Skip to content
Research Article Open access CC BY 3.0

An Improved Protocol for Extraction of Metagenomic DNA from High Humus, Alkaline and Saline Soil of Chinampa for T-RFLP Fingerprinting Analysis

Salvador Embarcadero-Jiménez, Feng Long Yang, Raquel Freye-Hernández, Yanelly Trujillo-Cabrera, Flor N. Rivera Orduña, Hong Li Yuan, En Tao Wang

Microbiology Research Journal International · pp. 821–830 · Published 8 Apr 2014

10.9734/BMRJ/2014/9670

Abstract

Aims: The principle aim of this study was to obtain high quality metagenomic DNA from the high humus-containing, alkaline soils of the chinampas, an artificial sustainable agro-ecosystem. Study Design: Different protocols reported previously were tested and were modified to extract the metagenomic DNA. Quality of the DNA samples was evaluated by amplification of 16S rRNA gene with PCR and T-RFLP (Terminal Restriction Fragment Length Polymorphism) analysis. Place and Duration of Study: This study was performed in Department of Microbiology, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional during 2011-2012. Methodology: Four soil samples were collected from two chinampas at the depth of 0-30 cm and 30-60cm. A protocol started with repeated prewashing before the direct cell lysis with lysozyme followed by SDS treatments, frozen and melting cycling was developed which combined the DNA isolation and purification procedures. The 16SrRNA genes were amplified from the extracted metagenomic DNAs and were used for T-RFLP fingerprinting analysis. Results: The 16SrRNA genes were amplified from all the DNA extracts corresponding to the four soil samples and were successfully used in the T-RFLP analysis, which generated 25 to 109T-RFs in the four soil samples digested separately with the restriction endonucleases HaeIII, HhaI and MspI. Conclusion: The protocol developed in the present study could generate high molecular weight and high quality metagenomic DNA from soils with high content of humic materials, for which the other reported protocols were not functioned. This soil harboured very diverse and unique bacterial communities belonging to at least nine phyla that might contribute to the high soil fertility.

Metagenomics DNA extraction humic acids T-RFLP fingerprinting bacterial communities

References (32)

  1. 1 PAST: PALEONTOLOGICAL STATISTICAL SOFTWARE PACKAGE FOR EDUCATION AND DATA ANALYSIS
  2. 2 How Many Species Are There on Earth and in the Ocean? [DOI]
  3. 3 DNA recovery from soils of diverse composition [DOI]
  4. 4 Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA [DOI]
  5. 5 Rapid Method for Coextraction of DNA and RNA from Natural Environments for Analysis of Ribosomal DNA- and rRNA-Based Microbial Community Composition [DOI]
  6. 6 Metagenomic Pyrosequencing and Microbial Identification [DOI]
  7. 7 Extraction of DNA from soil [DOI]
  8. 8 INVITED REVIEW: Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances [DOI]
  9. 9 Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR [DOI]
  10. 10 Characterisation of the soil bacterial community structure and composition of natural and constructed wetlands [DOI]
  11. 11 Simple and rapid method fordirect extraction of microbial DNA fromsoil for PCR [DOI]
  12. 12 Soil Microbial Community Analysis using Terminal Restriction Fragment Length Polymorphisms [DOI]
  13. 13 Strategies for accessing soil metagenome for desired applications [DOI]
  14. 14 Characterization of soil bacterial community structure and physicochemical properties in created and natural wetlands [DOI]
  15. 15 Functional analysis of metagenomes and metatranscriptomes using SEED and KEGG [DOI]
  16. 16 Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments [DOI]
  17. 17 Direct Extraction of DNA from Soils for Studies in Microbial Ecology [DOI]
  18. 18 Influence of intercropping and intercropping plus rhizobial inoculation on microbial activity and community composition in rhizosphere of alfalfa (Medicago sativaL.) and Siberian wild rye (Elymus sibiricusL.) [DOI]
  19. 19 Optimization of methods for detecting Mycobacterium avium subsp. paratuberculosis in environmental samples using quantitative, real-time PCR [DOI]
  20. 20 An Improved Protocol for DNA Extraction from Alkaline Soil and Sediment Samples for Constructing Metagenomic Libraries [DOI]
  21. 21 Extraction of DNA from soil using nanoparticles by magnetic bioseparation [DOI]
  22. 22 The bacterial community structure in an alkaline saline soil spiked with anthracene [DOI]
  23. 23 A rapid, cost-effective procedure for the extraction of microbial DNA from soil [DOI]
  24. 24 Factors influencing the composition of bacterial communities found at abandoned copper-tailings dumps [DOI]
  25. 25 Molecular characterization of soil bacterial communities in contrasting zero tillage systems [DOI]
  26. 26 An interlaboratory comparison of 16S rRNA gene‐based terminal restriction fragment length polymorphism and sequencing methods for assessing microbial diversity of seafloor basalts [DOI]
  27. 27 Diverse cellulolytic bacteria isolated from the high humus, alkaline-saline chinampa soils [DOI]
  28. 28 Rapid and simple methodology for isolation of high quality genomic DNA from coniferous tissues (Taxus baccata) [DOI]
  29. 29 METALES PESADOS, SALES Y SODIO EN SUELOS DE CHINAMPA EN MÉXICO HEAVY METALS, SALTS AND SODIUM IN CHINAMPA SOILS IN MÉXICO
  30. 30 Comparison of rapid DNA extraction methods applied to contrasting New Zealand soils [DOI]
  31. 31 Extraction and purification of microbial DNA from petroleum‐contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis [DOI]
  32. 32 Extraction of High Molecular Weight Genomic DNA from Soils and Sediments [DOI]

Cited by 6

An improved method for extraction of microbial DNA from alkaline-saline soil

Valentín Pérez-Hernández, Mario Hernández-Guzmán, César Valenzuela-Encinas · REVISTA TERRA LATINOAMERICANA · 2021

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

E. Ahmadi, M. Kowsari, D. Azadfar · Annals of Forest Science · 2018

Optimized genomic DNA extraction by a modified organic phenol- chloroform method without using PCR for best results

H. Ali, Sadaf Jafar, Qurat-ul-Ain · 2016

Bacterial communities estimated by pyrosequencing in the soils of chinampa, a traditional sustainable agro-ecosystem in Mexico

S. Embarcadero-Jiménez, F. N. Rivera-Orduña, E. Wang · Journal of Soils and Sediments · 2016

16S rRNA BASED T-RFLP ANALYSIS OF METHANOGENS IN BIOGAS PLANT WITH P. HYSTEROPHORUS L. AS SUBSTRATE -

R. Ramya, M. Shree · 2014

Showing 5 of 6 known citations — external sources report more than can currently be individually listed.

Related research

Article metrics

Real usage data collected on this platform.

68

Page views

0

PDF downloads

54

Outbound clicks

6

Citations

Views over time

Views by country

Approximate, from request IP at view time — not citizenship or institution. Countries with fewer than 5 views are grouped as "Other".

Traffic sources

Referring site, by host.

Views and downloads exclude known bots/crawlers. Citations combines this platform's own DOI-resolved index with each external source's own reported total — see Cited by above for individually listed citing works. Last refreshed 0 seconds ago.