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Addressing PCR Biases in Environmental Microbiology Studies

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Bioremediation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 599))

Abstract

Each step of a molecular environmental microbiology study is prone to errors, though the qualitative and quantitative biases of PCR amplification could result in the most serious biases. One has to be aware of this fact, and well-characterized PCR biases have to be avoided by using target-optimized PCR protocols. The most important tasks are primer and thermal profile optimization. We have shown that primer mismatches, even in the case of universal primers, can cause almost complete missing of common taxa from clone libraries, for example. Similarly high annealing temperatures can drastically distort community composition of the sample in the PCR product. Strategies of primer selection and PCR thermal profile design are discussed in detail.

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References

  1. von Wintzingerode, F., Gobel, U. B. and Stackebrandt, E. (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21, 213–229.

    Article  Google Scholar 

  2. Fischer, S. G., and Lerman, L. S. (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc. Natl. Acad. Sci. USA 80, 1579–1583.

    Article  CAS  Google Scholar 

  3. Frostegard, A., Courtois, S., Ramisse, V., Clerc, S., Bernillon, D., Le Gall, F., et al. (1999) Quantification of bias related to the extraction of DNA directly from soils. Appl. Environ. Microbiol. 65, 5409–5420.

    CAS  Google Scholar 

  4. Qiu, X. Y., Wu, L. Y., Huang, H. S., McDonel, P. E., Palumbo, A. V., Tiedje, J. M. and Zhou, J. Z. (2001) Evaluation of PCR-generated chimeras: Mutations, and heteroduplexes with 16S rRNA gene-based cloning. Appl. Environ. Microbiol. 67, 880–887.

    Article  CAS  Google Scholar 

  5. Porteous, L. A., and Armstrong, J. L. (1993) A simple mini-method to extract DNA directly from soil for use with polymerase chain reaction amplification. Curr. Microbiol. 27, 115–118.

    Article  CAS  Google Scholar 

  6. Steffan, R. J., and Atlas, R. M. (1991) Polymerase chain reaction: applications in environmental microbiology. Annu. Rev. Microbiol. 45, 137–161.

    Article  CAS  Google Scholar 

  7. Young, C. C., Burghoff, R. L., Keim, L. G., Minak-Bernero, V., Lute, J. R., and Hinton, S. M. (1993) Polyvinylpyrrolidone-agarose gel electrophoresis purification of polymerase chain reaction-amplifiable DNA from soils. Appl. Environ. Microbiol. 59, 1972–1974.

    CAS  Google Scholar 

  8. Zhou, J., Bruns, M. A., and Tiedje, J. M. (1996) DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316–322.

    CAS  Google Scholar 

  9. Sagova-Mareckova, M., Cermak, L., Novotna, J., Plhackova, K., Forstova, J., and Kopecky, J. (2008) Innovative methods for soil DNA purification tested in soils with widely differing characteristics. Appl. Environ. Microbiol. 74, 2902–2907.

    Article  CAS  Google Scholar 

  10. Saano, A., and Lindström, K. (1995) Small scale extraction of DNA from soil with spun column cleanup, in Molecular Microbial Ecology Manual (Akkermans, A. D. L., van Elsas, J. D., and DeBruijn, F. J. ed.), Kluwer, Dordrecht, pp. 1–6.

    Google Scholar 

  11. Lakay, F. M., Botha, A., and Prior, B. A. (2007) Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils. J. Appl. Microbiol. 102, 265–273.

    Article  CAS  Google Scholar 

  12. Robe, P., Nalin, R., Capellano, C., Vogel, T. M., and Simonet, P. (2003) Extraction of DNA from soil. Eur. J. Soil Biol. 39, 183–190.

    Article  CAS  Google Scholar 

  13. Forney, L. J., Zhou, X., and Brown, C. J. (2004) Molecular microbial ecology: land of the one-eyed king. Curr. Opin. Microbiol. 7, 210–220.

    Article  CAS  Google Scholar 

  14. Baker, G. C., Smith, J. J., and Cowan, D. A. (2003) Review and re-analysis of domain-specific 16S primers. J. Microbiol. Methods 55, 5541–555.

    Article  Google Scholar 

  15. Lane, D. J. (1991) 16S/23S rRNA sequencing, in Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt, E., and Goodfellow, M. ed.), Wiley, London, pp. 115–147.

    Google Scholar 

  16. Marchesi, J. R., Sato, T., Weightman, A. J., Martin, T. A., Fry, J. C., Hiom, S. J., et al. (1998) Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol. 64, 795–799.

    CAS  Google Scholar 

  17. Hongoh, Y., Yuzawa, H., Ohkuma, M., and Kudo, T. (2003) Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment. FEMS Microbiol. Lett. 221, 299–304.

    Article  CAS  Google Scholar 

  18. Ellis, R. J., Morgan, P., Weightman, A. J., and Fry, J. C. (2003) Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated soil. Appl. Environ. Microbiol. 69, 3223–3230.

    Article  CAS  Google Scholar 

  19. Santos, S. R., and Ochman, H. (2004) Identification and phylogenetic sorting of bacterial lineages with universally conserved genes and proteins. Environ. Microbiol. 6, 754–759.

    Article  CAS  Google Scholar 

  20. Case, R. J., Boucher, Y., Dahllof, I., Holmstrom, C., Doolittle, W. F., and Kjelleberg, S. (2007) Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl. Environ. Microbiol. 73, 278–288.

    Article  CAS  Google Scholar 

  21. Sipos, R., Székely, A. J., Palatinszky, M., Révész, S., Márialigeti, K., and Nikolausz, M. (2007) Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targeting bacterial community analysis. FEMS Microbiol. Ecol. 60, 341–350.

    Article  CAS  Google Scholar 

  22. Blackwood, C. B., Oaks, A., and Buyer, J. S. (2005) Phylum- and class-specific PCR primers for general microbial community analysis. Appl. Environ. Microbiol. 71, 6193–6198.

    Article  CAS  Google Scholar 

  23. Boon, N., De Windt, W., Verstraete, W., and Top, E. M. (2002) Evaluation of nested PCR-DGGE (denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants. FEMS Microbiol. Ecol. 39, 101–112.

    CAS  Google Scholar 

  24. Fierer, N., Jackson, J. A., Vilgalys, R., and Jackson, R. B. (2005) Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl. Environ. Microbiol. 71, 4117–4120.

    Article  CAS  Google Scholar 

  25. Baker, G. C., and Cowan, D. A. (2004) 16S rDNA primers and the unbiased assessment of thermophile diversity. Biochem. Soc. Trans. 32, 218–221.

    Article  CAS  Google Scholar 

  26. Purkhold, U., Pommerening-Roser, A., Juretschko, S., Schmid, M. C., Koops, H. P., and Wagner, M. (2000) Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Appl. Environ. Microbiol. 66, 5368–5382.

    Article  CAS  Google Scholar 

  27. Zak, D. R., Blackwood, C. B., and Waldrop, M. P. (2006) A molecular dawn for biogeochemistry. Trends Ecol. Evol. 21, 288–295.

    Article  Google Scholar 

  28. Táncsics, A., Szoboszlay, S., Kriszt, B., Kukolya, J., Baka, E., Márialigeti, K., and Révész, S. (2008) Applicability of the functional gene catechol 1,2-dioxygenase as a biomarker in the detection of BTEX-degrading Rhodococcus species. J. Appl. Microbiol. 105, 1026–1033.

    Article  Google Scholar 

  29. Hyndman, D. L., and Mitsuhashi, M. (2003) PCR Primer Design, in PCR Protocols (Bartlett, J. M. S., and Stirling, D. ed.), Humana, Totowa, NJ, pp. 89–99.

    Google Scholar 

  30. Ahn, J. H., Kim, M. C., Shin, H. C., Choi, M. K., Yoon, S. S., Kim, T., et al. (2006) Improvement of PCR amplification bias for community structure analysis of soil bacteria by denaturing gradient gel electrophoresis. J. Microbiol. Biotechnol. 16, 1561–1569.

    CAS  Google Scholar 

  31. Bru, D., Martin-Laurent, F., and Philippot, L. (2008) Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Appl. Environ. Microbiol. 74, 1660–1663.

    Article  CAS  Google Scholar 

  32. Okano, Y., Hristova, K. R., Leutenegger, C. M., Jackson, L. E., Denison, R. F., Gebreyesus, B., et al. (2004) Application of real-time PCR to study effects of ammonium on population size of ammonia-oxidizing bacteria in soil. Appl. Environ. Microbiol. 70, 1008–1016.

    Article  CAS  Google Scholar 

  33. Teske, A., and Sorensen, K. B. (2008) Uncultured archaea in deep marine subsurface sediments: have we caught them all? ISME J. 2, 3–18.

    Article  CAS  Google Scholar 

  34. Sekido, T., Bodelier, P. L., Shoji, T., Suwa, Y., and Laanbroek, H. J. (2008) Limitations of the use of group-specific primers in real-time PCR as appear from quantitative analyses of closely related ammonia-oxidising species. Water Res. 42, 1093–1101.

    Article  CAS  Google Scholar 

  35. Polz, M. F., and Cavanaugh, C. M. (1998) Bias in template-to-product ratios in multitemplate PCR. Appl. Environ. Microbiol. 64, 3724–3730.

    CAS  Google Scholar 

  36. Watanabe, K., Kodama, Y., and Harayama, S. (2001) Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting. J. Microbiol. Methods 44, 253–262.

    Article  CAS  Google Scholar 

  37. Loy, A., Arnold, R., Tischler, P., Rattei, T., Wagner, M., and Horn, M. (2008) probeCheck - a central resource for evaluating oligonucleotide probe coverage and specificity. Environ. Microbiol. 10, 2844–2848.

    Article  Google Scholar 

  38. Cole, J. R., Chai, B., Farris, R. J., Wang, Q., Kulam, S. A., McGarrell, D. M., et al. (2005) The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res. 33, D294–296.

    Article  CAS  Google Scholar 

  39. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Res. 32, 1363–1371.

    Article  CAS  Google Scholar 

  40. Brons, J. K., and van Elsas, J. D. (2008) Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers. Appl. Environ. Microbiol. 74, 2717–2727.

    Article  CAS  Google Scholar 

  41. Grunenwald, H. (2003) Optimization of Polymerase Chain Reactions, in PCR Protocols (Bartlett, J. M. S., and Stirling, D. ed.), Humana, Totowa, NJ, pp. 89–99.

    Chapter  Google Scholar 

  42. Abu Al-Soud, W., and Radstrom, P. (1998) Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl. Environ. Microbiol. 64, 3748–3753.

    CAS  Google Scholar 

  43. Arezi, B., Xing, W., Sorge, J. A., and Hogrefe, H. H. (2003) Amplification efficiency of thermostable DNA polymerases. Anal. Biochem. 321, 226–235.

    Article  CAS  Google Scholar 

  44. Tyler, K. D., Wang, G., Tyler, S. D. and Johnson, W. M. (1997) Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. J. Clin. Microbiol. 35, 339–346.

    CAS  Google Scholar 

  45. Cline, J., Braman, J. C., and Hogrefe, H. H. (1996) PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24, 3546–3551.

    Article  CAS  Google Scholar 

  46. Tebbe, C. C., and Vahjen, W. (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl. Environ. Microbiol. 59, 2657–2665.

    CAS  Google Scholar 

  47. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. (1991) 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19, 4008.

    Article  CAS  Google Scholar 

  48. Muyzer, G., de Waal, E. C., and Uitterlinden, A. G. (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695–700.

    CAS  Google Scholar 

  49. Ishii, K., and Fukui, M. (2001) Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR. Appl. Environ. Microbiol. 67, 3753–3755.

    Article  CAS  Google Scholar 

  50. Janse, I., Bok, J., and Zwart, G. (2004) A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. J. Microbiol. Methods 57, 279–281.

    Article  CAS  Google Scholar 

  51. Kainz, P. (2000) The PCR plateau phase − towards an understanding of its limitations. Biochim. Biophys. Acta 1494, 23–27.

    CAS  Google Scholar 

  52. Suzuki, M. T., and Giovannoni, S. J. (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62, 625–630.

    CAS  Google Scholar 

  53. Lueders, T., and Friedrich, M. W. (2003) Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl. Environ. Microbiol. 69, 320–326.

    Article  CAS  Google Scholar 

  54. Acinas, S. G., Sarma-Rupavtarm, R., Klepac-Ceraj, V., and Polz, M. F. (2005) PCR-induced sequence artifacts and bias: Insights from comparison of two 16S rRNA clone libraries constructed from the same sample. Appl. Environ. Microbiol. 71, 8966–8969.

    Article  CAS  Google Scholar 

  55. Jensen, M. A., and Straus, N. (1993) Effect of PCR conditions on the formation of heteroduplex and single-stranded-DNA products in the amplification of bacterial ribosomal DNA spacer regions. PCR-Methods Appl. 3, 186–194.

    CAS  Google Scholar 

  56. Shinde, D., Lai, Y., Sun, F., and Arnheim, N. (2003) Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)n and (A/T)n microsatellites. Nucleic Acids Res. 31, 974–980.

    Article  CAS  Google Scholar 

  57. Haqqi, T. M., Sarkar, G., David, C. S., and Sommer, S. S. (1988) Specific amplification with PCR of a refractory segment of genomic DNA. Nucleic Acids Res. 16, 11844.

    Article  CAS  Google Scholar 

  58. Dar, S. A., Kuenen, J. G., and Muyzer, G. (2005) Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities. Appl. Environ. Microbiol. 71, 2325–2330.

    Article  CAS  Google Scholar 

  59. Oros-Sichler, M., Gomes, N. C., Neuber, G., and Smalla, K. (2006) A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities. J. Microbiol. Meth. 65, 63–75.

    Article  CAS  Google Scholar 

  60. el Fantroussi, S., Mahillon, J., Naveau, H., and Agathos, S. N. (1997) Introduction of anaerobic dechlorinating bacteria into soil slurry microcosms and nested-PCR monitoring. Appl. Environ. Microbiol. 63, 806–811.

    CAS  Google Scholar 

  61. Nikolausz, M., Sipos, R., Révész, S., Székely, A., and Márialigeti, K. (2005) Observation of bias associated with re-amplification of DNA isolated from denaturing gradient gels. FEMS Microbiol. Lett. 244, 385–390.

    Article  CAS  Google Scholar 

  62. Levesque, M. J., La Boissiere, S., Thomas, J. C., Beaudet, R., and Villemur, R. (1997) Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction. Appl. Microbiol. Biotechnol. 47, 719–725.

    Article  CAS  Google Scholar 

  63. DeJournett, T. D., Arnold, W. A., and LaPara, T. M. (2007) The characterization and quantification of methanotrophic bacterial populations in constructed wetland sediments using PCR targeting 16S rRNA gene fragments. Appl. Soil Ecol. 35, 648–659.

    Article  Google Scholar 

  64. Temmerman, R., Masco, L., Vanhoutte, T., Huys, G., and Swings, J. (2003) Development and validation of a nested-PCR-denaturing gradient gel electrophoresis method for taxonomic characterization of bifidobacterial communities. Appl. Environ. Microbiol. 69, 6380–6385.

    Article  CAS  Google Scholar 

  65. Phillips, C. J., Harris, D., Dollhopf, S. L., Gross, K. L., Prosser, J. I., and Paul, E. A. (2000) Effects of agronomic treatments on structure and function of ammonia-oxidizing communities. Appl. Environ. Microbiol. 66, 5410–5418.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Microbiology, Eötvös Loránd University, Budapest, Hungary

    Rita Sipos, Anna Székely, Sára Révész & Károly Márialigeti

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  1. Rita Sipos
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  2. Anna Székely
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  4. Károly Márialigeti
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  1. School of Applied Sciences, Northumbria University, Ellison Place, Newcastle upon Tyne, NE1 8ST, United Kingdom

    Stephen P. Cummings

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Sipos, R., Székely, A., Révész, S., Márialigeti, K. (2010). Addressing PCR Biases in Environmental Microbiology Studies. In: Cummings, S. (eds) Bioremediation. Methods in Molecular Biology, vol 599. Humana Press. https://doi.org/10.1007/978-1-60761-439-5_3

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  • DOI: https://doi.org/10.1007/978-1-60761-439-5_3

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