AmpErase® Uracil N-Glycosylase (UNG), part of the GeneAmp® PCR Carry-over Prevention Kit, is a 26 kDa ultrapure, recombinant enzyme encoded by the E. coli uracil N-glycosylase gene, which has been inserted into an E. coli host to direct the expression of the wild type form of the enzyme. The enzyme removes any uracil incorporated into single- or double-stranded DNA.
GeneAmp® PCR Carry-over Prevention Kit
The GeneAmp® PCR Carry-over Prevention Kit (available separately) provides reagents for a simple yet powerful method of ensuring that PCR products cannot be reamplified in subsequent PCR amplifications, thereby preventing false positive results. Features of this kit:
• Enzymatic method stops PCR carryover contamination, which prevents false positive results
• Designed to degrade PCR products from previous PCR amplifications without degrading native nucleic acid templates, which improves amplification
• No interference with any PCR or real-time PCR application
• Optimized for use with GeneAmp® PCR core reagents and GeneAmp® instrument systems
Eliminate PCR carryover contamination
The kit uses an enzymatic reaction analogous to the restriction-modification and excision-repair systems of cells to specifically degrade PCR products from previous PCR amplifications in which dUTP has been incorporated, without degrading native nucleic acid templates. The method used to render PCR products susceptible to degradation involves substituting dUTP for dTTP in the PCR mixture, and pretreating all subsequent PCR mixtures with AmpErase® Uracil N-glycosylase (UNG) prior to PCR amplification. Products from PCR amplification contain uracil, and are readily distinguishable from native thymidine-containing DNA templates. Products from previous PCR amplifications are eliminated by excising uracil residues using UNG, and degrading the resulting abasic polynucleotide with heat. Although UNG is active on single- and double-stranded dU-containing DNA, ribouracil residues in RNA and dUTP itself are not substrates for UNG. AmpliTaq® DNA Polymerase, AmpliTaq Gold® DNA Polymerase, and the other components of the PCR mixture remain intact during UNG treatment, providing PCR amplification free of PCR product carryover. The dU-containing PCR product behaves like dT-containing DNA in blotting, cloning, sequencing, and most other post-PCR analyses.