Ribosomal RNA Gene Amplification and Sequencing
to Identify Unknown Microbes.
 
Jeffrey D. Newman
Lycoming College, Williamsport, PA 17701.


Abstract

A high percentage of undergraduate microbiology laboratory courses include some form of exercise in which students identify unknown microbes. In this investigative laboratory exercise, students sampled microbes from environments of their choice, isolated a single pure culture and conducted a series of biochemical tests for comparison to data in Bergey's Manual of Systematic Bacteriology.  This relatively common approach was supplemented with a more molecular strategy. Genomic DNA was isolated from the organism and a segment of the 16S rRNA gene was amplified by the polymerase chain reaction (PCR) using primers to highly conserved regions of the gene. The PCR products were subsequently sequenced using non-radioactive methods and compared to sequences accessible through the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/). In this pilot project (15 students), all of the students were successful in obtaining PCR products and 80% obtained sufficient DNA sequence for identification. The use of rRNA sequencing in this traditional laboratory exercise reflects the recent paradigm shift in our understanding of microbial phylogeny.

Objectives

Reinforce concepts discussed in class

Skill development  
 
 

Methods/Protocols

  • Results
  • Discussion
    This page was created or last modified on 5/13/98 by Jeff Newman .
     
    Assistant Professor Web page: http://lyco.lycoming.edu/~newman 
    Department of Biology Email: newman@lycoming.edu
    Lycoming College Phone: 717-321-4386
    Williamsport PA 17701 Fax: 717-321-4073
    © 1998  Jeffrey D. Newman