Terminating PCR: How to Control DNA Amplification Length

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SUMMARY

The discussion focuses on the termination of PCR (Polymerase Chain Reaction) and how to control the amplification length of DNA, specifically targeting a 2kb region. It is established that primers play a crucial role in determining the starting point of amplification, binding to the 5' end of the target region. Although polymerase may extend beyond the desired length during elongation, subsequent cycles will only amplify the region defined by the primers, effectively limiting the product to the target length after several cycles. This understanding clarifies the importance of primer design in PCR protocols.

PREREQUISITES
  • Understanding of PCR (Polymerase Chain Reaction) fundamentals
  • Knowledge of primer design and function in DNA amplification
  • Familiarity with DNA polymerase mechanisms
  • Basic concepts of DNA replication and amplification cycles
NEXT STEPS
  • Research optimal primer design techniques for PCR
  • Learn about different DNA polymerases and their properties
  • Explore methods for verifying PCR product length, such as gel electrophoresis
  • Investigate advanced PCR techniques, including quantitative PCR (qPCR)
USEFUL FOR

This discussion is beneficial for molecular biologists, genetic researchers, and laboratory technicians involved in DNA amplification and analysis, particularly those looking to optimize PCR protocols for specific target lengths.

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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What role primers play?
 
primers determine the 5' start of the new strand... not the 3' end?
 
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.
 
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!
 

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