Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA.
Yeast artificial chromosomes (YACs) are useful cloning vectors that have the capacity to carry large DNA inserts. The largest barrier to using such large DNA molecules in transformation experiments has been their physical instability in solution. We developed a new method of transforming yeast using chromosome-sized DNA. The method uses calcium alginate microbeads to immobilize high-density yeast chromosomal DNA. Chromosomal DNA immobilized on microbeads is physically stabilized compared with naked chromosomal DNA. The microbead-mediated transformation performed well, not only with respect to the transformation frequency with large DNA molecules (> 100 kb) but also in successful transformation using split chromosome DNA that exceeded 450 kb.
Osaka University, Osaka, Japan.
Atsushi
A
Mizukami
Eiji
E
Nagamori
Yukiko
Y
Takakura
Sachihiro
S
Matsunaga
Yoshinobu
Y
Kaneko
Shin'ichiro
S
Kajiyama
Satoshi
S
Harashima
Akio
A
Kobayashi
Kiichi
K
Fukui
D000327:Adsorption; D000464:Alginates / Q000737:chemistry*; D015825:Chromosomes, Fungal / Q000235:genetics; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D004271:DNA, Fungal / Q000008:administration & dosage*; D016503:Drug Delivery Systems / Q000379:methods*; D005240:Feasibility Studies; D018014:Gene Transfer Techniques; D020723:Glucuronic Acid / Q000737:chemistry*; D006603:Hexuronic Acids / Q000737:chemistry*; D012440:Saccharomyces / Q000187:drug effects* / Q000235:genetics*; D013499:Surface Properties; D014170:Transformation, Genetic / Q000235:genetics*
D000464:Alginates; D020099:Coated Materials, Biocompatible; D004271:DNA, Fungal; D006603:Hexuronic Acids; D020723:Glucuronic Acid