Nucleic Acid Extraction for Studying Plant-Microbe Interactions in Rhizosphere

  • Gautam Anand
  • Abhineet Sain
  • Virendra S. Bisaria
  • Shilpi SharmaEmail author


Studies on diversity of microbial community in the field of rhizosphere ecology vastly rely on nucleic acid markers for analysis. The extraction can be a tedious and complicated task owing to the vast heterogeneity present in soil in terms of organic and inorganic constituents, texture and moisture content, and also the huge repertoire of life forms that it nurtures. There is no universal method of extraction for all soil types. The various challenges presented by the soil constituents make it an ever-evolving process. Cell lysis is an inherent part of any extraction process, with the extraction methodology exerting a huge impact on purity and yield of nucleic acid. Different extraction methods employed so far can be classified under two main categories based on the step of lysis: indirect lysis and direct lysis methods. Humic acid is a persistent contaminant that has the maximum impact on nucleic acid quality, along with its interference with several downstream analyses. Several methods have been optimized for removal of soil organic content. With the realization of the importance of RNA component in providing a deeper insight into the functionality of the system, the co-extraction of DNA and RNA is a trending technique, with vast emphasis on the removal of humic acid, and purity of the extracted DNA/RNA.


Cell lysis Humic acid Co-extraction DNA RNA 



Funding received from DBT (BT/PR5499/AGR/21/355/2012) and SERB (YSS/2015/001437) is acknowledged.


  1. Amann RI, Ludwig W, Schleifer K (1995) Phylogenetic identification and in-situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedPubMedCentralGoogle Scholar
  2. Bakken LR, Lindahl V (1995) Recovery of bacterial cells from soil. In: Nucleic acids in the environment. Springer, Heidelberg/Berlin, pp 9–27Google Scholar
  3. Boivin-Jahns V, Ruimy R, Bianchi A et al (1996) Bacterial diversity in a deep-subsurface clay environment. Appl Environ Microbiol 62:3405–3412PubMedPubMedCentralGoogle Scholar
  4. Braid MD, Daniels LM, Kitts CL (2003) Removal of PCR inhibitors from soil DNA by chemical flocculation. J Microbiol Methods 52:389–393CrossRefPubMedGoogle Scholar
  5. Bruce KD, Hiorns WD, Hobman JL et al (1992) Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction. Appl Environ Microbiol 58:3413–3416PubMedPubMedCentralGoogle Scholar
  6. Bürgmann H, Pesaro M, Widmer F, Zeyer J (2001) A strategy for optimizing quality and quantity of DNA extracted from soil. J Microbiol Methods 45:7–20CrossRefPubMedGoogle Scholar
  7. Clegg CD, Ritz K, Griffiths BS (1997) Direct extraction of microbial community DNA from humified upland soils. Lett Appl Microbiol 25:30–33CrossRefPubMedGoogle Scholar
  8. Costa R, Gomes NCM, Milling A, Smalla K (2004) An optimized protocol for simultaneous extraction of DNA and RNA from soils. Braz J Microbiol 35:230–234CrossRefGoogle Scholar
  9. Courtois S, Frostegård A, Göransson P et al (2001) Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation. Environ Microbiol 3:431–439CrossRefPubMedGoogle Scholar
  10. Cullen DW, Hirsch PR (1998) Simple and rapid method for direct extraction of microbial DNA from soil for PCR. Soil Biol Biochem 30:983–993CrossRefGoogle Scholar
  11. Degrange V, Bardin R (1995) Detection and counting of Nitrobacter populations in soil by PCR. Appl Environ Microbiol 61:2093–2098PubMedPubMedCentralGoogle Scholar
  12. Dijkmans R, Jagers A, Kreps S et al (1993) Rapid method for purification of soil DNA for hybridization and PCR analysis. Microb Releases 2:29–34PubMedGoogle Scholar
  13. Duarte GF, Rosado AS, Seldin L et al (1998) Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community. J Microbiol Meth 32:21–29CrossRefGoogle Scholar
  14. Erb RW, Wagner-Dobler I (1993) Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol 59:4065–4073PubMedPubMedCentralGoogle Scholar
  15. Fægri A, Torsvik VL, GoksÖyr J (1977) Bacterial and fungal activities in soil: separation of bacteria and fungi by a rapid fractionated centrifugation technique. Soil Biol Biochem 9:105–112CrossRefGoogle Scholar
  16. Fang C, Xu T, Ye C et al (2014) Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil. World J Microbiol Biotechnol 30:783–789CrossRefPubMedGoogle Scholar
  17. Felske A, Engelen B, Nübel U, Backhaus H (1996) Direct ribosome isolation from soil to extract bacterial rRNA for community analysis. Appl Environ Microbiol 62:4162–4167PubMedPubMedCentralGoogle Scholar
  18. Frostegård ÅSA, Courtois S, Ramisse V et al (1999) Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbiol 65:5409–5420PubMedPubMedCentralGoogle Scholar
  19. Griffiths RI, Whiteley AS, Anthony G et al (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl Environ Microbiol 66:5488–5491CrossRefPubMedPubMedCentralGoogle Scholar
  20. Harry M, Gambier B, Bourezgui Y, Garnier-Sillam E (1999) Evaluation of purification procedures for DNA extracted from rich organic samples: interference with humic substances. Analusis 27:439–441CrossRefGoogle Scholar
  21. Hilger AB, Myrold DD (1991) Method for extraction of Frankia DNA from soil. Agric Ecosyst Environ 34(1-4):107–113Google Scholar
  22. Hirsch PR, Mauchline TH, Clark IM (2010) Culture-independent molecular techniques for soil microbial ecology. Soil Biol Biochem 42:878–887CrossRefGoogle Scholar
  23. Holben WE, Jansson JK, Chelm BK, Tiedje JM (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl Environ Microbiol 54:703–711PubMedPubMedCentralGoogle Scholar
  24. Hurt RA, Qiu X, Wu L et al (2001) Simultaneous recovery of RNA and DNA from soils and sediments simultaneous recovery of RNA and DNA from soils and sediments. Appl Environ Microbiol 67:4495–4503CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jackson CR, Harper JP, Willoughby D et al (1997) A simple, efficient method for the separation of humic substances and DNA from environmental samples. Appl Environ Microbiol 63:4993–4995PubMedPubMedCentralGoogle Scholar
  26. Jacobsen CS, Rasmussen OF (1992) Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin. Appl Environ Microbiol 58:2458–2462PubMedPubMedCentralGoogle Scholar
  27. Kreader CA (1996) Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 62:1102–1106PubMedPubMedCentralGoogle Scholar
  28. Kuske CR, Banton KL, Adorada DL et al (1998) Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl Environ Microbiol 64:2463–2472PubMedPubMedCentralGoogle Scholar
  29. Lindahl V, Bakken LR (1995) Evaluation of methods for extraction of bacteria from soil. FEMS Microbiol Ecol 16:135–142CrossRefGoogle Scholar
  30. Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63:4516–4522PubMedPubMedCentralGoogle Scholar
  31. Macdonald RM (1986) Sampling soil microfloras: dispersion of soil by ion exchange and extraction of specific microorganisms from suspension by elutriation. Soil Biol Biochem 18:399–406CrossRefGoogle Scholar
  32. Martin-Laurent F, Philippot L, Hallet S, Chaussod R, Germon JC, Soulas G, Catroux G (2001) DNA extraction from soils: old bias for new microbial diversity analysis methods. Appl Environ Microbiol 67(5):2354–2359Google Scholar
  33. Maarit Niemi R, Heiskanen I, Wallenius K, Lindström K (2001) Extraction and purification of DNA in rhizosphere soil samples for PCRDGGE analysis of bacterial consortia. J Microbiol Methods 45(3):155–165Google Scholar
  34. Mayr C, Winding A, Hendriksen NB (1999) Community level physiological profile of soil bacteria unaffected by extraction method. J Microbiol Meth 36:29–33CrossRefGoogle Scholar
  35. McIlroy SJ, Porter K, Seviour RJ, Tillett D (2009) Extracting nucleic acids from activated sludge which reflect community population diversity. Antonie van Leeuwenhoek Int J Gen Mol Microbiol 96:593–605CrossRefGoogle Scholar
  36. Mettel C, Kim Y, Shrestha PM, Liesack W (2010) Extraction of mRNA from soil. Appl Environ Microbiol 76:5995–6000CrossRefPubMedPubMedCentralGoogle Scholar
  37. Miller DN (2001) Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. J Microbiol Methods 44:49–58CrossRefPubMedGoogle Scholar
  38. Miller DN, Bryant JE, Madsen EL, Ghiorse WC (1999) Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol 65:4715–4724PubMedPubMedCentralGoogle Scholar
  39. Miskin IP, Farrimond P, Head IM (1999) Identification of novel bacterial linages as active members of microbial populations in a freshwater sediment using a rapid RNA extraction procedure and RT-PCR. Microbiology 145:1977–1987CrossRefPubMedGoogle Scholar
  40. Moran MA, Torsvik VL, Torsvik T, Hodson RE (1993) Direct extraction and purification of rRNA for ecological studies. Appl Environ Microbiol 59:915–918PubMedPubMedCentralGoogle Scholar
  41. Moré MI, Herrick JB, Silva MC et al (1994) Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Appl Environ Microbiol 60:1572–1580PubMedPubMedCentralGoogle Scholar
  42. Nannipieri P, Ascher J, Ceccherini MT et al (2003) Microbial diversity and soil functions. Eur J Soil Sci 54:655CrossRefGoogle Scholar
  43. Ogram A, Sayler GS, Barkay T (1987) The extraction and purification of microbial DNA from sediments. J Microbiol Meth 7:57–66CrossRefGoogle Scholar
  44. Ogram A, Sun W, Brockman FJ (1995) Isolation and characterization of RNA from low-biomass deep-subsurface sediments. Appl Environ Microbiol 61:763–768PubMedPubMedCentralGoogle Scholar
  45. Orsini M, Romano-Spica V (2001) A microwave-based method for nucleic acid isolation from environmental samples. Lett Appl Microbiol 33:17–20CrossRefPubMedGoogle Scholar
  46. Peršoh D, Theuerl S, Buscot F, Rambold G (2008) Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil. J Microbiol Meth 75:19–24CrossRefGoogle Scholar
  47. Picard C, Ponsonnet C, Paget E et al (1992) Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol 58:2717–2722PubMedPubMedCentralGoogle Scholar
  48. Pillai SD, Josephson KL, Bailey RL et al (1991) Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. Appl Environ Microbiol 57:2283–2286PubMedPubMedCentralGoogle Scholar
  49. Pitcher DG, Saunders NA, Owen RJ (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8:151–156CrossRefGoogle Scholar
  50. Porteous LA, Armstrong JL (1991) Recovery of bulk DNA from soil by a rapid, small-scale extraction method. Curr Microbiol 22:345–348CrossRefGoogle Scholar
  51. Porteous LA, Seidler RJ, Watrud LS (1997) An improved method for purifying DNA from soil for polymerase chain reaction amplification and molecular ecology applications. Mol Ecol 6:787–791CrossRefGoogle Scholar
  52. Purdy KJ, Embley TM, Takii S, Nedwell DB (1996) Rapid extraction of DNA and rRNA from sediments by a novel hydroxyapatite spin-column method. Appl Environ Microbiol 62:3905–3907PubMedPubMedCentralGoogle Scholar
  53. Ramsay AJ (1984) Extraction of bacteria from soil: efficiency of shaking or ultrasonication as indicated by direct counts and autoradiography. Soil Biol Biochem 16:475–481CrossRefGoogle Scholar
  54. Robe P, Nalin R, Capellano C et al (2003) Extraction of DNA from soil. Eur J Soil Biol 39:183–190CrossRefGoogle Scholar
  55. Romanowski G, Lorenz MG, Sayler G, Wackernagel W (1992) Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay. Appl Environ Microbiol 58:3012–3019PubMedPubMedCentralGoogle Scholar
  56. Roose-Amsaleg CL, Garnier-Sillam E, Harry M (2001) Extraction and purification of microbial DNA from soil and sediment samples. Appl Soil Ecol 18:47–60CrossRefGoogle Scholar
  57. Sagova-Mareckova M, Cermak L, Novotna J et al (2008) Innovative methods for soil DNA purification tested in soils with widely differing characteristics. Appl Environ Microbiol 74:2902–2907CrossRefPubMedPubMedCentralGoogle Scholar
  58. Saleh-Lakha S, Shannon KE, Goyer C, Trevors JT (2011) Challenges in quantifying microbial gene expression in soil using quantitative reverse transcription real-time PCR. J Microbiol Meth 85:239–243CrossRefGoogle Scholar
  59. Saleh-Lakha S, Miller M, Campbell RG, Schneider K, Elahimanesh P, Hart MM, Trevors JT (2005) Microbial gene expression in soil: methods, applications and challenges. J Microbiol Methods 63(1):1–19Google Scholar
  60. Selenska S, Klingmüller W (1991) Direct detection of nif-gene sequences of Enterobacter agglomerans in soil. FEMS Microbiol Lett 80:243–245CrossRefGoogle Scholar
  61. Sharma S, Mehta R, Gupta R, Schloter M (2012) Improved protocol for the extraction of bacterial mRNA from soils. J Microbiol Meth 91:62–64CrossRefGoogle Scholar
  62. Simonet P, Capellano A, Navarro E et al (1984) An improved method for lysis of Frankia with achromopeptidase allows detection of new plasmids. Can J Microbiol 30:1292–1295CrossRefGoogle Scholar
  63. Smalla K, Cresswell N, Mendonca Hagler LC et al (1993) Rapid DNA extraction protocol from soil for polymerase chain reaction mediated amplification. J Appl Bacteriol 74:78–85CrossRefGoogle Scholar
  64. Steffan RJ, Atlas RM (1988) DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl Environ Microbiol 54:2185–2191PubMedPubMedCentralGoogle Scholar
  65. Steffan RJ, Goksøyr J, Bej AK, Atlas RM (1988) Recovery of DNA from soils and sediments. Appl Environ Microbiol 54:2908–2915PubMedPubMedCentralGoogle Scholar
  66. Tebbe CC, 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 59:2657–2665PubMedPubMedCentralGoogle Scholar
  67. Thakuria D, Schmidt O, Mac Siúrtáin M et al (2008) Importance of DNA quality in comparative soil microbial community structure analyses. Soil Biol Biochem 40:1390–1403CrossRefGoogle Scholar
  68. Thornhill RH, Burgess JG, Matsunaga T (1995) PCR for direct detection of indigenous uncultured magnetic cocci in sediment and phylogenetic analysis of amplified 16S ribosomal DNA. Appl Environ Microbiol 61:495–500PubMedPubMedCentralGoogle Scholar
  69. Torsvik VL (1980) Isolation of bacterial DNA from soil. Soil Biol Biochem 12:15–21CrossRefGoogle Scholar
  70. Torsvik VL, Goksoyr J (1978) Determination of bacterial DNA in soil. Soil Biol Biochem 10:7–12CrossRefGoogle Scholar
  71. Torsvik V, Goksøyr J, Daae FL (1990) High diversity in DNA of soil bacteria. Appl Environ Microbiol 56:782–787PubMedPubMedCentralGoogle Scholar
  72. Towe S, Wallisch S, Bannert A et al (2011) Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils. J Microbiol Meth 84:406–412CrossRefGoogle Scholar
  73. Tsai YL, Olson BH (1991) Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol 57:1070–1074PubMedPubMedCentralGoogle Scholar
  74. Tsai YL, Park MJ, Olson BH (1991) Rapid method for direct extraction of mRNA from seeded soils. Appl Environ Microbiol 57:765–768PubMedPubMedCentralGoogle Scholar
  75. Turpin PE, Maycroft KA, Rowlands CL, Wellington EM (1993) An ion-exchange based extraction method for the detection of salmonellas in soil. J Appl Bacteriol 74:181–190CrossRefPubMedGoogle Scholar
  76. Van Elsas JD, Van Overbeek LS, Fouchier R (1991) A specific marker, pat, for studying the fate of introduced bacteria and their DNA in soil using a combination of detection techniques. Plant Soil 138:49–60CrossRefGoogle Scholar
  77. Van Elsas JD, Mantynen V, Wolters AC (1997) Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence. Biol Fertil Soils 24:188–195CrossRefGoogle Scholar
  78. Volossiouk T, Robb EJ, Nazar RN (1995) Direct DNA extraction for PCR-mediated assays of soil organisms. Appl Environ Microbiol 61:3972–3976PubMedPubMedCentralGoogle Scholar
  79. Young CC, Burghoff RL, Keim LG et al (1993) Polyvinylpyrrolidone-agarose gel electrophoresis purification of polymerase chain reaction-amplifiable DNA from soils. Appl Environ Microbiol 59:1972–1974PubMedPubMedCentralGoogle Scholar
  80. Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  • Gautam Anand
    • 1
  • Abhineet Sain
    • 1
  • Virendra S. Bisaria
    • 1
  • Shilpi Sharma
    • 1
    Email author
  1. 1.Department of Biochemical Engineering and BiotechnologyIndian Institute of Technology DelhiNew DelhiIndia

Personalised recommendations