Molecular assessment of S1 endonuclease-resistant snapback hairpin loops generated by DNA polymerase I during the in-vitro nick translation reaction

Research output: Contribution to journalArticle

Abstract

The in-vitro nick translation reaction used to label DNA to high specific activity also produces aberrant DNA structures known as "snapback" hairpin loops. Hairpin structures are precluded from participating in precise DNA-DNA hybridization interactions. Three nick translation systems were all found to yield significant quantities of snapback hairpins, as determined by their resistance to S1 endonuclease digestion following denaturation. The relative quantities of hairpins produced correlated with both the mass average size of the final DNA probe product synthesized as well as the overall rate of the nick translation reaction. Decreases in the amount of exogenous DNase I used in nick translation reactions produced significant decreases in the amount of hairpin loop structures formed. Hairpins could be effectively removed from nick-translated DNAs by employing hydroxylapatite column chromatography. Strategies to reduce hairpin formation during nick translation and the removal of hairpins from nick-translated DNAs are presented.

Original languageEnglish (US)
Pages (from-to)1-15
Number of pages15
JournalApplied Biochemistry and Biotechnology
Volume11
Issue number1
DOIs
StatePublished - Feb 1985

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DNA Polymerase I
Endonucleases
DNA
Single-Stranded DNA Breaks
Deoxyribonuclease I
DNA Probes
Durapatite
Chromatography
Digestion
Column chromatography
Denaturation
In Vitro Techniques
Labels

Keywords

  • chromatography of snapback DNA with hydroxylapatite
  • DNase I, generation of snapback hairpin DNA loops in response to
  • endonuclease, resistance of DNA to S1
  • hydroxylapatite chromatography, removal of snapback DNA by
  • labeled DNA probes
  • Nick translation
  • polymerase, generation of snapback hairpin DNA loops in response to
  • S1 endonuclease, resmstance of DNA to
  • snapback hairpin loops of DNA
  • translation, snapback hairpin DNA loops during nick

ASJC Scopus subject areas

  • Bioengineering
  • Biotechnology
  • Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Applied Microbiology and Biotechnology
  • Environmental Engineering
  • Molecular Biology

Cite this

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abstract = "The in-vitro nick translation reaction used to label DNA to high specific activity also produces aberrant DNA structures known as {"}snapback{"} hairpin loops. Hairpin structures are precluded from participating in precise DNA-DNA hybridization interactions. Three nick translation systems were all found to yield significant quantities of snapback hairpins, as determined by their resistance to S1 endonuclease digestion following denaturation. The relative quantities of hairpins produced correlated with both the mass average size of the final DNA probe product synthesized as well as the overall rate of the nick translation reaction. Decreases in the amount of exogenous DNase I used in nick translation reactions produced significant decreases in the amount of hairpin loop structures formed. Hairpins could be effectively removed from nick-translated DNAs by employing hydroxylapatite column chromatography. Strategies to reduce hairpin formation during nick translation and the removal of hairpins from nick-translated DNAs are presented.",
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