A polymerase chain reaction-mediated yeast artificial chromosome-splitting technology for generating targeted yeast artificial chromosomes subclones.
The yeast artificial chromosomes (YAC) system makes it possible not only to clone large DNA fragments but also to simplify the physical mapping and functional analysis of chromosomes and genomes through YAC manipulation. To manipulate large DNA fragments cloned into YACs, YAC fragmentation has been developed and also a new method called the YAC-splitting method was developed recently. Although the YAC fragmentation method can be used to delete DNA from one side of a YAC, the YAC-splitting method generates two smaller YACs, both of which are transmitted to daughter cells during mitotic growth. This chapter describes the YAC-splitting method improved by incorporating polymerase chain reaction-mediated chromosome splitting (PCS) technique and by adding yeast autonomously replicating sequence (ARS) to the system. The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event) with the Cre/loxP system for marker rescue. The improved YAC-splitting method can convert a targeted region of a eukaryotic chromosome within a YAC into a new replicating YAC.
Kim
Yeon-Hee
YH
Laboratory of Molecular Genetics, Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Sugiyama
Minetaka
M
Kaneko
Yoshinobu
Y
Fukui
Kiichi
K
Kobayashi
Akio
A
Harashima
Satoshi
S
D018244:Chromosomes, Artificial, Yeast; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods*; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal; D016521:Electrophoresis, Gel, Pulsed-Field; D015723:Gene Library; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004271:DNA, Fungal