Heat‐labile uracil‐DNA glycosylase: purification and characterization

Abstract

A uracil-DNA glycosylase (UNG) from a psychro-PCR [10]. philic marine bacterium (BMTU 3346) has been purified to apparent homogeneity. The enzyme has a molecular weight of LING degrades contaminating uracil-containing DNA leav-23 400 Do. It is stable in complex buffers (containing glycerol/ ing the natural (thymine-containing) target DNA unaffected. BSA), whereas it is heat-labile in dilute buffers (free ofFor this application usually the uracil-DNA glycosylase from stabilizers) with a half-life of 2 rain at 40°C. Due to the E. coli is used. However, it has been reported that the enzyme thermolability, uracil-DNA glycosylase is suitable for applieais not completely inactivated by heat denaturation, leading to tion in the carryover prevention technique showing less residual degradation of the PCR product affecting its integrity and activity and/or a slower reactivation rate than the usually applied yield. Furthermore, reactivation of UNG after PCR has UNG from Escheriehia coli.been discussed [12].In the present paper, the purification and characterization

Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii

“…No manipulation of amplicons is required at any step during real-time PCR, lessening the possibility of contamination problems. To further reduce contamination risks, UNG and dUTP were used for prevention of amplicon carryover (20). The entire assay has a turnaround time of Ͻ4 h, including DNA extraction, which further increases the potential utility of this procedure.…”

Effectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assays

“…The potential problem with residual UDG activity after PCR has led some investigators to use a heat-labile form of the enzyme (5,6). Incubations with heat-labile UDG (Roche Applied Science) at the manufacturer's recommended conditions of 25C for 10 min prevented detectable amplification in only 6 of 20 samples containing the TSPY PCR product (Table 1).…”

Filaggrin Genotyping

“…Severe preventative actions are strongly recommended, as are very stringent clean-up using a hypochlorite solution, discarding of all used reagents, UV irradiation of workspace for PCR setup in a dedicated laminar fl ow bench, and use of fi lter tips. In addition to physical separation of PCR setup facilities and PCR product analysis facilities is the use of uracil-DNA glycosylase (UNG) and substitution of dTTP with dUTP in the PCR process, an important preventive step [ 165 ]. This enables the digestion of any PCR products introduced into the sample, thus ensuring amplifi cation of genuine DNA and eliminating false positives.…”