Popular Applications for Specialized PCR: What to Know

In summary, the conversation discussed the use of Klenow fragment in DNA synthesis and its advantages over DNA polymerase III. Some popular applications of Klenow fragment include its use in electrophoretic mobility shift assay (EMSA) and DNA footprinting. It can also be used for synthesis of double-stranded DNA from single-stranded templates, filling in recessed 3' ends of DNA fragments, digesting away protruding 3' overhangs, and preparation of radioactive DNA probes. Klenow fragment has the advantage of being able to start synthesis from a primed single strand without the need for additional proteins, unlike DNA polymerase III which requires ssb protein.
  • #1
rockind78
78
0
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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  • #2
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 [Broken]

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:
  • #3
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?
 
  • #4
Klenow fragment can start synthesis from a primed single strand whereas DNA Pol III requires ssb protein in addition to a primed ss.
 

1. What is PCR and why is it important?

PCR stands for polymerase chain reaction, and it is a technique used in molecular biology to amplify a specific DNA sequence. This is important because it allows scientists to make multiple copies of a DNA sample, which is useful in many applications such as genetic testing, disease diagnosis, and DNA sequencing.

2. What are some popular applications for specialized PCR?

Specialized PCR techniques, such as real-time PCR, digital PCR, and multiplex PCR, have a wide range of applications. Some popular ones include genetic testing for hereditary diseases, detection of infectious agents, forensic analysis, and paternity testing.

3. What are the benefits of using specialized PCR over traditional PCR?

Specialized PCR techniques offer several advantages over traditional PCR, including increased sensitivity, specificity, and accuracy, as well as the ability to detect multiple targets in a single reaction. They also require less time and labor, making them more efficient and cost-effective.

4. What do I need to know before using specialized PCR?

Before using specialized PCR, it is important to have a good understanding of the technique and its principles. You should also be familiar with the specific equipment and reagents required for your chosen application. It is also essential to follow proper experimental design and optimization protocols to achieve reliable and reproducible results.

5. Are there any limitations or challenges associated with specialized PCR?

While specialized PCR techniques offer many benefits, there are also some limitations and challenges to consider. These may include the need for specialized equipment and reagents, as well as the potential for contamination and false positives. It is also important to carefully design and validate your experiments to avoid any potential bias or errors.

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