Colony PCR Specificity - (Sep/29/2010 )

Hi,

I recently ran the MAGE protocol to introduce a stop codon in a gene after its initial start codon as well as deleted the first following basepair in order to knock out the gene function.
The original gene went "...ATG GC." The mutation I introduced was supposed to make the sequence "...ATG TAA C." However, when I placed this latter sequence at the 3' end of my forward primer, colony PCR of a negative control gave a positive result, implying some lack of specificity. While it could be related to a low annealing temperature, this is impossible to test without a positive control. Since I have no positive control, I decided to redesign my forward primer to match the original, unmutated gene so that the presence of a band meant no knockout. Now I am still concerned about the possibility of low specificity and that I may get bands from what is actually a knockout strain. Does anyone have any recommendations for the determination of a good annealing temperature to maintain specificity of such a change in the strain? I have access to a gradient thermocycler.

Note that I have attached the picture from a prior gradient PCR if it is any help. The bands result from temperatures that decrease from left to right, and yet the band intensity decreases. The 6th well had a large amount of cell matter, so the streaking here is expected. The first five bands have visible, diminishing bands, the 6th has the streaking, and the 7th and 8th are barely visible. Annealing temperatures here decrease from 65 to 55 C.

Thanks.

The last 2 bp of primer should not match the old template.
If I understand the situation correctly you made a forward primer

original = ATG GC.

New(primer) = ATG TAA C

I noticed the primer ends with a C, which might base complement with the original sequence. (The TAA could curl up and allow the C to base pair).

I think the primer would work better if it ended in ............ATG TAA

perneseblue on Thu Sep 30 02:35:10 2010 said:

That makes sense. The second forward primer I have ordered is "ATG GCA A," (the original gene) so there should be a band for WT and no band for KO. Do you think there is any danger this time that an ATG GCA A might bind to the ATG TAA C? Two A's are not so strong as the C, so I'm hoping not.

My time and money are running short, so I'm hoping to not have to order a third primer. Thanks for your help.