Yeast gap repair without close restriction sites

[SyntonicC]

Sorry for the slightly vague title.

I am using random PCR mutagenesis to generate fragments that can be co-transformed into yeast with the cut vector which will then be gap-repaired via recombination. The problem is that I only want to randomly mutagenize a small portion of my gene (405 bp total) but the only unique restriction sites around this segment will generate a 2.8 kB fragment. So the result will be that the majority of the mutants I get will not be in my region of interest.

Are there any other alternatives that I have in this situation?

Thanks!

[mchlbrmn]

You can PCR/mutate your region of interest, and then use the PCR product as a primer set for a mutagenesis polymerization (circular polymerization around the entire plasmid). This would be the Qikchange (sorry, I never remember how they spell it) type reaction, and you can check their protocols, and calculate what concentration of your PCR product would be equal to a minimal 0.2 or 0.3 uM primer concentration. Dpn-I enzyme can then destroy the (methlyated) plasmid template, leaving only the new product.
If you don't want an entire plasmid, you can still use a plasmid template (methlyated by the bacteria) and PCR/ mutate your 405bp, then use the PCR product as a primer, along with an external primer at the end of the 2.6kb, in separate reactions to each end of the 2.6kb, gel purify the two overlapping products, then combine them along with only the two external primers to PCR the 2.6kb.
Can you understand?

Another alternative would be to do mutagenesis (the Quikchange style can work again) to add silent mutations to create restriction enzyme sites closer to where you want them.

3d method: PCR 3 segments, mutated, and two non mutated end sections. Then add klenow polymerase with only one dNTP so it runs backward until it reaches that dNTP base, and stops. This creates overhangs. Your PCR product segments are designed so they overlap for the overhang length, so the 3 segments can be ligated together. If the onverhangs are not palindromic (as they usually are) they can not self ligate so it should be easier to ligate 3 inserts together at once than with normal palindromic recognition sequence overhangs. Do you think this can work?

4: PCR two end segments, and ligate into a vector. You only need to find a 3nt 1/2 of a blunt end RE enzyme site at future the junction with your PCR/mutated segment. Much easier than finding an entire unique 6bp where you need it. The two ends are ligated into the vector in such a way that they create a new, or regenerate the same, RE site at the junction, where you can in the next step cut and ligate in the mutated piece. Cohesive overhang RE ends can be used at the ends of the 2.6 kb to ligate into vector. If you can add or mutate some nt.s it should be easy to figure out. If not, you shop for junctions of the two "wing" segments to regenerate a RE site when ligated together that when cut at the next stage provides a site to insert the mutated segment.

I'd better stop.

[mchlbrmn]

Another method. It's a variant on the last one above when I assumed you couldn't mutate your gene. This one uses silent mutations to create a cloning site for the mutated segment:

PCR the two "wings" of the gene on either side of the mutated center section. PCR to make silent mutations in the primer at the end of each wing adjacent to the mutated center. These silent mutations will create two new unique restriction sites. One site (present in both overlapping primers) is used to ligate the two sections together into a vector (containing the remainder of the gene, if I understand you), also using the restriction sites you have already located at the other ends. You now have a vector containing your gene but lacking the mutated center section, however it contains two restriction sites near each other into which the mutated section can be ligated, one that you already used in the previous ligation, and another one engineered into only one primer. It is now relatively simple to cut the vector with the two engineered restriction sites, and ligate in the randomly mutated section to complete the gene, with one section containing mutations.

[SyntonicC]

Thank you for your suggestions!

I need to think about which approach will be the most appropriate for me. I have about 4 other things going on right now that are my top priority but once those items are complete I will get back to this and post any more issues I run into. Thanks again!