Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast.
With efficient homologous recombination in Saccharomyces cerevisiae, a rapid in vivo cloning technique has been available. Here we demonstrated that 30 bp of a homologous sequence at each end of a DNA fragment is sufficient to integrate the fragment into a linearized plasmid in yeast. To obtain a high yield of recombination transformants, however, more than 60 bp are desirable. Interestingly, we observed that 20 bp of homology at one end of a DNA fragment is sufficient for efficient recombination provided that the other end contains 80 bp of homologous sequence. Some applications, including high-throughput transferring of EST inserts to the yeast expression systems for the Human Genome Project, are discussed.
Hua
S B
SB
GeneNet Group, CLONTECH Laboratories, Inc., Palo Alto, California 94303, USA. sbhua@clontech.com
Qiu
M
M
Chan
E
E
Zhu
L
L
Luo
Y
Y
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D017931:DNA Primers; D016159:Tumor Suppressor Protein p53