Assembling Large DNA Segments in Yeast
As described in a different chapter in this volume, the uracil-specific excision reaction (USER) fusion method can be used to assemble multiple small DNA fragments (∼0.75-kb size) into larger 3-kb DNA segments both in vitro and in vivo (in Escherichia coli). However, in order to assemble an entire synthetic yeast genome (Sc2.0 project), we need to be able to assemble these 3-kb pieces into larger DNA segments or chromosome-sized fragments. This assembly into larger DNA segments is carried out in vivo, using homologous recombination in yeast. We have successfully used this approach to assemble a 40-kb chromosome piece in the yeast Saccharomyces cerevisiae. A lithium acetate (LiOAc) protocol using equimolar amount of overlapping smaller fragments was employed to transform yeast. In this chapter, we describe the assembly of 3-kb fragments with an overlap of one building block (∼750 base pairs) into a 40-kb DNA piece.
Key wordsSynthetic yeast USER fusion DNA assembly Large DNA fragments
This work was supported by grants from National Science Foundation (MCB0718846) to JDB, JSB, and SC; from Microsoft to JSB and JDB; and from National Institutes of Health (GM077291) to SC. HM was a recipient of a fellowship from the Fondation pour la Recherche Medicale (FRM).
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