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Isolation of PCR-quality Genomic DNA from Soils Impacted with Extra Heavy Crude Oil

Laynet A. Puentes Laynet A. Puentes
YR Yusibeska L. Ramos
YI Ysvic A. Inojosa
César E. Rivera César E. Rivera
AS Angela De Sisto

Published: Jun 20, 2019 DOI: 10.21769/BioProtoc.3265 Views: 3900

Edited by: Alexandros Alexandratos

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Abstract

For the study of microbial communities in samples of soils impacted with extra heavy crude oil, it is necessary to perform molecular analyses. Due to the difficulty of oil matrix handling, there are very few protocols reported in writing. Also, one can only observe a very low concentration of DNA. That’s why it is required to have an effective protocol to conduct studies in this type of matrix. This protocol includes steps of cell lysis by saline buffer with ionic/non-ionic detergents, and enzymatic digestion with lysozyme and proteases, complemented with organic extraction and alcohol precipitation. Additionally, it requires purification to eliminate the inhibitory substances of the extract that cause PCR inhibition. The method of DNA extraction proposed in this study is easy to handle and low cost. It allows the extraction of DNA from different bacteria and fungi, associated with soil contaminated with extra heavy crude.

Keywords: Metagenomic search Microorganisms search Crude Oil search Soil search Contamination search PCR search

Background

On bioremediation, the identification of helpful bacteria is not always easy. Kathiravan et al. (2015) developed a way of extracting total genomic DNA from samples of farmland. However, working with crude oil can be a little bit more problematic. The metagenomic DNA obtained must be purified, since humic acid contaminants and proteins have serious negative effects on the DNA polymerase (Wang et al., 2013). In the same way, hydrocarbon-contaminated soils present an even greater challenge. Because, in addition to the usual soil contaminants, traces of organic substances and heavy metals remain in the DNA extracts and reduce PCR amplification efficiency, by degrading or capturing nucleic acids, or by deactivating DNA polymerase (Fortin et al., 2004). Polymerase chain reaction (PCR), is used in order to detect genes involved in metabolic pathways of interest and obtain an analysis of soil microbial diversity. The PCR is a necessary study to remedy and restore oil contaminated soils through environmentally friendly biological technologies (Lozano-A et al., 2008).

In order to bypass the PCR inhibitors in DNA extraction from soil, different methodologies are employed to eliminate them: washing steps, thermal shocks, chemical lysis with detergents, and an enzymatic digestion step that frequently employs lysozyme and proteinase K to quicken the process and increase the DNA yield. However, these steps tend to make DNA extraction protocols time-consuming, costly, tiresome and laborious.

The objective of this study was to evaluate the protocol of Kathiravan et al. (2015) modified to match a soil matrix highly contaminated with extra heavy oil. The protocol includes a cell lysis by means of saline buffer with ionic/non-ionic detergents, and enzymatic digestion with lysozyme and proteases, plus a purification step to remove the contaminants. The quality and purity of the DNA were validated by the amplification efficiencies of several genes of interest for PCR. It was also tested if this DNA extraction proposed is easy to handle and low cost.

Materials and Reagents

  1. Pipette tips 
  2. Eppendorf tubes 1.5 ml
  3. Porcelain Mortar and pestle 500 ml
  4. Reusable Stainless Pellet Pestles, 1.5 ml (Thomas Scientific, catalog number: 749515-0000)
  5. 2 mm 100 200 micron sand mesh ss oil size test slotted soil testing filter sieve/sediment sieves
  6. Proteinase K (Invitrogen, catalog number: 25530-015) 
  7. Lysozyme (Sigma, catalog number: 235-747-3)
  8. Hexadecyltrimethylammonium Bromide (CTAB) (Sigma, catalog number: 52366)
  9. SDS (Sigma-Aldrich, catalog number: L3771)
  10. Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (Sigma, catalog number: P3803)
  11. Chloroform:Isoamyl Alcohol 24:1 (Sigma, catalog number: C0549)
  12. Isopropanol (Sigma, catalog number: I0398)
  13. Ethanol (Sigma, catalog number: V0T0041)
  14. GoTaq Flexi DNA Polymerase (Promega, catalog number: M829)
  15. dNTP Set (100 mM) Solution (Invitrogen, catalog number 10-297-018) 
  16. Agarose D-1 LE GQT (Scientific Trade Corp, catalog number: 8015)
  17. FavorPrep Gel/PCR Purification mini kit (Favorgen, catalog number: FAGCK001-1)
  18. 100 bp DNA Ladder (Promega, catalog number: G2101)
  19. 1 kb DNA Ladder (Sigma-Aldrich, catalog number: D0428-1VL)
  20. Na2EDTA (Sigma-Aldrich, catalog number: 6381-92-6)
  21. NaH2PO4 (J.T. Baker, catalog number: 10049-21-5)
  22. NaCl (Sigma-Aldrich, catalog number: S-6191)
  23. Tris-Base (1 M) (Promega, catalog number: 77-86-1)
  24. Boric Acid AR/ACS (Loba Chemie, catalog number: 10043-35-3)
  25. EDTA (Sigma-Aldrich, catalog number: E9884)
  26. MgCl2
  27. Ethidium bromide
  28. Tris-HCl
  29. DNA extraction buffer (see Recipes)
  30. Buffer TBE 5x (see Recipes)
  31. Agarose Gel (see Recipes)

Equipment

  1. Analytical balance (Ohaus adventurer AR2140, catalog number: 67850)
  2. Eppendorf pipettes
  3. Thermomixer (Eppendorf 5350, catalog number: 5350-000.013)
  4. Vortex (Heidolph Reax Top, catalog number: 541-10000-01-0)
  5. Centrifuge (Eppendorf 5415 D, catalog number: 5425-000.014)
  6. Centrifuge (Eppendorf 5424, catalog number: 5424-000.614)
  7. Concentrator (Eppendorf 5301, catalog number: 5301-000.016)
  8. Mastercycler ep gradient (Eppendorf 5341, catalog number 5341-00-108)
  9. Horizontal electrophoresis system (Mini-Sub Cell GT Cell, and Power Pac Basic Power Supply) (Bio-Rad, catalog number: 1640300)
  10. Bio photometer (Eppendorf 6131, catalog number 6131-000.012) 
  11. Gel Documentation Systems (Bio-Rad Universal Hood II, catalog number: 1708195EDU)

Procedure

Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Microbial genetics > DNA > DNA detection and isolation

Molecular Biology > DNA > DNA extraction

Environmental science > Bacterium

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