Skip to main content

Advertisement

Log in

Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance

  • Methods and protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Denaturing gradient gel electrophoresis (DGGE) is a powerful technique to reveal the community structures and composition of microorganisms in complex natural environments and samples. However, positive and reproducible polymerase chain reaction (PCR) products, which are difficult to acquire for some specific samples due to low abundance of the target microorganisms, significantly impair the effective applications of DGGE. Thus, nested PCR is often introduced to generate positive PCR products from the complex samples, but one problem is also introduced: The total number of thermocycling in nested PCR is usually unacceptably high, which results in skewed community structures by generation of random or mismatched PCR products on the DGGE gel, and this was demonstrated in this study. Furthermore, nested PCR could not resolve the uneven representative issue with PCR products of complex samples with unequal richness of microbial population. In order to solve the two problems in nested PCR, the general protocol was modified and improved in this study. Firstly, a general PCR procedure was used to amplify the target genes with the PCR primers without any guanine cytosine (GC) clamp, and then, the resultant PCR products were purified and diluted to 0.01 μg ml−1. Subsequently, the diluted PCR products were utilized as templates to amplify again with the same PCR primers with the GC clamp for 17 cycles, and the products were finally subjected to DGGE analysis. We demonstrated that this is a much more reliable approach to obtain a high quality DGGE profile with high reproducibility. Thus, we recommend the adoption of this improved protocol in analyzing microorganisms of low abundance in complex samples when applying the DGGE fingerprinting technique to avoid biased results.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410

    Article  CAS  PubMed  Google Scholar 

  • Cao H, Li M, Hong Y, Gu J-D (2011) Diversity and abundance of ammonia-oxidizing archaea and bacteria in polluted mangrove sediment. Syst Appl Microbiol 34(7):513–523. doi:10.1016/j.syapm.2010.11.023

    Article  CAS  PubMed  Google Scholar 

  • Cébron A, Coci M, Garnier J, Laanbroek HJ (2004) Denaturing gradient gel electrophoretic analysis of ammonia-oxidizing bacterial community structure in the lower Seine River: impact of Paris wastewater effluents. Appl Environ Microbiol 70(11):6726–6737. doi:10.1128/aem.70.11.6726-6737.2004

    Article  PubMed Central  PubMed  Google Scholar 

  • Chappell PD, Moffett JW, Hynes AM, Webb EA (2012) Molecular evidence of iron limitation and availability in the global diazotroph Trichodesmium. ISME J 6(9):1728–1739

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Halm H, Lam P, Ferdelman TG, Lavik G, Dittmar T, LaRoche J, D'Hondt S, Kuypers MMM (2012) Heterotrophic organisms dominate nitrogen fixation in the South Pacific Gyre. ISME J 6(6):1238–1249

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Han P, Li M, Gu J-D (2013) Biases in community structures of ammonia/ammonium-oxidizing microorganisms caused by insufficient DNA extractions from Baijiang soil revealed by comparative analysis of coastal wetland sediment and rice paddy soil. Appl Microbiol Biotechnol 97(19):8741–8756. doi:10.1007/s00253-013-5169-2

    Article  CAS  PubMed  Google Scholar 

  • Heiri O, Lotter AF, Lemcke G (2001) Loss on ignition as a method for estimating organic and carbonate content in sediments: reproducibility and comparability of results. J Paleolimnol 25(1):101–110

    Article  Google Scholar 

  • Hirsch PR, Mauchline TH, Clark IM (2010) Culture-independent molecular techniques for soil microbial ecology. Soil Biol Biochem 42(6):878–887

    Article  CAS  Google Scholar 

  • Hussain Q, Liu Y, Jin Z, Zhang A, Pan G, Li L, Crowley D, Zhang X, Song X, Cui L (2011) Temporal dynamics of ammonia oxidizer (amoA) and denitrifier (nirK) communities in the rhizosphere of a rice ecosystem from Tai Lake region, China. Appl Soil Ecol 48(2):210–218. doi:10.1016/j.apsoil.2011.03.004

    Article  Google Scholar 

  • Kowalchuk GA, Stephen JR, De Boer W, Prosser JI, Embley TM, Woldendorp JW (1997) Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl Environ Microbiol 63(4):1489–1497

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kowalchuk GA, Bodelier PLE, Heilig GHJ, Stephen JR, Laanbroek HJ (1998) Community analysis of ammonia-oxidising bacteria, in relation to oxygen availability in soils and root-oxygenated sediments, using PCR, DGGE and oligonucleotide probe hybridisation. FEMS Microbiol Ecol 27(4):339–350

    Article  CAS  Google Scholar 

  • Kuypers MMM, Sliekers AO, Lavik G, Schmid M, Jorgensen BB, Kuenen JG, Sinninghe Damsté JS, Strous M, Jetten MSM (2003) Anaerobic ammonium oxidation by anammox bacteria in the Black Sea. Nature 422(6932):608–611

    Article  CAS  PubMed  Google Scholar 

  • Li M, Cao H-L, Hong Y-G, Gu J-D (2011a) Seasonal dynamics of anammox bacteria in estuarial sediment of the Mai Po Nature Reserve revealed by analyzing the 16S rRNA and hydrazine oxidoreductase (hzo) genes. Microbes Environ 26(1):15–22

    Article  CAS  PubMed  Google Scholar 

  • Li M, Cao H, Hong Y, Gu J-D (2011b) Spatial distribution and abundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in mangrove sediments. Appl Microbiol Biotechnol 89(4):1243–1254. doi:10.1007/s00253-010-2929-0

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Li M, Hong Y-G, Cao H-L, Gu J-D (2011c) Mangrove trees affect the community structure and distribution of anammox bacteria at an anthropogenic-polluted mangrove in the Pearl River Delta reflected by 16S rRNA and hydrazine oxidoreductase (HZO) encoding gene analyses. Ecotoxicology 20(8):1780–1790. doi:10.1007/s10646-011-0711-4

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Malik S, Beer M, Megharaj M, Naidu R (2008) The use of molecular techniques to characterize the microbial communities in contaminated soil and water. Environ Int 34(2):265–276

    Article  CAS  PubMed  Google Scholar 

  • Mullis KB (1990) The unusual origin of the polymerase chain reaction. Sci Am 262(4):56–61

    Article  CAS  PubMed  Google Scholar 

  • Muyzer G (1999) DGGE/TGGE a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2(3):317–322

    Article  CAS  PubMed  Google Scholar 

  • Muyzer G, de Waal EC, Uitterlinden AG (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(3):695–700

    CAS  PubMed Central  PubMed  Google Scholar 

  • Nicolaisen MH, Ramsing NB (2002) Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. J Microbiol Methods 50(2):189–203. doi:10.1016/s0167-7012(02)00026-x

    Article  CAS  PubMed  Google Scholar 

  • Park SJ, Park BJ, Rhee SK (2008) Comparative analysis of archaeal 16S rRNA and amoA genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments. Extremophiles 12(4):605–615

    Article  CAS  PubMed  Google Scholar 

  • Paul JH, Cazares L, Thurmond J (1990) Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments. Appl Environ Microbiol 56(6):1963–1966

    CAS  PubMed Central  PubMed  Google Scholar 

  • Pires ACC, Cleary DFR, Almeida A, Cunha Â, Dealtry S, Mendonça-Hagler LCS, Smalla K, Gomes NCM (2012) Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples. Appl Environ Microbiol 78(16):5520–5528. doi:10.1128/aem.00386-12

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Qin H, Yuan H, Zhang H, Zhu Y, Yin C, Tan Z, Wu J, Wei W (2013) Ammonia-oxidizing archaea are more important than ammonia-oxidizing bacteria in nitrification and NO3 --N loss in acidic soil of sloped land. Biol Fertil Soils 49(6):767–776. doi:10.1007/s00374-012-0767-1

  • Saiki R, Scharf S, Faloona F, Mullis K, Horn G, Erlich H, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230(4732):1350–1354. doi:10.1126/science.2999980

    Article  CAS  PubMed  Google Scholar 

  • Saiki R, Gelfand D, Stoffel S, Scharf S, Higuchi R, Horn G, Mullis K, Erlich H (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491. doi:10.1126/science.2448875

    Article  CAS  PubMed  Google Scholar 

  • Schmid M, Walsh K, Webb R, Rijpstra WI, van de Pas-Schoonen K, Verbruggen MJ, Hill T, Moffett B, Fuerst J, Schouten S, Damsté JSS, Harris J, Shaw P, Jetten M, Strous M (2003) Candidatus “Scalindua brodae”, sp. nov., Candidatus “Scalindua wagneri”, sp. nov., two new species of anaerobic ammonium oxidizing bacteria. Syst Appl Microbiol 26(4):529–538

    Article  CAS  PubMed  Google Scholar 

  • Sheffield VC, Cox DR, Lerman LS, Myers RM (1989) Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci U S A 86(1):232–236

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Steffan R, Atlas R (1991) Polymerase chain reaction: applications in environmental microbiology. Ann Rev Microbiol 45(1):137–161

    Article  CAS  Google Scholar 

  • Streit WR, Schmitz RA (2004) Metagenomics—the key to the uncultured microbes. Curr Opin Microbiol 7(5):492–498

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739. doi:10.1093/molbev/msr121

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tourna M, Freitag TE, Prosser JI (2010) Stable isotope probing analysis of interactions between ammonia oxidizers. Appl Environ Microbiol 76(8):2468–2477. doi:10.1128/aem.01964-09

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Videira S, Pereira e Silva M, Souza Galisa P, Dias A, Nissinen R, Divan V, Elsas J, Baldani J, Salles J (2013) Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop. Plant Soil 373(1-2):737–754. doi:10.1007/s11104-013-1828-4

    Article  CAS  Google Scholar 

  • Wang Y-F, Gu J-D (2014) Effects of allylthiourea, salinity and pH on ammonia/ammonium-oxidizing prokaryotes in mangrove sediment incubated in laboratory microcosms. Appl Microbiol Biotechnol 98(7):3257–3274. doi:10.1007/s00253-013-5399-3

  • Wang Y-F, Feng Y-Y, Ma X, Gu J-D (2013) Seasonal dynamics of ammonia/ammonium-oxidizing prokaryotes in oxic and anoxic wetland sediments of subtropical coastal mangrove. Appl Microbiol Biotechnol 97(17):7919–7934. doi:10.1007/s00253-012-4510-5

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wang Y-F, Li X-Y, Gu J-D (2014a) Differential responses of ammonia/ammonium-oxidizing prokaryotes in mangrove sediment to amendment of acetate and leaf litter. Appl Microbiol Biotechnol 98(7):3165–3180. doi:10.1007/s00253-013-5318-7

    Google Scholar 

  • Wang J, Wang W, Gu J-D (2014b) Community structure and abundance of ammonia-oxidizing archaea and bacteria after conversion from soybean to rice paddy in albic soils of Northeast China. Appl Microbiol Biotechnol. 98(6):2765−2778. doi:10.1007/s00253-013-5213-2

  • Weidler GW, Gerbl FW, Stan-Lotter H (2008) Crenarchaeota and their role in the nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps. Appl Environ Microbiol 74(19):5934–5942. doi:10.1128/aem.02602-07

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63(10):3741–3751

    CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgements

This research was supported by a Ph.D. studentship (Y-FW) from the Graduate School of The University of Hong Kong and Environmental and Conservation Fund grant (15/2011). Additional financial support of this project was from Environmental Toxicology Education and Research Fund of this laboratory. We would like to thank Ms. Jessie Lai and Kelly Lau for their support in chemical analysis and Dr. Meng Li for the assistance in field sampling.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ji-Dong Gu.

Additional information

Yong-Feng Wang and Fang-Qiu Zhang contributed to this work equally.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, YF., Zhang, FQ. & Gu, JD. Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance. Appl Microbiol Biotechnol 98, 5655–5663 (2014). https://doi.org/10.1007/s00253-014-5734-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-5734-3

Keywords

Navigation