Site-directed deletion mutagenesis using phagemid vectors and genetic selection.
Oligonucleotide-directed mutagenesis was used along with the dut and ung genetic selection method of Kunkel to introduce large site-specific deletions into cDNAs cloned into phagemid vectors. We find that large deletions can be achieved with an efficiency equal to that of single point mutations, with a very low frequency of aberrent clones. To facilitate screening of clones, E. coli strain DH5 alpha was used as the recipient host cell to genetically select for deletion mutants. Comparisons were made to deletion mutagenesis without genetic selection, and to reactions utilizing two oligonucleotide primers simultaneously. The low frequency of deletion mutants observed without genetic selection renders random screening for deletion mutant clones cumbersome. The results provide representative expectations and a useful guide for those contemplating the construction of deletion mutants.
Wang
L M
LM
University of Texas, Health Science Center.
Geihl
D K
DK
Choudhury
G G
GG
Minter
A
A
Martinez
L
L
Weber
D K
DK
Sakaguchi
A Y
AY
D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012641:Selection, Genetic
D004247:DNA