Use of a new retrieving adaptor in the cloning of a synthetic human insulin A-chain gene

Richard C. Scarpulla*, Saran Narang, Ray Wu

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

A DNA duplex encoding the A-chain of human insulin was constructed from eight chemically synthesized oligomers by enzymatic ligation to form a partial duplex followed by repair synthesis to complete the complementary strands. After sequential addition of translation start and stop signal adaptors the assembly was cloned in pBR322. To regenerate the end of the coding sequence by precise removal of extraneous nucleotides a new method using a synthetic retrieval adaptor was developed. The procedure included filling in the cohesive ends of the EcoRI site by repair synthesis, ligating a symmetrical adaptor having an MboII recognition sequence to the resulting blunt end, cutting with MboII and removing the single protruding 3′-nucleotide using the 3′ exonuclease activity of DNA polymerase I. Synthetic oligomers useful for ligation to a synthetic insulin C-chain gene were added to the retrieved end of the gene. Sequence analysis established that retrieval adaptors of this type may be used for precise excision of up to eight nucleotides from the end of a cloned DNA fragment.

Original languageEnglish (US)
Pages (from-to)356-365
Number of pages10
JournalAnalytical Biochemistry
Volume121
Issue number2
DOIs
StatePublished - Apr 1982

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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