In contrast, we observed many instances in which high ΔCq values were associated with highly efficient reactions. This can be explained by the presence of thermolabile inhibitors that diminish with cycling; they may inhibit during early cycles of PCR, but the amplification efficiency will eventually recover and produce only a delayed Cq value. Alternatively, inhibitors that degrade or capture DNA (e.g., nucleases and DNA binding substances such as silica) may reduce the number of amplifiable IPC templates in an otherwise efficient PCR. This observation illustrates the importance of using an IPC for inhibition testing; without it (i.e., using efficiency calculations alone) these Cq shifts and large drops in ER would remain undetected.
Amplification of damaged templates may create the same pattern due to a reduction in amplification efficiency in the early cycles of PCR until newly synthesized/undamaged templates predominate (33). This is unlikely to affect our IPC, but may be an unavoidable source of error when quantifying damaged DNA relative to a pristine standard.
With repeat inhibition tests, we were able to optimize the PCR facilitator combinations and dilutions that result in the highest possible ER for each extract. For the bone and most soil samples, dilution could be avoided altogether; in contrast, inhibition in the feces and hair extracts could not be overcome with BSA and additional Taq, but instead required that 1/50 or even 1/100 dilutions be used in PCR. Although the resulting ER values are indeed optimal within the parameters of this study, the values are so low for these diluted extracts that additional PCR facilitators should be considered before subsequent PCR runs are attempted. If the values cannot be improved this way, additional purification or even alternative extraction procedures may be beneficial. As we have demonstrated, inhibition testing can also be useful in evaluating these protocols.
Although we recommend testing every extract for inhibition, it may not be necessary to test every combination of PCR facilitators. Some general trends exist within sample types using a particular extraction method: for instance, compared with BSA, additional Taq appeared to provide limited improvement for PCR results from our feces and soil extracts. This suggests that their major inhibitors do not act directly on the enzyme, provided that enough Taq was used to observe such an effect. In contrast, improvements in solely Taq-supplemented hair and bone reactions indicate that direct inhibitors of the enzyme are present. It is not surprising that BSA was able to overcome inhibition in all sample types, based on its combined enzyme-stabilizing and inhibitor-binding (34) abilities.
If the samples are to be used in subsequent quantitative comparisons, several considerations must be made to ensure accuracy. For threshold-based quantification, reaction conditions producing equivalent amplification efficiencies between samples and/or standards should be favored, even if ER is low (e.g., with dilution). If this is not feasible, differences in efficiency can be applied as correction factors (13). Relative measurements of efficiency are sufficient in the former situation, while the latter requires highly accurate, absolute values. Alternatively, the recently published Cy0 method (21) obviates the need to assess amplification efficiency separately from quantification, and may be particularly useful when efficiency is highly variable; furthermore, ΔCy0 values determined from IPC reactions should be analogous to ΔCq, with their incorporation in ER calculations producing a convenient, single-parameter correction factor.
The accuracy of correction factors derived from IPC data also depends on the original assumption that the IPC and target assays are equally affected by inhibition. Intuitively, large differences in the amplicon sizes should be avoided; however, no particular sequence characteristics are clear predictors of inhibition susceptibility (11). Huggett et al. (11) suggest that assay performance be compared in the presence of a suspected inhibitor; however, this approach may be ineffective when the inhibitors are of unknown types and amounts, and additionally impractical when inhibitors vary between samples. If inhibition susceptibility is instead related to competition for reaction components (10), it should be sufficient to demonstrate that the assays behave similarly (i.e., within the accuracy requirements of the experiment) under a variety of conditions (e.g., concentration gradients of Mg2+, primers, and facilitators).
Based on the data presented here, we suggest that inhibition testing be regularly incorporated in procedures involving low amounts of DNA, such as in the forensic and ancient DNA fields. While the results are generally helpful for improving PCR success, they should be considered essential in experiments involving qPCR comparisons and when interpreting negative results. These tests should incorporate an IPC, which allows for simultaneous measurement of amplification efficiency and ΔCq. When employed routinely, this form of pre-PCR processing ensures both maximum access to template DNA and more accurate quantification of template amounts.
This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (grant no. 299103-2004), the Canadian Research Chairs program (to H.P.), the Social Sciences and Humanities Research Council of Canada (grant no. 410-2004-0579), the Government of Ontario Early Researcher Award program (to H.P.), the Ontario Graduate Scholarship program (to C.E.K.), and McMaster University.
The authors thank Kirsti Bos, Alison Devault, Debi Poinar, John Okello, and Jaymi Zurek for helpful discussion; and Duane Froese, Grant Zazula, Bernard Buigues, and Felisa Smith for providing the samples.
The authors declare no competing interests.
Correspondence Address correspondence to Hendrik N. Poinar, McMaster Ancient DNA Centre, Department of Anthropology, McMaster University, 1280 Main Street West, Hamilton ON, L8S 4L9, Canada. email: [email protected]References
1.) Wilson, I.G. 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63:3741-3751.
2.) Rossen, L., P. Nørskov, K. Holmstrøm, and O.F. Rasmussen. 1992. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int. J. Food Microbiol. 17:37-45.
3.) Ramakers, C., J.M. Ruijter, R.H. Deprez, and A.F. Moorman. 2003. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339:62-66.
4.) Cloud, J.L., W.C. Hymas, A. Turlak, A. Croft, U. Reischl, J.A. Daly, and K.C. Carroll. 2003. Description of a multiplex Bordetella pertussis and Bordetella parapertussis LightCycler PCR assay with inhibition control. Diagn. Microbiol. Infect. Dis. 46:189-195.
5.) Sutlovic, D., M. Definis Gojanovi, S. Andelinovi, D. Gugi, and D. Primorac. 2005. Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid. Croat. Med. J. 46:556-562.
6.) Deagle, B.E., J.P. Eveson, and S.N. Jarman. 2006. Quantification of damage in DNA recovered from highly degraded samples--a case study on DNA in faeces. Front. Zool. 3:11.
7.) Nolan, T., R.E. Hands, W. Ogunkolade, and S.A. Bustin. 2006. SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. Anal. Biochem. 351:308-310.
8.) Swango, K.L., M.D. Timken, M.D. Chong, and M.R. Buoncristiani. 2006. A quantitative PCR assay for the assessment of DNA degradation in forensic samples. Forensic Sci. Int. 158:14-26.
9.) Niederstätter, H., S. Köchl, P. Grubwieser, M. Pavlic, M. Steinlechner, and W. Parson. 2007. A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA. Forensic Sci. Int.; Genet. 1:29-34.
10.) Stahlberg, A., P. Aman, B. Ridell, P. Mostad, and M. Kubista. 2003. Quantitative real-time PCR method for detection of B-lymphocyte monoclonality by comparison of kappa and lambda immunoglobulin light chain expression. Clin. Chem. 49:51-59.
11.) Huggett, J.F., T. Novak, J.A. Garson, C. Green, S.D. Morris-Jones, R.F. Miller, and A. Zumla. 2008. Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon. BMC Res Notes 1:70.
12.) Bustin, S.A., V. Benes, J.A. Garson, J. Hellemans, J. Huggett, M. Kubista, R. Mueller, T. Nolan. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55:611-622.
13.) Liu, W., and D.A. Saint. 2002. A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal. Biochem. 302:52-59.
14.) Liu, W., and D.A. Saint. 2002. Validation of a quantitative method for real time PCR kinetics. Biochem. Biophys. Res. Commun. 294:347-353.
15.) Tichopad, A., A. Dzidic, and M.W. Pfaffl. 2002. Improving quantitative real-time RT-PCR reproducibility by boosting primer-linked amplification efficiency. Biotechnol. Lett. 24:2053-2056.
16.) Bar, T., A. Stahlberg, A. Muszta, and M. Kubista. 2003. Kinetic Outlier Detection (KOD) in real-time PCR. Nucleic Acids Res. 31:e105.
17.) Peirson, S.N., J.N. Butler, and R.G. Foster. 2003. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res. 31:e73.
18.) Tichopad, A., M. Dilger, G. Schwarz, and M.W. Pfaffl. 2003. Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res. 31:e122.
19.) Rutledge, R.G. 2004. Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications. Nucleic Acids Res. 32:e178.
20.) Kontanis, E.J., and F.A. Reed. 2006. Evaluation of real-time PCR amplification efficiencies to detect PCR inhibitors. J. Forensic Sci. 51:795-804.
21.) Guescini, M., D. Sisti, M.B. Rocchi, L. Stocchi, and V. Stocchi. 2008. A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition. BMC Bioinformatics 9:326.
22.) Rutledge, R.G., and D. Stewart. 2008. Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR. BMC Mol. Biol. 9:96.
23.) Shain, E.B., and J.M. Clemens. 2008. A new method for robust quantitative and qualitative analysis of real-time PCR. Nucleic Acids Res. 36:e91.
24.) Swillens, S., B. Dessars, and H.E. Housni. 2008. Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data. Anal. Biochem. 373:370-376.
25.) Ruijter, J.M., C. Ramakers, W.M. Hoogaars, Y. Karlen, O. Bakker, M.J. van den Hoff, and A.F. Moorman. 2009. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 37:e45.
26.) Rådström, P., R. Knutsson, P. Wolffs, M. Lövenklev, and C. Löfström. 2004. Pre-PCR processing: strategies to generate PCR-compatible samples. Mol. Biotechnol. 26:133-146.
27.) Willerslev, E., A.J. Hansen, J. Binladen, T.B. Brand, M.T. Gilbert, B. Shapiro, M. Bunce, C. Wiuf. 2003. Diverse plant and animal genetic records from Holocene and Pleistocene sediments. Science 300:791-795.
28.) Boom, R., C.J. Sol, M.M. Salimans, C.L. Jansen, P.M. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495-503.
29.) Rohland, N., and M. Hofreiter. 2007. Comparison and optimization of ancient DNA extraction. BioTechniques 42:343-352.
30.) Poinar, H.N., C. Schwarz, J. Qi, B. Shapiro, R.D.E. MacPhee, B. Buigues, A. Tikhonov, D.H. Huson. 2006. Metagenomics to paleog-enomics: large-scale sequencing of mammoth DNA. Science 311:392-394.
31.) Gilbert, M.T., L.P. Tomsho, S. Rendulic, M. Packard, D.I. Drautz, A. Sher, A. Tikhonov, L. Dalen. 2007. Whole-genome shotgun sequencing of mitochondria from ancient hair shafts. Science 317:1927-1930.
32.) Gilbert, M.T., T. Haselkorn, M. Bunce, J.J. Sanchez, S.B. Lucas, L.D. Jewell, E. Van Marck, and M. Worobey. 2007. The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?. PLoS One 2:e537.
33.) Sikorsky, J.A., D.A. Primerano, T.W. Fenger, and J. Denvir. 2004. Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method. Biochem. Biophys. Res. Commun. 323:823-830.
34.) Kreader, C.A. 1996. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62:1102-1106.
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