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• W47445083 abstract "BioTechniquesVol. 36, No. 1 BenchmarksOpen AccessEffectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assaysKenneth E. Pierce & Lawrence J. WanghKenneth E. Pierce*Address correspondence to: Kenneth Pierce, Department of Biology MS-008, Brandeis University, Waltham, MA 02454-9110, USA. e-mail: E-mail Address: pierce@brandeis.eduBrandeis University, Waltham, MA, USASearch for more papers by this author & Lawrence J. WanghBrandeis University, Waltham, MA, USASearch for more papers by this authorPublished Online:6 Jun 2018https://doi.org/10.2144/04361BM04AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail PCR is sufficiently sensitive to detect single copy genes from single cells. This extreme sensitivity is also the basis of one of its potential problems; even a single product molecule from a previous amplification can lead to a false positive result. Substituting dUTP for dTTP during PCR and treating subsequent reactions with uracil-DNA glycosylase (UDG) prior to amplification is one strategy for limiting carryover contamination (1). However, total elimination of contaminants is not always accomplished using this technique, particularly where PCR product length is short (2), which is a common situation in realtime PCR assays. In addition, there is the possibility, particularly when the initial sample contains only one or a few target molecules, that inclusion of UDG may reduce amplification efficiency and thereby delay or prevent detection.As a first step toward implementing a general protocol for using UDG at the level of single copy genes in single cells, we investigated conditions for preventing contamination during amplification of a 133-bp segment within the multicopy testis-specific protein gene (TSPY) from single male lymphocytes. Cell lysis and real-time PCR with molecular beacons were carried out as described previously (3), except that the extension step of thermal cycling was increased to 30 s and the dTTP was replaced with dUTP at a 3-fold higher concentration. The resulting PCR product contained 27 uracil residues in the sense strand and 28 uracil residues in the antisense strand.Initial experiments compared the efficiency of amplification in the presence of dUTP or dTTP in terms of the mean detection cycle (CT) value (i.e., the point at which the molecular beacon fluorescence intensity reaches a threshold of 200 U) and the final fluorescence after 45 cycles. Amplification plots are shown in Figure 1A. The mean CT values of 33.9 and 34.4 for dUTP and dTTP samples, respectively, were in the range of values previously observed using single lymphocytes, reflecting the presence of approximate 30 copies of the TSPY gene on the Y chromosome (3). The differences in mean CT value and mean final fluorescence between the two sample groups were not statistically significant (P > 0.05). Thus, substitution of dUTP (at 3× concentration) for dTTP did not reduce amplification efficiency for this sequence. Similarly, the use of dUTP did not significantly alter amplification efficiency of a 175-bp segment of U2, another multicopy gene (3) (data not shown). The U2 PCR product contained 26 uracil residues in the sense strand and 37 uracil residues in the antisense strand.Figure 1. Molecular beacon fluorescence detection of testis-specific protein gene (TSPY) amplification during real-time PCR.(A) TSPY amplification in samples containing single male lymphocytes and either dTTP (dark green) or dUTP (red), but no uracil-DNA glycosylase (UDG). (B) TSPY amplification in samples containing single male lymphocytes in the presence (blue) or absence (red) of UDG. All samples contain dUTP. (C) TSPY amplification in samples with diluted TSPY PCR product (averaging about three molecules per sample) following a 10-min incubation at 37°C with UDG (purple) or without UDG (red). (D) TSPY amplification in samples with diluted TSPY PCR product (averaging about 10 molecules per sample) following a 10-min (green) or 30-min (blue-green) incubation at 30°C with heat-labile UDG or without heat-labile UDG (red).Next, we tested the effect of UDG (from Roche Applied Science, Indianapolis, IN, USA) on the efficiency of TSPY and U2 amplification, since some enzyme activity remains after 10 min at 95°C (a typical inactivation step) (4). Theoretically, residual enzyme activity might degrade a fraction of the PCR product during each annealing step of the subsequent PCR and might, thereby, delay or prevent signal detection. Samples (all containing dUTP) with or without UDG were incubated at 95°C for 10 min prior to thermal cycling. Real-time detection of TSPY amplification is shown in Figure 1B. Mean CT values and final fluorescence were not significantly altered by the presence of UDG (P > 0.05). Similarly, amplification of U2 was not significantly affected by the presence of UDG (data not shown). It should be noted that the annealing temperature used for these amplifications was 58°C, and additional tests may be warranted in cases where annealing temperatures are closer to 50°C, at which UDG has maximal activity (5).The ability of UDG to degrade PCR products prior to amplification was tested using TSPY PCR product molecules generated with dUTP. The goal of these experiments was to determine whether a high percentage of samples could be completely “cleared” of product molecules using various protocols with UDG. Initial conditions employing a 10-min incubation at 25°C prior to PCR, as recommended by the manufacturer, were found to be inadequate. Three of eight samples generated detectable TSPY product from residual target molecules (Table 1). Subsequent tests employed a 10-min incubation at 37°C. All four samples without UDG, but only 1 of 24 samples with UDG, showed detectable amplification (Figure 1C). The mean CT value of 37.3 in samples without UDG indicates an average of about three target molecules in each of the tested samples, while the positive signal in the sample with UDG had a CT value of 38.9 and was therefore likely due to amplification from a single undigested PCR product. Similar results were obtained using the 37°C protocol with PCR product amplified from the U2 gene. Reamplification was observed in only 1 of 24 samples containing UDG. Thus, the treatment at the higher temperature was successful, although not absolute in preventing amplification from PCR product generated using dUTP. Higher temperatures were not tested, but might further decrease the percentage of samples with undigested product.Table 1. Ability of UDG and Heat-Labile UDG to Prevent Amplification from TSPY PCR Products Containing UracilThe 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 25°C for 10 min prevented detectable amplification in only 6 of 20 samples containing the TSPY PCR product (Table 1). An unacceptably high percentage of samples also generated detectable product even when the enzyme concentration was doubled or the incubation temperature increased to 30° or 37°C. However, increasing the duration of a 30°C incubation to 30 min did decrease the percentage of samples generating product from the PCR product molecules. In one experiment using that protocol, only one of eight samples generated a detectable signal, and that CT value suggested amplification from a single target (blue-green line in Figure 1D). Eight samples treated for only 10 min all generated amplification, although the range of CT values was above that of untreated controls (green and red lines, respectively, in Figure 1D), indicating that some of the estimated 10 target molecules in each sample were degraded prior to amplification. A subsequent experiment using the 30-min incubation on samples containing an average of only three PCR product molecules still resulted in detectable amplification in 4 of 20 samples, indicating that the starting number of molecules was not the sole reason for the poorer results with the heat-labile enzyme. In all, 23 of 28 (82%) samples incubated at 30°C for 30 min with heatlabile UDG were successfully cleared of the PCR product molecules. Long incubation at higher temperatures was not tested because of the short half-life of the enzyme, which is 2 min at 40°C according to the manufacturer.In summary, dUTP and UDG can be used in real-time PCR applications without affecting the efficiency of amplification and thus the ability to detect and quantify targets of interest, even when those targets are few in number. It may be important to maintain relatively high annealing temperatures when UDG is present in order to avoid effects of residual enzyme activity during amplification. Incubating samples with UDG at 37°C prior to PCR greatly reduces the probability of contamination, although it does not guarantee complete removal of PCR product molecules from all samples. Since the nucleotide sequence and the choice of PCR buffer are likely to affect the degradation of products, we recommend similar tests be carried out for any assay in which avoiding contamination is critical. Such tests are definitely warranted for the use of heat-labile UDG. None of the protocols tested here with that enzyme proved satisfactory for the complete removal of PCR product molecules. While it may be possible to obtain a satisfactory protocol, in particular where those molecules are longer, it may require much higher concentrations of the heat-labile enzyme and/or long incubation times that increase expense and duration of the assay. In any case, users should always remember that either enzyme should be an adjunct to, rather than a replacement for, strict handling protocols aimed at avoiding contamination.AcknowledgmentThis research was supported by grants from Brandeis University and Hamilton Thorne Biosciences, Inc.References1. Longo, M.C., M.S. Berninger, and J.L. Hartley. 1990. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93:125–128.Crossref, Medline, CAS, Google Scholar2. Espy, M.J., T.F. Smith, and D.H. Persing. 1993. Dependence of polymerase chain reaction product inactivation protocols on amplicon length and sequence composition. J. Clin. Microbiol. 31:2361–2365.Crossref, Medline, CAS, Google Scholar3. Pierce, K.E., J.E. Rice, J.A. Sanchez, C. Brenner, and L.J. Wangh. 2000. Real-time PCR using molecular beacons for accurate detection of the Y chromosome in single human blastomeres. Mol. Hum. Reprod. 6:1155–1164.Crossref, Medline, CAS, Google Scholar4. Thornton, C.G., J.L. Hartley, and A. Rashtchian. 1992. Utilizing uracil DNA glycosylase to control carryover contamination in PCR: characterization of residual UDG activity following thermal cycling. BioTechniques 13:180–184.Medline, CAS, Google Scholar5. Jaeger, S., R. Schmuck, and H. Sobek. 2000. Molecular cloning, sequency, and expression of the heat-labile uracil-DNA glycosylase from a marine psychrophilic bacterium, strain BMTU3346. Extremophiles 4:115–122.Crossref, Medline, CAS, Google Scholar6. Sobek, H., M. Schmidt, B. Frey, and K. Kaluza. 1996. Heat-labile uracil-DNA glycosylase: purification and characterization. FEBS Lett 388:1–4.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByComputational design of a thermolabile uracil-DNA glycosylase of Escherichia coliBiophysical Journal, Vol. 413Computational Study on DNA Repair: The Roles of Electrostatic Interactions Between Uracil-DNA Glycosylase (UDG) and DNA6 August 2021 | Frontiers in Molecular Biosciences, Vol. 8Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons16 October 2018 | International Journal of Molecular Sciences, Vol. 19, No. 10Awareness of mutational artefacts in suboptimal DNA samples: possible risk for therapeutic choices26 April 2018 | Expert Review of Molecular Diagnostics, Vol. 18, No. 5Label-free and sensitive detection of uracil-DNA glycosylase using exponential real-time rolling circle amplification1 January 2018 | Analytical Methods, Vol. 10, No. 20Reactive Lymphoid Hyperplasia or Tubercular Lymphadenitis: Can Real-Time PCR on Fine-Needle Aspirates Help Physicians in Concluding the Diagnosis?15 May 2018 | Acta Cytologica, Vol. 62, No. 3A highly specific strategy for in suit detection of DNA with nicking enzyme assisted amplification and lateral flowSensors and Actuators B: Chemical, Vol. 253Amplification Product Inactivation6 August 2012No Evidence of Persisting Measles Virus in Peripheral Blood Mononuclear Cells From Children With Autism Spectrum Disorder1 October 2006 | Pediatrics, Vol. 118, No. 4Validation of molecular-diagnostic techniques in the parasitological laboratoryVeterinary Parasitology, Vol. 136, No. 2Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR11 April 2005 | Virology Journal, Vol. 2, No. 1Advances in Real‐Time PCR: Application to Clinical Laboratory DiagnosticsImproved Methods for Extracting RNA from Exfoliated Human Colonocytes in Stool and RT-PCR Analysis1 November 2004 | Digestive Diseases and Sciences, Vol. 49, No. 11-12Diagnostic Microbiology Using Real-Time PCR Based on FRET TechnologyAmplification Product Inactivation Vol. 36, No. 1 Follow us on social media for the latest updates Metrics Downloaded 1,984 times History Received 23 July 2003 Accepted 21 October 2003 Published online 6 June 2018 Published in print January 2004 Information© 2004 Author(s)AcknowledgmentThis research was supported by grants from Brandeis University and Hamilton Thorne Biosciences, Inc.PDF download" @default.
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