Community DNA Extraction from Bacterial Colonies
Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Traditional methods of anal…
1. Bacterial Community DNA Extraction
- To begin the procedure, weigh out 100 g of sieved soil. Add this to a polypropylene vessel, and add 100 mL of extraction buffer comprised of Tris buffer amended with EDTA to promote the release of bacteria from the soil matrix, then shake by hand.
- Next, weigh 100 g of glass beads, and add these to the mixing vessel. Agitate the sample for 5 min using a bead beating device or mechanical wrist-action shaker for 15 min. Add 10 mL 20% sodium dodecyl sulfate, or SDS, to the mixture, then agitate for an additional minute. Incubate at a high temperature of 60 - 65 °C for 60 min.
- Equally distribute the sample among separate 50-mL tubes, and centrifuge for 10 min at 6,000 x g. Transfer the supernatant from the tubes to a single sterile container. Next, repeat the extraction on the soil pellet as previously described, using a fresh volume of extraction buffer.
- Next, add the total volume of processed supernatant, approximately 200 mL, to a clean 50-mL tube filled to half volume with a solution of 30% polyethylene glycol and 1.6 M sodium chloride. Invert the bottles several times by hand to mix, and incubate at room temperature for 2 h. Centrifuge samples at 10,000 x g for 20 min to pellet the DNA.
- Remove the supernatant carefully from the centrifuge tube, leaving behind the partially purified nucleic acid pellet. Add 20 mL of TE Buffer and 1.5 mL of a 7.5 M potassium acetate solution to resuspend the pellet, then vortex. Place the suspension on ice for 5 min. Centrifuge at 16,000 x g for 30 min at 4 °C to precipitate proteins and polysaccharides.
- Next, add an RNAse and proteinase K to the sample, mix gently by hand, and let sit for moment. Add an equivalent volume of phenol:chloroform:isoamyl alcohol (ratio mixture of 25:24:1) to the suspension to be extracted, and mix gently by hand. Centrifuge the preparation for 10 min at 13,000 x g. Carefully remove the vessel from the centrifuge, and note the two layers.
- The bottom, heavier layer is comprised of the phenol:chloroform:isoamyl alcohol and extracted debris, and the top layer is the aqueous and contains the DNA. Place the aqueous phase into a sterile vessel, add an equivalent volume of isopropanol, and invert gently to initiate DNA precipitation. Incubate the suspension at room temperature for 2 h. Pellet the purified DNA by centrifugation at 16,000 x g for 30 min. Carefully remove the supernatant as the pelleted DNA may or may not be visible at the bottom of the vessel, and then resuspend in 1 mL of TE Buffer.
- Using a spectrophotometer or DNA/RNA quantification fluorimeter, measure the level of DNA extracted from the sample. The amount of DNA is estimated from the 260 nm reading. An absorbance reading of 1.0 is equivalent to 50 µg of DNA per mL of solution. If the concentration is too high for accurate readings, dilute the suspension 1 to 10, or 1 to 100 using molecular grade water.
- The purity of DNA is estimated from the ratio of the reading at 260 nm to that at 280 nm. A value > 1.7 indicates relatively pure DNA. The maximum theoretical value is 2.0.
Bacterial community DNA extraction is a process by which DNA is obtained from multiple bacterial species within a community during a single extraction procedure.
Traditional analyses of microbial communities in soil have usually involved cultural assays, utilizing dilution and plating methodology on different selective media. However, many bacteria grow poorly under laboratory conditions or on the specific growth media conditions selected, meaning they may be missed or severely underrepresented.
Recently, extracting community DNA from soil bacterial samples has allowed for a more comprehensive sampling of bacterial communities. This non-culture based approach is thought to be more representative of the actual communities present than traditional culture based methods.
This video will demonstrate a non-culture method of bacterial community DNA extraction, how to check the quality and quantity of extracted DNA, and explore how this DNA may be utilized to study bacterial diversity.
DNA extraction from soil can be conducted in one of two ways. In the fractionation method, cells are first separated from the soil matrix prior to extraction of the genetic material. The sample is then subjected to successive cycles of blending and slow centrifugation in order to collect intact cells in a pellet.
Lysozymes are?then added to the cells, and the suspension is incubated. Lysozymes are?enzymes that break down bacterial cell walls. Once the cell wall structure has been compromised, the DNA may then be liberated for purification. However, a second method of community DNA extraction, the in situ method, has been shown to yield greater DNA concentration.
Here, a mass of soil is combined with an equivalent volume of Tris-EDTA extraction buffer and glass beads, and mixed aggressively to facilitate separation of cells from the soil particles. A detergent is then added, generally sodium dodecyl sulfate, or SDS, and the sample is further blended to promote lysing of the cells and release of their contents, including DNA.
Incubation at a high temperature is then undertaken to lyse any remaining bacterial cells. Samples are centrifuged, and a polyethylene glycol extraction and incubation is performed on the supernatant in order to precipitate the DNA, which is then centrifuged into a pellet.
The DNA is resuspended in TE Buffer and potassium acetate in order to further wash the DNA of proteins and polysaccharides, then centrifugation is carried out to pellet these undesirable components. The aqueous supernatant containing the DNA is removed, and subjected to a phenol-chloroform extraction and isopropanol precipitation to further clean and concentrate the DNA. Following a room temperature incubation period of two hours, the DNA is centrifuged and resuspended in TE Buffer for storage until analysis.
Now that we are familiar with the concepts and processes behind bacterial community DNA extraction, let's take a look at how it is carried out in the laboratory.
To begin the procedure, weigh out 100 g of sieved soil. Add this to a polypropylene vessel, and add 100 mL of extraction buffer comprised of Tris buffer amended with EDTA to promote the release of bacteria from the soil matrix, then shake by hand.
Next, weigh 100 g of glass beads, and add these to the mixing vessel. Agitate the sample for 5 min?using a bead beating device or mechanical wrist-action shaker. Add 10 mL 20% sodium dodecyl sulfate, or SDS, to the mixture, then agitate for an additional minute. Incubate at a high temperature for 60 min.
Equally distribute the sample among separate 50 mL tubes, and centrifuge for 10 min?at 6,000 x g. Transfer the supernatant from the tubes to a single sterile container. Next, repeat the extraction on the soil pellet as previously described, using a fresh volume of extraction buffer.
Next, add the total volume of processed supernatant to a clean 50-mL tube filled to half volume with a solution of 30% polyethylene glycol and 1.6 M sodium chloride. Invert the bottles several times by hand to mix, and incubate at room temperature for 2 h. Centrifuge samples at 10,000 x g for 20 min?to pellet the DNA.
Remove the supernatant carefully from the centrifuge tube, leaving behind the partially purified nucleic acid pellet. Add 20 mL of TE Buffer and 1.5 mL of a 7.5 M potassium acetate solution to resuspend the pellet, then vortex. Place the suspension on ice for 5 min. Centrifuge at 16,000 x g for 30 min at 4 ?C to precipitate proteins and polysaccharides.
Next, add an RNAse and proteinase K to the sample, mix gently by hand, and let sit for moment. Add an equivalent volume of phenol:chloroform:isoamyl alcohol to the suspension to be extracted, and mix gently by hand. Centrifuge the preparation for 10 min at 13,000 x g. Carefully remove the vessel from the centrifuge, and note the two layers.
The bottom, heavier layer is comprised of the phenol:chloroform:isoamyl alcohol and extracted debris, and the top layer is the aqueous and contains the DNA. Place the aqueous phase into a sterile vessel, add an equivalent volume of isopropanol, and invert gently to initiate DNA precipitation. Incubate the suspension at room temperature for 2 h. Pellet the purified DNA by centrifugation at 16,000 x g for 30 min. Carefully remove the supernatant as the pelleted DNA may or may not be visible at the bottom of the vessel, and then resuspend in 1 mL of TE Buffer.
Using a spectrophotometer or DNA/RNA quantification fluorimeter, measure the level of DNA extracted from the sample. DNA/RNA fluorimeters will output DNA levels in units of nanograms per milliliter. If the concentration is too high for accurate readings, dilute the suspension 1 to 10, or 1 to 100 using molecular grade water.
Once DNA is obtained from a bacterial community, this can be utilized in a number of different ways. Some of those applications are explored here.
Spectrophotographic analyses of the DNA extracted from community DNA can provide an insight into the number of bacterial cells present in a given soil sample. The estimated quantity of DNA in ng per mL of solution can be related back to the total volume of DNA extracted in solution, to give the total amount of DNA per g of soil. Knowing the theoretical value of DNA per cell, the total number of cells per g of soil can be calculated.
For more targeted applications, DNA extracted from bacterial communities can be subjected to PCR to determine if a particular species is present within the community. For example, scientists may want to identify whether soil samples contain specific pathogens, such as Clostridium perfringens or Bacillus anthracis.
Finally, to obtain a more comprehensive understanding of the bacteria present in a community, DNA samples can be subjected to "omic" and bioinformatic characterization that allow for deeper analysis of the original bacteria within the sample. ?Omics? describes a range of technologies that explore roles, relationships, and actions of molecules in organisms or communities. This includes the studies of genes and their function, or ?Genomics?, and ?Proteomics?, the study of proteins and their roles.?For example, sequencing of 16S?RNA from the samples can allow a metagenomic determination of specific species within the community, giving a more detailed estimate of diversity. This approach can give scientists a better understanding of the species makeup of a community, and what roles they may be undertaking.
You've just watched JoVE's introduction to bacterial community DNA extraction. You should now understand how to extract DNA from a bacterial community, how to check the quality of this DNA, and how this DNA can be used for investigations of bacterial community composition. Thanks for watching!
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Q1: Why is community DNA extraction better than traditional culture-based methods for studying soil bacteria?
Community DNA extraction is a non-culture-based approach that captures DNA from multiple bacterial species in a single procedure, providing a more representative sample of actual communities. Traditional culture-based methods miss bacteria that grow poorly in laboratory conditions, capturing less than 1% of soil bacteria. DNA extraction reveals the true diversity and genetic potential of microbial communities.
Q2: What are the two main methods for extracting community DNA from soil?
The fractionation method separates cells from soil using blending and centrifugation, then uses lysozymes to break down cell walls before DNA extraction. The in situ method, which yields greater DNA concentration, combines soil with extraction buffer and glass beads, then adds detergent and heat to lyse cells directly in the soil matrix, releasing DNA for purification.
Q3: How do you measure the quality and quantity of extracted community DNA?
DNA concentration is measured using a spectrophotometer or DNA/RNA quantification fluorimeter, which outputs results in nanograms per milliliter. If the concentration is too high for accurate readings, dilute the sample 1:10 or 1:100 using molecular grade water. These measurements allow calculation of total DNA per gram of soil and estimation of bacterial cell numbers.
Q4: What role does phenol-chloroform extraction play in community DNA purification?
Phenol-chloroform extraction removes proteins, polysaccharides, and other contaminants from the DNA sample. The mixture separates into two layers during centrifugation: the bottom layer contains phenol-chloroform and debris, while the top aqueous layer contains purified DNA. This step is critical for obtaining clean DNA suitable for downstream molecular analysis.
Q5: How can community DNA be used to identify specific bacterial pathogens in soil?
DNA extracted from bacterial communities can be subjected to PCR to determine if particular species are present. Scientists can target specific pathogens such as Clostridium perfringens or Bacillus anthracis using detecting environmental microorganisms with the polymerase chain reaction and gel electrophoresis to confirm their presence in soil samples.
Q6: What is metagenomic analysis and how does it reveal bacterial community composition?
Metagenomic analysis uses omics technologies, including 16S RNA sequencing, to characterize genes and proteins in bacterial communities without culturing. Sequencing 16S RNA allows identification of specific species present and their relative abundance, providing a detailed estimate of community diversity and the functional roles different bacteria may undertake in the ecosystem.
Q7: Why is the in situ DNA extraction method preferred over the fractionation method?
The in situ method yields greater DNA concentration than the fractionation method because it lyses cells directly within the soil matrix using detergent and high-temperature incubation. This approach minimizes DNA loss during cell separation and extraction steps, making it more efficient for obtaining sufficient DNA from complex soil bacterial communities for comprehensive analysis.