Unwanted frameshift after overlap extension PCR

Go to the profile of marilynliddell
Jun 07, 2018
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Hi everyone,

I did an overlap extension PCR to introduce a couple of point mutations in the middle of a gene sequence. The construct did not express any protein when transfected. Later, I sequenced the construct to find that there has been a frameshift, exactly at that point where one of the amino acids was mutated, thereby leading to truncation, etc.
I intend to do the whole thing all over again, but I'm confused what step to start with. I am confused whether the frameshift was introduced in the Overlap PCR step (where two initial products are thermocycled for a few cycles before adding the flanking primers) or in one of the earlier PCRs which amplified the two products to be spliced together. Is it the overlap extension step where there is a higher chance of a shift being introduced?

Since I haven't encountered this problem before, I am unsure if I should just do the overlap extension again (I have the two smaller PCR products), or start from scratch. The primers seem to be fine.

Help!

2 Comments

Go to the profile of Roberto Rosati
Roberto Rosati 5 months ago

Hi,
If I understood correctly, you did overlap extension PCR, did the cloning, and then just randomly selected one clone without sequencing it, and went on transfecting? That's a bit scary.
Mutations can indeed happen... the safe approach is to sequence your inserts after any PCR step.

Go to the profile of mchlbrmn
mchlbrmn 5 months ago

Hello, I'll add thoughts, although I'm probably too late.
As R.rosati says, it's necessary to sequence a new construct before using it.
You probably do not need to repeat the experiment if you still have the bacterial plate used, as you can simply pick several more clones and sequence them to find a good one. It is not unusual to find a bad clone with a mutation. This can be a PCR error, or perhaps an artifact from the DNA secondary structure. If you don't have the bacteria, but still have the ligation (or assembly reaction), you can retransform and pick more colonies.
First, you should double check the primer design for a mistake. If that was the case, all clones could have the problem.