Popular Applications for Specialized PCR: What to Know

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Discussion Overview

The discussion centers around the popular applications of specialized PCR techniques, with a focus on specific uses and comparisons between different DNA synthesis methods.

Discussion Character

  • Exploratory, Technical explanation, Conceptual clarification

Main Points Raised

  • One participant inquires about specific popular applications of specialized PCR.
  • Another participant mentions the use of PCR for synthesizing double-stranded DNA for electrophoretic mobility shift assays (EMSA) and DNA footprintings, listing several applications including synthesis from single-stranded templates and preparation of radioactive DNA probes.
  • A follow-up question is raised regarding the advantages of using Klenow fragment for synthesizing double-stranded DNA from single-stranded DNA compared to using DNA polymerase III.
  • It is noted that Klenow fragment can initiate synthesis from a primed single strand, while DNA polymerase III requires single-stranded binding protein in addition to a primed single strand.

Areas of Agreement / Disagreement

Participants express varying levels of understanding and curiosity about the applications and mechanisms of specialized PCR, but no consensus is reached on the advantages of one method over another.

Contextual Notes

The discussion includes assumptions about the knowledge of PCR techniques and specific enzyme functions, which may not be universally understood by all participants.

rockind78
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I know what it is, but my question is: What are some (specifically) popular applications? I am guessing some sort of specialized PCR? Any thoughts would be appreciated.
 
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I know we use it to double strand DNA for electrophoretic mobility shift assay (EMSA) and DNA footprintings.

From the following site
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/klenow.html

1. Synthesis of double-stranded DNA from single-stranded templates
2. Filling in recessed 3' ends of DNA fragments
3. Digesting away protruding 3' overhangs
4. Preparation of radioactive DNA probes
 
Last edited by a moderator:
Thank you Ian, that is just what I wanted to know! :smile: This brings me to my next question I guess, being, what advantage would there be in utilizing JUST klenow fragment in synthesis of DS DNA fragments from SS DNA versus DNA polymerase III?
 
Klenow fragment can start synthesis from a primed single strand whereas DNA Pol III requires ssb protein in addition to a primed ss.
 

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