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.
Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD, USA.
Héloïse
H
Muller
Narayana
N
Annaluru
Joy Wu
JW
Schwerzmann
Sarah M
SM
Richardson
Jessica S
JS
Dymond
Eric M
EM
Cooper
Joel S
JS
Bader
Jef D
JD
Boeke
Srinivasan
S
Chandrasegaran
D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005818:Genetic Engineering / Q000379:methods*; D016681:Genome, Fungal / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D004247:DNA