Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides

Gustavo Caetano-Anollés, Brant J. Bassam, Peter M. Gresshoff

Research output: Contribution to journalArticlepeer-review

Abstract

DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5′ terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3′ terminus. Increasing annealing temperatures from 15° to 70° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3′-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.

Original languageEnglish (US)
Pages (from-to)157-165
Number of pages9
JournalMGG Molecular & General Genetics
Volume235
Issue number2-3
DOIs
StatePublished - Nov 1 1992
Externally publishedYes

Keywords

  • DNA amplification fingerprinting (DAF)
  • PCR
  • Primer-template interactions
  • Single oligonucleotide primer

ASJC Scopus subject areas

  • Genetics

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