Analyse mutant clone - (Oct/02/2009 )

Hello,
I generated a PCR based insert clone that contains a S130A mutation. I tarnsformed it and got a few colonies. I mini-prepped some of the colonies and want to check for my insert. The best and the obvious way in to sequence it. Is there any other way? There was no special restriction site 'generated' because of the resulting nucleotide change. Some tell me one can check a mutation by PCR. How is that possible? Or can you suggest any other way? Even an abstract/theorical mode is worth knowing.
Regards!

Hey,

Chk if u kill any restriction site while engineering the mutant?
Best,
TC

Rituparna on Oct 3 2009, 07:52 AM said:

Thanks. I checked if I deleted/created any restriction site during engineering the mutant. No I did not create or delete any. What could I do apart from sequencing?

Thanks,
R

First, did you check your transformants for inserts of the appropriate size? No sense sequencing anything until you're reasonably sure you've got something worth sequencing... You can do a restriction digest of your plasmid clone DNA and compare it to similarly digested vector-only DNA to see if you've got an insert of appropriate size, or do a PCR on your clone to detect the insert.

Why do you not want to do sequencing?

I will do the sequencing but was curious if there were other ways of knowing if my mutant insert is there. MY mutant was generated from the wild type insert, so I think there should be another way of knowing if the mutation that was generated by using standard PCR took effect in the "wild type' to give me my mutant insert.
R

You could pick a primer that has the missing base(s) at the 3' end. In a PCR reaction, this primer should fail, but it should work in the wild-type plasmid.

The problem with this approach is the possibility of false negatives -- PCR reactions that fail to produce an amplicon for reasons other than the missing 3' bases.

Thanks Homebrew. There is no missing base in the 3' end; just a substituted base. So if I use the primer whose 3' corresponds to the mutant, the mutant insert should be amplified. But this same primer should not amplify the wild type (as wild type has different 3'. Is that correct?

Thanks!

That's correct -- and the issue of false negatives still applies. Keep the PCR conditions tight and don't overdo it on the number of cycles, and you'll probably be okay as a screen. As a kind of control, you could buy two primers, one with the wild-type base in the 3' position and one with the mutant base there, and run both of them against your putative mutant, each coupled with the same third primer as partner -- one set should produce an amplicon, and the other should fail.