Chemical ligation of DNA: the first non-enzymatic assembly of a biologically active gene

Nucleic Acids Res. 1991 Aug 11;19(15):4247-51. doi: 10.1093/nar/19.15.4247.

Abstract

An artificial gene comprising 183 base pairs has been assembled by template-directed condensation of 35- to 53-membered oligodeoxyribo nucleotides with cyanogen bromide as a condensing agent. The reaction is complete within several minutes at 0 degrees C in buffer. The resulting mini-gene was cloned and expressed in vivo and in vitro. We have also found that the polymerase inhibition technique (toe-printing) is a good way to ascertain that translation initiation complexes form in the case of single-stranded DNAs as well. Thus, along with the fully chemical assembly of synthetic genes, a rapid and sufficiently reliable method for determining their ribosome-binding properties was developed.

MeSH terms

  • Base Sequence
  • Cloning, Molecular
  • Cyanogen Bromide / chemistry
  • DNA, Single-Stranded / metabolism
  • DNA-Directed DNA Polymerase / metabolism
  • Genes, Synthetic*
  • Molecular Sequence Data
  • Oligonucleotides / chemical synthesis*
  • Polydeoxyribonucleotides / chemical synthesis*
  • Protein Biosynthesis / physiology
  • Ribosomes / metabolism
  • Temperature
  • Transcription, Genetic / physiology

Substances

  • DNA, Single-Stranded
  • Oligonucleotides
  • Polydeoxyribonucleotides
  • DNA-Directed DNA Polymerase
  • Cyanogen Bromide