PCR using different primers to generate same DNA sequence - explanation please :) (Feb/25/2006 )
I've been trying to understand this, maybe u can help me out .
For my project, i had to determine the presence of a particular gene in different serotypes of the same bacteria using PCR. A primer set was chosen( reverse and forward), and PCR was done. This amplified DNA product in all serotypes of the bacteria except for a few serotypes.
Explanation for this is that it could be that the gene were not present in those serotypes or that there might a genetic polymorphism in the primers used, so different primers may need to be used.
Therefore, different set of primers were chosen to be used in repeat PCR for the serotypes that did not work initially in the experiment above. My supervisor said that if it still doesnt work, then you can safely say that the gene is not present in the bacteria of that specific serotype.
Can someone explain to me how this really works (eg. polymorphism, gene not present if 2nd primers dont work)????? Thanks!!!
It is very difficult to conclude whether a gene is absent based on a negative PCR. In fact, even the presence of a PCR product does not mean a gene is there, if by "there" you mean active and functional -- a positive PCR product could be detecting the presence of an inactive gene; one that has acquired a stop codon in the middle of it, for example.
Amplification of a PCR product means only that there is DNA that matches your primer sequences, and that these matching segments are separated by an expected amount of DNA. Further experiments are required to conclude anything further.
There are many reasons why a PCR reaction might fail, only one of which is that the gene isn't there. As I said before, it's difficult to conclude anything from a negative PCR, except that it didn't work.
One of your primers is located in the gene where their could be a pm or a deletion. If you continue to move down (or up) from the site, you are still in the deleted area. If you would have moved the primers and got product I would safely say there is a polymorphism. Try making a new set of primers. Your forward primer (current) make it reverse and go upstream and create a new forward primer. Next, take your reverse primer and make it forward and create a new reverse primer. It may be that only one primer is in the deleted area or the polymorphic site. This may help you identify the area of interest. Also, it just depends how large the deletion is!