PCR "cloning" would this be possible?

[Ammie]

Theoretically it (the following) would work, wouldn't it? (Not so experienced...)

First step:
I have a plasmid with an insert. I want to mutate two nucleotides. I order four primers, the first primer (A- forward) is design from the area of the first nt with the mutant nt in the middle of the primer. The second primer (B-reverse) is from the second area with the second mutant nt in the middle om the primer. When used together in PCR they give a product that is a part of the insert with the desired mutated nucleotides.

The third primer (C- reverse) is complementary to A, and the fourth primer (D-forward) is complementary to B. When they are used together they give a PCR product of the rest of the insert together with the plasmid-sequence.

Second step:
I use a lot of starting material of the both PCR products and in equal amounts of copies/ends and add them together with a polymerase (pfu Ultra maybe), appropriate buffer, water and dNTPs. Now they will anneal to eachother, because they are overlapping in each end and they are elongated giving a circle with two nicks (at different places). No amplification occur.

(I have made a little picture to illustrate it (were not allowed to have it larger))

Than I can use this mixture for transfection, and the "ligated" products give colonies...

So, what do you think?

[danfive]

Remember that synthetic oligos (primers) lack 5'-terminal phosphate groups required for ligation.