Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA.
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.
Pang
J
J
Department of Pediatrics, Johns Hopkins University, Baltimore, Maryland 21205.
Modlin
J
J
Yolken
R
R
D016477:Artifacts; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D045647:DNA Glycosylases; D015342:DNA Probes / Q000378:metabolism; D003856:Deoxyuracil Nucleotides; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D005189:False Positive Reactions; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D051981:Uracil-DNA Glycosidase
D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D012367:RNA, Viral; C027078:deoxyuridine triphosphate; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase