20.109(S13):Context-setting and primer design (Day1)

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(Introduction)
(Introduction)
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==Introduction==
==Introduction==
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*overall motivation
+
In this module, you'll perform two investigations of pathogens that can infect birds. Through bird stool, these pathogens can be transferred to the environment –– and in some cases infect other animals. Perhaps the most well-known avian pathogen with zoonotic potential (i.e., 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.
-
Identifying pathogens in humans can suggest appropriate course of treatment. Identifying pathogens in other species can be both intellectually interesting and have applications for human health
+
Prof. Runstadler, the lecturer for this module, studies phylogenetic relationships among avian flus [cite 2012 virology paper]. Tracking viral mutations/evolution, infection of different bird species (including co-infection by multiple strains), and the trafficking patterns of those birds may provide information useful for predicting the next flu pandemic and commencing vaccine production in time. Your own phylogenetic analysis will consist of comparing bacterial communities in two distinct bird populations. (We admit, not as flashy as studying the flu!) More about the significance of that research next time.
-
Molecular identification of pathogens as powerful and perhaps less subjective than the identification according to morphology and phenotype
+
Your other research project will pertain to an unusual fungus called microsporidia. (background parts move up here.)
-
Requires a highly conserved gene. Something essential and little tolerance of mutation. Hence rRNA.
+
Specifically, today you will attempt to design PCR primers that improve upon the sensitivity and/or specificity of the state-of-the-art in microsporidia identification. This experience will give you a sense of the importance of methods for successful scientific investigation. Traditionally, many pathogens were identified by imaging techniques for morphology and culturing techniques for probing phenotype. Recently, molecular identification has emerged as a powerful and perhaps less subjective technique. Specific sequences in the pathogens' DNA can be targeted by unique primers and amplified in a polymerase chain reaction (PCR). Notably, there are conflicting reports as to whether sequence-based identification is any more sensitive than imaging when it comes to microsporidia. (cite that paper)
-
(Clarridge review)
+
Identification according to sequence requires a highly conserved gene. Ribosomal RNA is an excellent candidate for this strategy, as it is essential and little tolerant of mutation. Depending on the pathogen, different subunits or also the internal transcribed sequence might be the most reliable or otherwise preferred sequences[UGH sentence revise]. In the case of microsporidia, we will start with primers that target the 5' end of the small subunit rRNA. You will have the option of targeting a different portion of the rRNA or a coat protein unique to microsporidia [<font color=red>NOT CERTAIN YET</font color>, as described in the protocols below.
-
*background about small subunit rRNA
+
Techniques for identifying pathogens can be applied to human samples (feces, urine, tissue scrapings, etc.), animal samples, or in some cases even environmental samples such as dirt. Identifying pathogens in already infected humans can sometimes suggest an appropriate course of treatment, for example by providing knowledge of the pathogen's antibiotic resistance profile. Identifying pathogens in other species can be both intellectually interesting and have applications for human health. With respect to microsporidia, knowing which human–infecting species are also capable of infecting other hosts may suggest synodic relationships and environmental or health recommendations thoroughly for immunocompromised individuals such as testing reservoir waters and avoiding certain pets.
-
 
+
*things to cite somewhere above:
-
*bacteria-specific background
+
**Clarridge review re: 16S rRNA importance in general (if bacteria-focused, hmmm)
 +
** Mathis et al review re: zoonotic potential of &mu;SP
 +
**Garcia re: &mu;SP identification
 +
** some of the more primer-specific &mu;SP papers, perhaps in protocols below
*microsporidia-specific background
*microsporidia-specific background
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Over 1000 species, with 14 infecting humans
Over 1000 species, with 14 infecting humans
-
Not a time of full genome sequence information
+
Not a ton of full genome sequence information
(Broad initiative)
(Broad initiative)
-
Cites for bird infections and zoonotic potential
+
*primer design basic background here or directly in protocols?
-
Parallels to avian flu
+
likely the latter. and definitely should be explaining full PCR cycle on D2, not waiting till D3
-
 
+
-
*primer design basic background here or directly in protocols?
+
==Protocols==
==Protocols==

Revision as of 16:16, 14 December 2012

20.109(S13): Laboratory Fundamentals of Biological Engineering

Home        Schedule Spring 2013        Assignments       
DNA Engineering        Protein Engineering        Cell Engineering              

Contents

Introduction

In this module, you'll perform two investigations of pathogens that can infect birds. Through bird stool, these pathogens can be transferred to the environment –– and in some cases infect other animals. Perhaps the most well-known avian pathogen with zoonotic potential (i.e., 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.

Prof. Runstadler, the lecturer for this module, studies phylogenetic relationships among avian flus [cite 2012 virology paper]. Tracking viral mutations/evolution, infection of different bird species (including co-infection by multiple strains), and the trafficking patterns of those birds may provide information useful for predicting the next flu pandemic and commencing vaccine production in time. Your own phylogenetic analysis will consist of comparing bacterial communities in two distinct bird populations. (We admit, not as flashy as studying the flu!) More about the significance of that research next time.

Your other research project will pertain to an unusual fungus called microsporidia. (background parts move up here.)

Specifically, today you will attempt to design PCR primers that improve upon the sensitivity and/or specificity of the state-of-the-art in microsporidia identification. This experience will give you a sense of the importance of methods for successful scientific investigation. Traditionally, many pathogens were identified by imaging techniques for morphology and culturing techniques for probing phenotype. Recently, molecular identification has emerged as a powerful and perhaps less subjective technique. Specific sequences in the pathogens' DNA can be targeted by unique primers and amplified in a polymerase chain reaction (PCR). Notably, there are conflicting reports as to whether sequence-based identification is any more sensitive than imaging when it comes to microsporidia. (cite that paper)

Identification according to sequence requires a highly conserved gene. Ribosomal RNA is an excellent candidate for this strategy, as it is essential and little tolerant of mutation. Depending on the pathogen, different subunits or also the internal transcribed sequence might be the most reliable or otherwise preferred sequences[UGH sentence revise]. In the case of microsporidia, we will start with primers that target the 5' end of the small subunit rRNA. You will have the option of targeting a different portion of the rRNA or a coat protein unique to microsporidia [NOT CERTAIN YET, as described in the protocols below.

Techniques for identifying pathogens can be applied to human samples (feces, urine, tissue scrapings, etc.), animal samples, or in some cases even environmental samples such as dirt. Identifying pathogens in already infected humans can sometimes suggest an appropriate course of treatment, for example by providing knowledge of the pathogen's antibiotic resistance profile. Identifying pathogens in other species can be both intellectually interesting and have applications for human health. With respect to microsporidia, knowing which human–infecting species are also capable of infecting other hosts may suggest synodic relationships and environmental or health recommendations thoroughly for immunocompromised individuals such as testing reservoir waters and avoiding certain pets.

  • things to cite somewhere above:
    • Clarridge review re: 16S rRNA importance in general (if bacteria-focused, hmmm)
    • Mathis et al review re: zoonotic potential of μSP
    • Garcia re: μSP identification
    • some of the more primer-specific μSP papers, perhaps in protocols below
  • microsporidia-specific background

Over 1000 species, with 14 infecting humans

Not a ton of full genome sequence information

(Broad initiative)

  • primer design basic background here or directly in protocols?

likely the latter. and definitely should be explaining full PCR cycle on D2, not waiting till D3

Protocols

Part 1: Lab practical

You and your partner may work together on the lab practical. (Note: this will not be the case for future quizzes.) You are of course welcome to give different answers should you disagree.

Part 2: Explore existing microsporidia-specific primers

Part 3: Design novel microsporidia-specific primers

For next time

Reagent list

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