How many mismatches in point mutation primers? - (Dec/05/2005 )
I'm trying to insert a His(8)-tag in a vector using point mutagenesis with Stratagenes Quikchange kit. I can't find any information about how many bases I can change at the same time.
Is there any one out there who has the slightest idea about this?
I guess I could try to change as many bases as possible as long as the recommended primer conditions are met, but it would be nice to hear if anyone has practical experience of this.
Thanks in advance!
I am no PCR expert, but I have done more than one mutagenic PCR reaction in which I successfully inserted a tag into a sequence. For this I used the Quikchange XL kit; I would guess that doing a His tag would just as successful. To be clear, though, I did not mutate any existing nucleotides, but rather used PCR to make an insertion. To do this, I designed primers to flank the desired insertion site with about 30-40 nucleotides on each side. I used such long primers on the recommendation of someone else, and it worked. I have no idea how much shorter on each side you can go.
The answer is context-dependent. The critical aspect is going to be the length and strength of the number of matching bases. For point mutations, you can use the following equation to get a good estimate of whether your construct will amplify...
Tm= 81.5 + 0.41x(%GC) - 675/N - (%mismatch)
Where % GC is 100 x (#G + #C)/total bases
N = total length
% mismatch is 100x # nonmatching bases/N
If the Tm value is over 80, it should work fine (I've never had a Tm over 80 not work). You also want to make sure you have approximately equal bases on either side of the mismatches, and try to minimize hairpins and dimers if you can.
If you're making a large insertion, though, it may not matter. What you're ultimately looking to do is introduce a section into a construct. If you make a long primer with complementary ends on either side, it may not prime both ends, but that doesn't really matter. So long as the 3' end binds as its supposed to, it will amplify, and drag along the 5' end for the next round of amplification (when it will be recognized by your antisense primer, creating the construct you want). So, you can essentially treat it like a regular gene amplification reaction - put whatever the heck you want on the 5' end, and just make sure the 3' end makes a stronger bond (ie, more bases after the mismatch than before).
If you are just mutating one amino acid at a time (mutating 2-3 bases), the regular Quikchange kit works like a champ, just about every clone is the correct mutant. The more bases/sites you mutate, the lower the efficiency. If you want to make a lot of mutations, you can either do it in steps with intermediate mutant clones (which could be interesting in some studies but probably not in your case with the his tag) or use the Multi-site mutagenesis kit and do up to 5 sites in one shot (which btw has a 30% efficiency).
I've never tried the multi-site kit so I can't comment on it. Another thing to consider is how long your primers are going to end up being and how much it is going to cost. You can get away with fudging a little bit on the primer parameters with length, but I always make sure the Tm and %GC content and ending with G/C and not had problems.
If strataweenie got rid of the calculator, this labgroup has their own:
http://depts.washington.edu/bakerpg/primer...primertemp.html