Terminating PCR: How to Control DNA Amplification Length

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In summary, the conversation discusses the termination of PCR and how to ensure that only a specific region of DNA is amplified. It is explained that primers play a role in determining the start of the new strand and that two primers are typically used. The length of the amplified DNA copies from the second and third generation is also mentioned. It is clarified that although the polymerase may go beyond the region of interest, the primer will only bind to the start of the region in the next cycle, resulting in only the region of interest being left after a few cycles.
  • #1
nokia8650
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I have a question regarding PCR which I was unable to find the answer to (maybe because its obvious and I am just not thinking of the reason!) How is PCR terminated? For instance, say you wish to amplify a 2kb length of dna, how does one ensure that just this 2kb region is amplified as opposed to a 100kb region for instance - how do you make sure that the polymerase doesn't just keep copying the template strand in a given cycle. I know that there are the denaturation, annealing and elongation phases, but I just don't understand how the elongation phase is terminated at the right point.

Thanks
 
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  • #2
What role primers play?
 
  • #3
primers determine the 5' start of the new strand... not the 3' end?
 
  • #4
Do you use, or two primers? Why?

Think what is length of a copies from the second & third DNA generation. To simplify things imagine you are throwing away all earlier generations before starting next cycle.

That is not exactly the case, but it doesn't matter much.
 
  • #5
I started to reply about how i still didnt understand, but then as I tried explaining why I saw the reason! Although the polymerase may go beyond the region of interest, on the next cycle, the primer will only bind to the start of the region of interest, and so after a few cycles, youll be left with just the region of interest.

Thanks so much!
 

1. What is PCR and why is it important in DNA amplification?

PCR stands for Polymerase Chain Reaction and it is a laboratory technique used to amplify specific DNA sequences. It is an important tool in molecular biology as it allows researchers to make multiple copies of a specific DNA region, making it easier to study and analyze.

2. How does PCR work to amplify DNA?

PCR uses a process of heating and cooling cycles to amplify DNA. The DNA sample is first heated to denature it, separating the double-stranded DNA into single strands. Then, DNA polymerase enzyme is used to add nucleotides to the single strands, creating two new double-stranded DNA molecules. This process is repeated multiple times, resulting in a significant increase in the amount of DNA.

3. What is the purpose of terminating PCR and how is it done?

The purpose of terminating PCR is to control the length of the amplified DNA fragments. This is important because it allows for more precise amplification of specific DNA regions. Termination can be achieved by using specific primers that bind to the ends of the DNA region of interest, or by adding special nucleotides that halt the PCR reaction at a specific point.

4. How can I determine the ideal length for terminating PCR?

The ideal length for terminating PCR will depend on the specific DNA region you are amplifying and the purpose of your experiment. It is important to consider the size of the DNA fragment you want to amplify and the accuracy of the PCR process. Generally, a length of 150-300 base pairs is recommended for most applications.

5. Are there any risks or limitations to terminating PCR?

There are a few potential risks and limitations to terminating PCR. One of the main risks is the potential for contamination, which can lead to inaccurate results. It is important to follow proper laboratory protocols and use sterile techniques to minimize the risk of contamination. Additionally, terminating PCR may not be suitable for amplifying very long DNA fragments, as it can be difficult to control the length accurately. It is important to carefully consider the limitations and potential risks before using terminating PCR in your experiments.

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