Difference between revisions of "20.109(S14):Microbial DNA extraction (Day1)"
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− | ==Introduction== | + | ==Introduction== |
− | + | Please begin by reading the [[20.109%28S14%29:Module_1 | '''Module 1 overview'''.]] | |
− | + | Today we'll begin our primary experiment, a phylogenetic analysis of bird gut microbiota. Through birds' stool, their resident microbes can be transferred to the environment and in some cases infect other animals. Perhaps the most well-known pathogenic avian microbe with zoonotic potential (potential for inter-species transmission) is the flu virus. For your safety, all the samples we will work with have been screened to exclude those carrying human pathogenic flu strains. However, we will be able to mine much of the same intellectual content that we could were we studying flu directly. | |
− | + | Investigations in disease ecology, or "the ecological study of host-pathogen interactions within the context of their environment and evolution" ([http://www.nature.com/scitable/knowledge/library/disease-ecology-15947677 '''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 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3115932/ '''this paper'''] and [http://www.ncbi.nlm.nih.gov/pubmed/22192630 '''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 [http://www.ncbi.nlm.nih.gov/pubmed/15489351 '''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. | |
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− | [[Image: | + | [[Image:S14-M1 experiment-overview.jpg|thumb|450px|center|'''Bird gut microbiota experiment overview.''']] |
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− | Returning to today's specific work, each of you will extract a DNA pool from a single bird | + | 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 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC167874/pdf/621102.pdf '''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 |