Can I make four substitutions (mutagenesis) using PCR at a time? - help me out, pls :) (Oct/05/2005 )

Hi, I need your help again. thanks in advance

say, I have an original sequence here:

ttcctcaccaatgttccgtacaagcgaatagAa gAa CTcctctataaaatttccctt

The underlined codons encode amino acids 'EEL', I want to mutate these three amino acids to 'AAA'. So, I need to substitute at least four bases in the original DNA sequence,

In the DNA sequence, the first underlined A needs to be replaced with a C

and the second A also needs to be replaced with a C also.

The underlined CT needs to be subsituted by GC.

From reading a book titled PCR Cloning Technique, I know I should put the mismatches in the center of a primer. For 4 substitutions, there should be at least 20 bases at each side of the mismatches. I am wondering if I can get such a long primer to work in the PCR mutagenesis experiments. Anybody ever had such experiences? I used the Primer Generator to design the mismatched primer, but got no response from their server, it might be too many substitutions that cause the slow response from Server?

alternatively, do I need to do two PCRs to generate the 4 substitions, two substitutions in each PCR?

I would very much appreciate your help/ideas/suggestions....sorry if I confuse you.

Thank you!
IVYTONY

Do you need the bases 5' to the mutation? What if you just engineered the mutation into the 5' end of the primer, much like you'd do to put a restriction site on a primer, and let it go?

So, in other words, go with a primer like AagAaCTcctctataaaatttccctt.

The amount of full-length primer in your sample drops off the longer your primer gets -- making a primer of 47 or so bases might be tough.

But, I'm just chatting here, as I've never tried this (well, I've certainly put a lot of 5' restriction sites on primers, but I haven't attempted what you're trying to do). Let's see what others have to say...

Hi IVYTONY,

You are dealing with a plasmid right?
I have once deleted 10 bp on plasmid via mutagenesis, so I don't expect any problem for you case.
Yes the primers were really important, there should be at least 20 bases at each side of the mismatches. you can check the instruction of those mutagenesis kit for guide lines about primer design.

good luck:)

Maybe I'm misunderstanding the experiment. My question, essentially, is why do you need 20 bp 5' to the mutation? Can't you just let the non-homologous bases that are the mutation be the 5' end of the primer, and let it drift free during annealing, much like you do when adding a restriction site to the 5' end of a primer?

Clearly, I'm missing some key point here...

I recently inserted 4 bp change in one Site directed mutagenesis reaction.

The reason why you want extra long primers, is that when you do this, after the PCR you have circular nicked DNA (and not linear). Transforming this in e. coli will render it circular (non-nicked). You can read about this on stratagene's website (quickchane site directed mutagenesis kit if I remember the name correctly, you can easily buy the PCR-enzyme, DpnI and competent cells separately).

Not to beat this to death, but how does the length of a primer influence the form of the product you'll get? If you use a ~45 bp primer, you'll get circular nicked DNA, but if you use a ~25 bp primer you won't?

If there is enough overlap between your primers, you wouldn't get too much problems.

Anyway, if you're doing 4 substitutions at one time, and you of course still need your ends to bind fine to your DNA, you will have a lengthy primer no matter what you do.