A "double adaptor" method for improved shotgun library construction

Anal Biochem. 1996 Apr 5;236(1):107-13. doi: 10.1006/abio.1996.0138.


The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

Publication types

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

MeSH terms

  • Base Sequence
  • Cloning, Molecular / methods*
  • Genetic Vectors
  • Genomic Library*
  • Humans
  • Molecular Sequence Data
  • Oligonucleotides / chemistry
  • Polymerase Chain Reaction
  • X Chromosome


  • Oligonucleotides

Associated data

  • GENBANK/AF007128
  • GENBANK/AF007129
  • GENBANK/AF007130
  • GENBANK/AF007131
  • GENBANK/AF007132
  • GENBANK/AF007133
  • GENBANK/AF007134
  • GENBANK/AF007135
  • GENBANK/AF007136
  • GENBANK/AF007137
  • GENBANK/AF007138
  • GENBANK/AF007139
  • GENBANK/AF007140
  • GENBANK/AF007141
  • GENBANK/AF007142
  • GENBANK/AF007143
  • GENBANK/AF007144
  • GENBANK/AF007145
  • GENBANK/AF007146
  • GENBANK/AF007147
  • GENBANK/AF007148
  • GENBANK/AF007149
  • GENBANK/AF007150
  • GENBANK/AF007151
  • GENBANK/AF007152
  • GENBANK/AF007153
  • GENBANK/AF007154