Yeast-mediated Cloning and Oligonucleotide Stitching

Yeast-mediated cloning is very similar in principle to Gibson cloning, but instead of an in vitro reaction with purified enzymes, it takes advantage of the powerful recombination abilities of yeast. Similar to Gibson, this method can efficiently fuse two (or more) fragments of dsDNA that have 30 or more bases of overlapping homology. One major advantage is that much larger final products can be generated (up to 100kb) compared to other cloning methods that utilize bacteria where it becomes progressively more difficult to clone plasmids larger than 10kb. Another advantage is the ability to perform oligonucleotide stitching, in which pieces of DNA that share no end homology can still be fused together in a seamless manner. To accomplish this, you just need to introduce into the yeast the two (or more) fragments of DNA that you would like fused along with custom ordered DNA oligos of 60-80bp in length, with 30-40bp of homology to the ends of the two fragments that you would like to fuse. A major disadvantage is that you need to be set up to grow, transform and purify DNA from yeast.

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