Application of hydroxylamine hydrochloride for post-PCR sterilization.
A major problem in the application of polymerase chain reaction (PCR) in diagnostic laboratories is contamination with exogenous nucleic acid, especially aerosolized amplicon, from previous PCR. Although several pre- and post-PCR sterilization techniques have been proposed, an optimal sterilization technique is not yet available. Hydroxylamine hydrochloride is a mutagenic agent that binds to and chemically modifies DNA. In the present study PCR was performed on DNA extracted from Herpes simplex virus (HSV) and Borrelia burgdorferi with two sets of primers that amplified a 92 bp sequence unique to HSV DNA polymerase gene and a 156 bp sequence unique to B. burgdorferi Osp-A gene (35 cycles). Following the amplification, PCR products were treated with 0-500 mM hydroxylamine hydrochloride and incubated at room temperature for 30 min. One microlitre of each hydroxylamine treated PCR product was reamplified for an additional 35 cycles. Pre- and post-hydroxylamine treated PCR products were separated by electrophoresis in 3% agarose gel. Hydroxylamine, at a concentration of 250 mM or higher, was found to effectively modify PCR products and prevent their amplification in subsequent PCR.
Department of Laboratory Medicine, University of Connecticut Medical Center, Farmington 06030.
J
J
Aslanzadeh
D016477:Artifacts; D001483:Base Sequence; D015748:Borrelia burgdorferi Group / Q000235:genetics; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D015342:DNA Probes; D004269:DNA, Bacterial / Q000187:drug effects; D004279:DNA, Viral / Q000187:drug effects; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018139:Simplexvirus / Q000235:genetics; D013242:Sterilization / Q000379:methods*
D015342:DNA Probes; D004269:DNA, Bacterial; D004279:DNA, Viral; D006898:Hydroxylamines; D019811:Hydroxylamine; D004247:DNA