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The first step in the polymerase chain reaction is?

The first step in the polymerase chain reaction is? - PCR - Polymerase Chain Reaction Forum

The first step in the polymerase chain reaction is? - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 11-29-2011, 01:16 PM
Pipette Filler
 
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Default The first step in the polymerase chain reaction is?



a. primer extension
b. denaturation
c. annealing
d. cooling
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Old 11-29-2011, 01:16 PM
Ben Ben is offline
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The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

The steps are as follows:
1)Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 9498 C for 2030 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

2)Annealing step: The reaction temperature is lowered to 5065 C for 2040 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

3)Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 7580 C,[10][11] and commonly a temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

4) Final elongation: This single step is occasionally performed at a temperature of 7074 C for 515 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
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Old 11-29-2011, 01:16 PM
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B. Denaturing
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Old 11-29-2011, 01:31 PM
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Default Re: The first step in the polymerase chain reaction is?

To second Greg :-)
Denaturing at about 95 degrees.
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