Repeat of desired mutation in cloning product - (Oct/12/2012 )

Hi everyone, I have been attempting a single amino acid substitution for the longest time and noticed that I keep getting a repeat of the desired mutation in my product. However, it's interesting in that the repeat is very specific, and I believe that it corresponds with my primers (rather than being the mutation + random sequence + mutation).

For context, I want to turn a leucine into an inert alanine. This mutation would occur about 30 amino acids after the first restriction site I want to use to cut out the insert, and 20 amino acids before the second restriction site. So in order to ensure that these restriction sites are incorporated into the cloning product, I designed a forward primer that starts a few amino acids ahead of the desired mutation, and a reverse that starts a few amino acids after, so there is overlap between the two primers. I put both the forward and reverse primers into a PCR reaction, with the idea that this would double the odds of having a linear PCR product that extended enough to reach the next restriction site, and then do a blunt-end ligation. What I think is happening though is that the products from the forward and reverse reactions are ligating to each other. What the clone ends up looking like (and I've tried two different mutations) is that I get the target sequence, then my mutation, then it continues and abruptly goes back to another point in the sequence, then my mutation appears, then the sequence continues. It matches up perfectly with a sequence going through to the start of the reverse primer, then product from the forward primer starts.

However, I imagine that if this occurred, the clone would be twice the size of the PCR products, but they both end up around 5 kb.

I guess given these results, I have 2 questions: (1) what mechanism causes the products of the fwd and rev primer to ligate to each other, is it just more energetically favorable? And (2) If I want to reach the two restriction sites, how can my cloning strategy be improved?

Thank you so much for hearing me out!
C

I'm not sure I'm following how your cloning strategy works. Do you have a picture of that type of cloning? I'll just say what I do for introducing amino acid substitutions, and hopefully that'll help you figure out what might make your PCRs turn out the way they are.

I've always used two sets of primers, unless the mutation is so close to a restriction site that I can just include the new sequence in the primer (within maybe 10 bp of the restriction site). Otherwise, I'll make two pieces, purify them (Qiagen PCR Purification), then mix them with only the outside primers in a bridge PCR.

The first piece will use a primer annealing to the desired 5' restriction site and a reverse primer with your mutation, with 10 bases past the mutation site (so in the bridge PCR, it'll anneal well to the other piece). The second piece will have a forward primer with your desired mutation (again with 10 bases past the mutation site) and the reverse primer at the other 3' restriction site.

After you purify these products, I usually just use 1 ul of each piece (probably way more than necessary), and use the outside forward and reverse primers (with your 5' and 3' restriction sites in them) to amplify the whole final product with your mutation in the middle.

I'll usually PCR purify, then do my restriction digest, and then gel purify the product, as you want to separate the full-length product from the pieces. Then ligate into your cut vector as usual.

Does that make sense? Or am I completely misunderstanding the cloning you need to do?