerase. As mentioned, the polymerase attaches to the primer and attaches base pairs in a 3' to 5' direction. The terms 3' and 5' are in reference to the carbons of the sugar molecule in the sugar-phosphate backbone structure of the DNA strand. As the polymerase attaches nucleotides to the new strand in complement to the mother strand, they form hydrogen bonds and reform the original structure following the primer sequence. The mixture can be reheated to continue the cycle as long as there are enough primers and nucleotides to continue synthesizing new strands.

 

 

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