Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases.
A method for creating a group of deletion subclones for DNA sequencing by partial digestion of M13 bacteriophage constructions is outlined. The M13 construct is linearized at a unique site and then subjected to partial digestion with a frequent-cutting restriction endonuclease. The insert is truncated at different locations. The vector DNA is also partially digested. The products of a single partial digestion are repaired, recircularized by ligation, and used for bacterial transfection to generate subclones with a spectrum of deletions in the insert; most deletions in the vector DNA will disrupt vital viral genes and will thus disappear in the transfection. The subclones are sorted by size by gel electrophoresis of single-stranded viral DNA. This method is simpler and thus may be more reliable than established subcloning schemes.
Lamperti
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ED
Department of Cell Biology, University of Massachusetts Medical School, Worcester.
Villa-Komaroff
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D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D005821:Genetic Techniques; D015183:Restriction Mapping; D014162:Transfection
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004262:DNA Restriction Enzymes