20.109(S14):Microbial DNA extraction (Day1)

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20.109(S14): Laboratory Fundamentals of Biological Engineering

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Please begin by reading the Module 1 overview.


Investigations in disease ecology, or "the ecological study of host-pathogen interactions within the context of their environment and evolution" (via Scitable), help determine how pathogens transmit and cause disease, persist, and evolve in host organisms as different as humans and birds. Pathogen, host, and environment all play roles in defining the natural history of disease. In studies of pathogens that cause zoonotic disease, researchers are particularly interested in defining major influences on pathogen distribution, transmission, and evolution. The Runstadler lab currently studies the disease ecology of influenza viruses in several groups of birds and other animals (see for example this paper and this one), a research area that can provide information useful for predicting the next flu pandemic and designing effective vaccines that will be produced in time. Researchers track viral mutations/evolution, infection of different bird species (including co-infection by multiple strains), and the trafficking patterns of those birds; they often visualize the data by phylogenetic trees and related methods. Your own phylogenetic analysis will consist of comparing bacterial communities in two distinct bird populations. (We admit, not as flashy as studying the flu!)

Recent evidence in the burgeoning field of the microbiome suggests that the internal host environment may be a significant factor in the susceptibility/resistance profiles of individuals, populations, or species to disease pathogens. Different species of birds, although in the same family or genera taxonomically, may utilize vastly different environments and travel through flyways that are separated by thousands of miles. The associated microbial communities and potential pathogens these species encounter and carry may therefore be different, and even be a significant reason why one species is a host for a given pathogen and the other is not. As a part of Module 1, we will utilize an early approach to community profiling to gain a snapshot of the differences between gull populations in South Boston: we'll have access to samples from male and female ring-billed gulls collected at Carson Beach, and samples from male herring gulls collected at the South Bay parking lot. Should we expect these birds to be equivalent hosts for viruses? For fungi? For the bacteria that we will study?

To identify the bacteria cohabiting with individual birds, we'll preferentially extract microbial (rather than animal) DNA from bird cloacal swabs and sequence a conserved region. Genes encoding ribosomal RNA (rRNA) are excellent candidates for this strategy: they are essential for life and thus the organism is unlikely to survive rRNA gene mutation. Depending on the pathogen, the small or large subunit or the internal transcribed sequence (ITS) might be the most reliable sequence for identification. We will use primers that amplify a large fragment, or amplicon, of the 16S rRNA gene sequence; this rRNA is part of the 30S small subunit. The clinical potential of 16S rRNA-based sequencing for bacterial infections is described in the linked review by Dr. Jill Clarridge.

We must amplify the 16S rRNA gene sequences in a polymerase chain reaction (PCR), both to isolate the DNA of interest and to prepare large quantities of it. The PCR will result in a pool of 16S sequences representing different species of bacteria present approximately in proportion to their composition in the bird stool. That is, a species that is abundantly present in stool is more likely to have its DNA amplified during the PCR than is a low concentration species. The pool of 16S fragments can be cloned into a DNA vector and transformed into laboratory bacteria to produce isolated colonies. Each colony should contain identical copies of 16S DNA from a single species of bacteria, thus allowing us to deconvolve our pool of DNA and analyze individual sequences. How? Briefly, we'll perform a second DNA extraction from each of several colonies and determine the sequences through a method that resembles PCR. The individual steps will make more sense as we complete each of them, and an overview of the process as a whole is shown below.

Bird gut microbiota experiment overview.
Bird gut microbiota experiment overview.

Returning to today's specific work, each of you will extract a DNA pool from a single bird cloacal sample using a commercial kit. Unlike most mammals, which have different excretory paths for urine and feces, birds have both their urine and feces excreted through a single opening called the cloaca. The cloacal swab is a somewhat vexing material from which to extract DNA, because many enzyme inhibitors (including materials that inhibit polymerase) are present. As described in the paper by Carol Kreader, inhibitors in feces include bile salts and environmental inhibitors such as humic compounds present in water and dirt. Chemicals that degrade DNA may be present, which is especially troubling when one wants to amplify a low concentration DNA. The DNA extraction kit contains two reagents that degrade or bind up inhibitors – buffer ASL and the InhibiTEX tablets – but unfortunately their exact contents and mechanisms of action are proprietary.

Many reagents and steps in the purification are transparent in their composition and mechanism. For example, we know that ASL is a lysis reagent, and that we can increase the ratio of microbial:animal DNA recovered by performing lysis at a high temperature such as 70 °C. We also know that proteinase K is used to digest proteins, some of which themselves may act as PCR inhibitors or nucleases. After initial digestion and binding, the samples are purified on silica spin columns, which are also well understood.


We’ve made two minor modifications to the manufacturer's protocol today, due to the nature of our samples and our particular research question, respectively. One is using a somewhat lower volume of sample than recommended because our bird coalcal sample is more concentrated than the human stool the kit was designed for. The second is using chitinase in addition to proteinase K, and completing the enzymatic digestion at a lower temperature and for a longer time than recommended. Adding chitinase is useful for opening up pathogens with chitin walls, including the fungus microsporidia. More about the potential utility of this step next time!


Part 1: Begin DNA extraction from bird cloacal swab

The following protocol requires many tube changes. Be sure that each tube is clearly labeled with your sample number and initials to avoid swapping samples with anyone. There are a few other steps you can take to avoid cross-contamination: switch pipet tips at every step, keep only one tube open at a time, and avoid getting liquids on the lip of any tube or column.

Before beginning this protocol, check the maximum spin speed of your centrifuge. Some centrifuges reach 20,000 g and others reach only 16,000 g, and the time for centrifugation will have to be adjusted proportionally. Note that rpm stands for rotations per minute while rcf stands for relative centrifugal force. It is rcf that is equivalent to g-force, not rpm, because differently sized rotors will impart different forces at the same rotational speed.

  1. Obtain your 100 μL bird cloacal sample from the ice bucket nearest you, according to the number you are assigned on today's Talk page.
    • The solution will be homogeneous but somewhat thick.
  10. In a fresh 1.5 mL tube, dispense 15 μL proteinase K. Only then should you add 200 μL of the supernatant from step 9 followed by 200 μL of buffer AL, pipetting to mix each time. Finally, add 15 μL chitinase.
  11. Vortex for 15 sec (until solution is homogeneous) and incubate in the 56 °C oven for about 1.5 hours.

Part 2: Lab practical


Afterward, you can work on your notebook and start preparing for Part 3.

Part 3: Complete DNA extraction from bird cloacal swab

  2. Add 200 μL of ethanol, mix by briefly vortexing, and transfer to a QIAamp spin column (atop its 2 mL capless collection tube).
    • Be sure to label the column rather than the tube with your sample number and initials.
  4. Move the spin column to a fresh capless collection tube, add 500 μL buffer AW1, and spin 1 or 1.5 min as needed.
  6. In the meantime, trim the cap off a fresh 1.5 mL eppendorf tube using small scissors that have been wiped down with 70% ethanol. Save the cap! Prepare a sticky label (in your team color) for the top: write the date, your sample ID, and your initials. You should also label the side of each tube, at least with a short unique identifier, so you don't lose track of which sample is which in the following step.
    • This step completely removes remaining ethanol that could interfere with future steps.

For next time

  4. Finish your EHS training if you haven't already.

Reagent list

  • QIAamp DNA Stool Mini Kit from Qiagen
    • Therefore 0.4 U are added to each extraction
  • Ethanol (200 proof)

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