Nucleic Acid Isolation from Ecological Samples—Vertebrate Gut Flora

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Abstract

The utility of DNA molecules in identifying and characterizing intestinal microorganisms depends on methods that facilitate access to DNA of sufficient purity, quantity, and integrity. An efficient and unbiased extraction of DNA is thus critical to the validity of the subsequent analysis of the prevalence and diversity of the DNA sources in the sample. The highly heterogeneous composition of the diet of vertebrates makes DNA isolation challenging for this environment. Here, we consider the key steps involved in DNA isolation from vertebrate gut microflora including sample homogenization, lysis of bacterial cells, and extraction and precipitation of DNA. A detailed protocol for DNA isolation of the microbial contents of intestine and feces is also provided. In addition, we refer to commercially available methods for DNA extraction from the vertebrate gut flora.

Introduction

The gastrointestinal (GI) tract (gut) of vertebrates harbors complex microbial communities that provide essential functions for the host, including food digestion, breakdown of toxic substances, production of essential vitamins, and prevention of gut colonization by microbial pathogens (Mackie 1999, Wang 1996, Wang 2002).

Traditional methods for identifying and characterizing bacteria in the gut flora are based on various cultivation techniques (e.g., selective media), biochemical tests (e.g., enzymatic assays, Biolog plates), morphological examination (e.g., microscopy), and analysis of fatty acid production (e.g., fatty acid methyl ester [FAME] and phospholipid fatty acid [PFLA]) (Tannock 1999, Wang 1996, Zoetendal 2004). More recently, DNA-based methods for identifying and characterizing intestinal microorganisms have been adopted. These methods include 16S ribosomal DNA (rDNA)–targeted polymerase chain reaction (PCR) amplification and sequencing, and PCR-based community profiling techniques such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and terminal restriction fragment length polymorphism. (T-RFLP) (Gong 2002, Mackie 1999, Matsuki 2002, Simpson 1999, Withford 1998, Zhu 2002). DNA-based methods require that DNA can be extracted from the GI tract in sufficient quantity, free from excess fragmentation and contaminants, and in a way that is representative of the true species distribution and abundance in the specific environment. Incomplete bacterial cell lysis, DNA sorption to particulate material, enzymatic or physical degradation, and chemical modifications are some common problems that can negatively affect the efficiency of DNA extraction (Monterio 1997, Santini 2001, Zoetendal 2001). The variable composition of the diet of vertebrates makes nucleic acids isolation challenging for this heterogeneous environment and excludes the adoption of a single protocol (Blaut 2002, Mackie 1999).

Several methods for the extraction of DNA from the vertebrate gut have been described (Anderson 2003, Avgustin 1994, Bruce 1992, Tsai 1991, Wilson 1996, Li 2003, Pryde 1999, Santini 2001, Simpson 1999, Stahl 1988, Wood 1998, Zhu 2002). In addition, a number of commercial kits are available (Table I).

Here, we summarize the various steps involved in DNA isolation from vertebrate gut microflora and provide a detailed protocol for DNA isolation of intestinal contents and feces. The extraction and purification procedures described in this chapter have, with modifications, been shown to be suitable for identification of microorganisms in the gut flora of different animals, like chickens, pigs, and cattle (Anderson 2003, Gong 2002, Li 2003, Withford 1998).

Section snippets

Steps in DNA Isolation

The basic steps in the extraction of nucleic acids from vertebrate gut flora for the purpose of molecular analysis are summarized in Fig. 1. DNA isolation protocols found in the literature provide alternative approaches and combinations of these steps. Below, the steps are considered in more detail to allow system-specific optimization of the DNA isolation procedure.

Preparation of Sample before DNA Extraction

  • 1.

    Suspend 1–10 g of sample (depending on bacterial density) in 30 or 70 ml of sterile saline (0.85% NaCl) containing 0.1% Tween 80.

  • 2.

    Centrifuge at low speed (200g) for 5 min at 4° to remove larger particulate matter. Collect the upper phase, transfer the supernatant to a clean sterile tube, and keep on ice. Save the pellet.

  • 3.

    Resuspend the pellet in saline containing 0.1% Tween 80 and repeat centrifugation.

  • 4.

    Repeat step 3 three times and pool the supernatants. Pellets may now be discarded.

  • 5.

    Centrifuge the

Further Considerations

The DNA yield and quality obtained using several of the published methods have been compared (Anderson 2003, Holland 2000, Li 2003, McOrist 2002, Santini 2001). Anderson and Lebepe-Mazur (2003) compared 19 noncommercial methods that are used for nucleic acid extraction of gut microflora and concluded that four of the methods were preferable with regards to DNA yield and purity. One of these methods, with modifications, has been described in this chapter (Withford et al., 1998). Some studies

Acknowledgements

The authors acknowledge financial support from the Research Council of Norway (140890⧸720 and 140870⧸130). We thank Jessica L. Ray for comments on the manuscript.

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