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INTRODUCTION TO BIOINFORMATICS BACTERIAL IDENTIFICATION USING 16s rDNA SEQUENCE ANALYSIS
 
 
Authors          Audiences          Overview           Materials          Resources           Future Directions
 

 


Authors


Stephanie Dellis
College of Charleston

 
   
 


Possible Audiences:

mid- to upper-level undergraduates, can be adapted for other audiences  

 
 


Brief Overview:

Teaching Goals:

    A) Reading of primary scientific literature.
    B) Familiarity with the NCBI site and knowledge of the BLAST tools.
    C) Mastery of molecular skills including gel electrophoresis, PCR, and sequencing principles.
    D) Ability to use sequence data in a genome-wide context (such as determining relationships among organisms).
    E) Ability to communicate the theory, methodology, and results of this analysis to scientific peers. This is accomplished in two ways:
    1. Small groups of students (2-4) find a scientific article that uses 16s rDNA sequencing to answer a scientific question. The key points of the article, and their critique of the article, are presented to the lab using a power pint presentation.
    2. Scientific poster session. A larger class group (5-6) prepares a poster of the lab results and presents their poster to the other lab students.
 

 
   
 


Project Materials:

Outline:

  1. Read primary literature describing the use of 16s rDNA analysis in a clinical setting. Since many students have medical aspirations, this article is of interest to them. The article is critiqued on scientific content as well as the format of a scientific paper.

    The paper is handed out the same week that we do the isolation of our bacterial DNA and run the PCR reaction. "Ribosomal DNA Sequencing for Identification of Aerobic Gram-positive Rods in the Clinical Laboratory (an 18-month evaluation)." by P.P. Bosshard, S. Abels, R. Zbinden, E.C. Bottger, and M. Altwegg; Sept 2003; J Clinical Micro Vol 41: 4134-4140.

  2. PCR product is purified by running an agarose gel, cutting out the DNA band, and isolating the DNA from the gel slice using a commercial kit (Quiagen). DNA concentration is measured spectrophotometrically and the samples are taken to the CofC Grice Marine Lab for sequencing on a capillary sequencer. Gels should be loaded and run before lab starts. Since this is a relatively short lab experimentally, the processes of PCR and capillary sequencing, or the primary literature article, can be discussed.

  3. DNA sequences are returned and analyzed. By reading the scientific paper, the students are more familiar with the analysis and the criterion used for sequence identification. Two databases, NCBI and RDB, are used for sequence identification. Questions are posed on the relative merits of each database and the identification of the microbes. The combined sequence data is used to develop a phylogenetic tree of relationships among the identified bacteria using the Biology Workbench.  

 
 


Resources and References:

  1. "Ribosomal DNA Sequencing for Identification of Aerobic Gram-positive Rods in the Clinical Laboratory (an 18-month evaluation)." by P.P. Bosshard, S. Abels, R. Zbinden, E.C. Bottger, and M. Altwegg; Sept 2003; J Clinical Micro Vol 41: 4134-4140.
    A handout with questions is given with the paper. Answers carry a point value and are collected, then we discuss the paper as a class.

  2. Handouts:(a) background info and protocol for DNA isolation and amplification (b) kit instructions for isolating DNA from gel slice (c) guidelines for group presentation of a journal article utilizing the 16s rDNA identification technique (d) guidelines for using FinchTV and BLAST (e) guidelines for constructing a phylogenetic tree using Biology Workbench FinchTV free program for translating chromatogram to letter sequence. Students can make changes to chromatogram.

  3. NCBI BLASTn

  4. Biology Workbench  

 
   
 


Future Directions:

5. Future plans are to develop additional genomics investigations, possibly without a traditional lab component. The current wet lab on Southern analysis will be replaced by a bioinformatics investigation, possibly one published in “Microbes Count!’ or developed during a BioQuest workshop.  

 
 


Attachments


- 16s Paper Qs.doc
- 16s rDNAhandout.pdf
- DNA Sequencing Handout.pdf
- BioQuest1.ppt