fusion PCR

[Ammie]

Anyone that have any experiences with fusion PCR?

I have many overlapping fragment at about 1000bp that I want to ligate. Some authors write that this method is better than cut and paste with restriction enzymes and ligase.

It sounds easy in theory but it is in real world?

[blcr11]

I’m not sure I’m familiar with the term “fusion PCR.” From your description it sounds like a synthetic gene application where you use PCR to assemble a gene from overlapping oligonucleotides. I think the technology sounds great. I want to use it myself, but it hasn’t worked very well for me—yet. There are a number of protocols out there for doing this—I’ve tried a couple of them, alas, without much success. The basic problem is that I never see any full-length, amplified final product—just small mw bands and smears up to (and sometimes beyond) where I expect to see a clear band. I figure I’m missing something subtle about the protocols and/or I’m making mistakes when I dilute out the oligos, or there’s something wrong with the oligos I’ve ordered, either in my design or in the way they’ve been processed. There are companies that will make the gene for you—for a fee; it will cost 1-2 USD per basepair. There are webservers and free software that can help you design your oligonucleotides, too. Search Pubmed or google "codon optimization" or "synthetic gene" and you'll get a sampling of what's out there.

Of course, it this isn’t what you’re talking about, this is all nonsense to you.

[justinjun]

Anyone that have any experiences with fusion PCR?

I have many overlapping fragment at about 1000bp that I want to ligate. Some authors write that this method is better than cut and paste with restriction enzymes and ligase.

It sounds easy in theory but it is in real world?

Yes, that what I'm doing everyday.
It's easy way to stitch two fragments which have overlapping region together(or what you called as fusion or ligation, whatever).
But, there is one thing you should pay attention to: the melting temperature should be similar or higher than the Tm of your primers (the better way: two Tm is close to each other). Otherwise, when u running PCR, the overlapping region cannot anneal together. Of coz u cannot get any correct amplicon or u just get some low MW smears.
About primer design, there are tons of free-program you can choose. Usually, I like to use Primer3 (web-based,freeeeeeee)

[Ammie]

I made a little trial... mixed two products together and I got a third. (and some weak unspecific bands) Did not look at the melting temperature of the overlapping sequences, only the primers. But I will do that now.

I have used primer3 to design primers earlier.

But why do people use restrictionenzymes and ligase when this seems to be much more easy?

Thanks for the tips justinjun, do you have any more?

[justinjun]

I made a little trial... mixed two products together and I got a third. (and some weak unspecific bands) Did not look at the melting temperature of the overlapping sequences, only the primers. But I will do that now.

I have used primer3 to design primers earlier.

But why do people use restrictionenzymes and ligase when this seems to be much more easy?

Thanks for the tips justinjun, do you have any more?

Check out this paper: "PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans".
After digest this paper, u'll find out the answer by yourself.

[Dannyboy]

I do this process routinely and have very good success with the method I have developed. It uses IDTs Oligo Analyzer and Microsoft Word to design primers for making fusions, that are injected and analyzed by microscopy for GFP expression. I have a protocol typed up if anyone is still looking for this kind of information.

[Dakota]

I would appreciate the protocol, if you're willing to give it out, with many thanks. I have spent the last month trying to figure out why my fusion is not sticking; I'm about to give up on it, but before I do I want to try redesigning primers, and seeing if I can get some other primer to take. Any tips anyone may have, I'd be glad to hear.

[Totor]

Hello Dannyboy ,
I am also interested by your protocol, for designing 5' tails of oligos, for PCR fusions of multiple fragments, while avoiding unwanted cross-talks between fragment assembly.
Best,
Totor

[jackliu]

I'm doing this these days,but I always got a smear when fusing "A"(~250bp) to "B"(~3K).I tried many times but still failed to get the sequence wanted.I'd appreciate the protocol and any suggestions. Thanks a lot.

[JackBean]

3 kbp are fairly a lot. Do you have proper protocol for amplification of long pieces?

[jackliu]

3 kbp are fairly a lot. Do you have proper protocol for amplification of long pieces?

I use a High-fidelity DNA Polymerase--KOD-Plus-(TOYOBO CO., LTD.).Long targets ranging in size from 0.6 to 3.6 kb were amplified well according to its product introduction. I followed their protocol and there was no problem in amplification of 3kb pieces.Just stuck in the junction of the two pieces.

[JackBean]

KOD Plus, that seems fine. I guess you can't try you primer pair with some positive control, can you? How many steps in cycling are you using?