Polymerase chain reaction: Difference between revisions
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The '''polymerase chain reaction''' (PCR) is a technique used | The '''polymerase chain reaction''' (PCR) is a biochemical technique frequently used in molecular biology, and law enforcement, to amplify the amount of DNA present in a sample. Typically, the reaction is repeated a few dozen times, during which the DNA content is doubled at each cycle. The technique requires the DNA to be amplified, a DNA [[poylmerase]] enzyme, such as Taq-polymerase, to elongate primers, two DNA primer sequences that are complimentary to the ends of the sample DNA, and DNA [[nucleotide]]s to be incorporated into the new strands of DNA. | ||
==Process== | ==Process== | ||
A PCR reaction consists of three stages: denaturation, annealing and extension. This cycle is repeated until enough DNA is produced, | A PCR reaction consists of three stages: denaturation, annealing and extension. This cycle is repeated until enough DNA is produced, generally after 30-40 cycles. | ||
===Denaturation=== | ===Denaturation=== | ||
In the first step, the denaturation, the temperature is raised to above 90° C. Due to the high temperature, the hydrogen bonds between the two strands of DNA break and the strands are separated. Now the desired target DNA is made accessible for the next stage | In the first step, the denaturation, the temperature is raised to above 90° C. Due to the high temperature, the hydrogen bonds between the two strands of DNA break and the strands are separated. Now the desired target DNA is made accessible for the next stage. | ||
===Annealing=== | ===Annealing=== | ||
Revision as of 14:02, 25 May 2008
The polymerase chain reaction (PCR) is a biochemical technique frequently used in molecular biology, and law enforcement, to amplify the amount of DNA present in a sample. Typically, the reaction is repeated a few dozen times, during which the DNA content is doubled at each cycle. The technique requires the DNA to be amplified, a DNA poylmerase enzyme, such as Taq-polymerase, to elongate primers, two DNA primer sequences that are complimentary to the ends of the sample DNA, and DNA nucleotides to be incorporated into the new strands of DNA.
Process
A PCR reaction consists of three stages: denaturation, annealing and extension. This cycle is repeated until enough DNA is produced, generally after 30-40 cycles.
Denaturation
In the first step, the denaturation, the temperature is raised to above 90° C. Due to the high temperature, the hydrogen bonds between the two strands of DNA break and the strands are separated. Now the desired target DNA is made accessible for the next stage.
Annealing
In the next stage, the annealing, the temperature is cooled down between 40 and 60° C. The precise temperature is very important and should be determined before every reaction. After it is cooled down, the primers will bind to the DNA. Primers are short single strands of DNA that are complementary to the 3’ end of the target DNA.
Extension
In the third stage, the extension, the temperature is raised again, most of the time to 72° C, the optimal temperature of Taq-polymerase. The polymerase will bind to the primers and copy the target DNA in one direction, to the 5’ end. When the polymerase is done, two partially double-stranded DNA molecules are produced.