A direct selection strategy for shotgun cloning and sequencing in the bacteriophage M13.
A new cloning strategy is described which utilizes direct selection of recombinants for shotgun sequencing in the filamentous bacteriophage M13. Direct selection is accomplished by insertional inactivation of the M13 gene X protein, a powerful inhibitor of phage-specific DNA synthesis when overproduced. An extra copy of gene X was inserted into the intergenic region of M13 and placed under the control of the bacteriophage T7 gene 10 promoter and RBS. Random fragments are cloned into the EcoRV cloning site of the new gene X cistron and recombinants are selected in an E. coli male strain producing T7 RNA polymerase. Cloning efficiencies obtained with M13-100 or phosphatase-treated M13mp19 vector are comparable. The direct selection capability of M13-100 was demonstrated to have the following advantages: (a) consistently achieved high ratios of recombinants to religated vector in the libraries, averaging about 500:1 (0.2% background), and (b) the elimination of the need for phosphatase treatment of the vector to reduce background. The direct selection strategy significantly improves the efficiency of shotgun library construction in M13, and should therefore facilitate the cloning aspects of large scale sequencing projects.
Guilfoyle
R A
RA
Department of Chemistry, University of Wisconsin, Madison 53706.
Smith
L M
LM
D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli; D005814:Genes, Viral; D005822:Genetic Vectors; D015698:Genomic Library; D008969:Molecular Sequence Data; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics
D017931:DNA Primers; D014764:Viral Proteins; D015678:Viral Structural Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases