Friday, April 18, 2014

PCR methods

PCR demonstration:

Method: Polymerase chain reaction (PCR).

About: PCR allows for amplification of a particular DNA sequence.

What: Ampliifcation of a gene X sequence from genomic DNA.

Ok lets start. Polymerase chain reaction is a commonly used technique. The theory behind it as well aas practical part of PCR are very simple. For PCR reaction we need:
- thermostabile polymerase - which can stand temperature up to 95 degrees Celsius,
- primers - short DNA oligonucleotides that determine DNA sequence to be amplified,
- DNA template - contains target DNA,
- buffer - supply perfect conditions for polymerase activity.

As I mentioned before theory of PCR reaction is very simple. Cartoon below represents major steps in PCR protocol:
The steps 2 - 4 are repeated between 15 - 30 times in order to obtain a workable amount of the DNA product. Time of the PCR experiment depends on the product size. After the PCR reaction small amount of the reaction mix (approximatelly 5-10%) is analysed by the agarose gel electrophoresis. Example of the gel is shown on the picture below:

As you can observe we did amplify two different DNA sequences. The positive control reaction was set up with primers that previously amplified the desired sequence from the same DNA template (product size 2.5 kb). As expected there is no signal in the negative control lane as negative control reaction did not contain the DNA polymerase. The band in the P lane (the experiment PCR reaction) need to have an expected size. Our gene of interest is 3.9 kb long.

As you can see this way we can very easily ampllify a DNA sequence of interest in order to study its sequence or cellular function.

I hope you enjoyed.
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