Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA

Jenny Pang, John Modlin, Robert Yolken

Research output: Contribution to journalArticle

Abstract

The inadvertent carryover of amplified fragments of nucleic acids (amplicons) is a potential source of contamination in the polymerase chain reaction. Recently, a method has been developed to generate amplicons with deoxyuracil triphosphate (dUTP) and to specifically hydrolyze these amplicons with uracil-DNA glycosylase (UNG) following the completion of the assay. We evaluated this system for the specific amplification of RNA from coxsackievirus A3 and B3. We found that RNA from both viruses could be amplified with dUTP, although the use of this triphosphate in place of TTP resulted in some loss of assay sensitivity. We also found that the dUTP-containing amplicons could be efficiently hydrolyzed by UNG, resulting in a 10,000,000-fold reduction in amplicon concentration with little effect on the native nucleic acid. The dUTP-UNG method has a great deal of potential for reducing amplicon contamination during the routine performance of nucleic acid amplification reactions.

Original languageEnglish (US)
Pages (from-to)251-256
Number of pages6
JournalMolecular and Cellular Probes
Volume6
Issue number3
DOIs
StatePublished - Jun 1992

Keywords

  • Polymerase chain reaction
  • contamination
  • coxsackievirus
  • viral diagnosis

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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