REQ: PCR primers - help wanted

[Harry Witchel]

9605574b at udcf.gla.ac.uk () wrote:
>To whoever may be of help,
>
>I am currrently using a set of six primers in order to make deletions in the 
>gene for a human enzyme. All but one of my primers is working using the same 
>conditions. The one which isn't working is a G+C rich (66%) 30mer with a 
>melting temperature of 75.7 degrees C. There is a 12 degree difference in 
>melting temp. between this one and one of its pairs. Is this of any 
>significance?
>
>I have tried varying the denaturation times and the extension times. My 
>annealing temperature is 50 C which may be slightly low. There is also the 
>possibility that my primer is rather large and may have a secondary structure, 
>however I do not see any bands which may indicate primer dimerisation.
>
>Thanx
>
>
>FAB  ;-> 

Dear FAB --
   A 12 degree difference in melting temperatures certainly can have a 
big influence, but it would depend on the precise situation -- it is 
virtually impossible (despite the sales of many computer programs) to 
predict a priori whether a particular primer set will work with a 
particular template.  You mention that you do not see primer dimer bands, 
but you do not say whether you see any bands of the wrong size (which 
means your reaction is not stringent enough) or if you see absolutely no 
bands (which means that your reaction is too stringent).  I will assume 
you saw no bands at all (reaction too stringent), but if you saw a bunch 
of bands at the wrong size, then you have a reaction which is not 
stringent enough.  For a reaction that is too stringent, you can lower 
the annealing temperature, increase the cycle number, or design internal 
primers for a second PCR reaction.
   Your post sounds like a request for methods about PCR optimisation.  I 
am certain you are aware of all the possible ways to vary PCR including: 
change temperature, change the number of cycles, change magnesium, use a 
hot start, use a different enzyme, add 10% DMSO, use lowered dGTP and add 
dITP, use 7 deaza 2 deoxyguanosine, etc..  However, I would recommend as 
a first step that you change primers.  If you already know that one of 
the primers works well, you might keep that one and vary the other one, 
or you might change both primers.  While it might be best to go for a 
completely different region, if your choices are restricted by 
restriction sites and the mutations you want, you might try just moving 
the primer over by 5 or six bases -- PCR is very sensitive to precisely 
which 3' bases happen to be in the reaction.
   You do not mention whether all the PCR products you are going for are 
the same size.  Some researchers find that they have a certain size where 
reactions tend to be successful (usually short reactions <200-500) and 
that longer reactions are more difficult.  Highly GC rich regions and odd 
pairings of temperatures can interfere with a reaction's success.  You 
mention that your other PCR reactions are working, so your template is 
probably OK.
   Again, your choices are reaction optimisation and changing primers.
Good luck and mail me if you need more info:
Harry