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Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments

Authors
Journal
Gene
0378-1119
Publisher
Elsevier
Publication Date
Volume
243
Identifiers
DOI: 10.1016/s0378-1119(99)00508-9
Keywords
  • Bridge Oligonucleotides
  • Multifragment Ligation
  • Sequence-Specific Ligation
  • Site-Specific Ligation
  • Thermostable Ligation

Abstract

Abstract A novel DNA assembly method, chain reaction cloning (CRC), is described. CRC enables the ordered assembly of multiple DNA fragments in a single step. The power of the technique was demonstrated by the directed in vitro assembly of a plasmid comprised of six DNA fragments from a pool of 12 available fragments. The odds of obtaining the correct plasmid clone in a single step, using conventional techniques, is less than 1 in 191 000 000. Using CRC, the desired plasmid was recovered at a frequency of one in two. Ligation is no longer the rate limiting step in cloning, and limitless possibilities exist for the reconstruction of complex genomes.

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