Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases

Anal Biochem. 1990 Feb 15;185(1):187-93. doi: 10.1016/0003-2697(90)90278-h.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophages / genetics*
  • Base Sequence
  • Chromosome Deletion*
  • Cloning, Molecular
  • DNA Restriction Enzymes / metabolism*
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • DNA, Viral / genetics
  • DNA, Viral / metabolism*
  • Electrophoresis, Agar Gel
  • Genetic Techniques
  • Restriction Mapping
  • Transfection

Substances

  • DNA, Single-Stranded
  • DNA, Viral
  • DNA Restriction Enzymes