INTRODUCTION:

    The 16s ribosomal DNA gene is present in all prokaryotes as a component of the prokaryotic ribosome. The 16s rDNA  been used extensively to determine taxonomy and phylogeny (evolutionary relationships) among bacteria. Thus the comparison of 16s rDNA sequence can show evolutionary relatedness among microorganisms. This work was pioneered by Carl Woese, who proposed the three Domain system of classification - Archaea, Bacteria, and Eucarya - based on such sequence information.

    The 16s rDNA sequence has hypervariable regions, where sequences have diverged over evolutionary time. These are often flanked by strongly-conserved regions. Primers are designed to bind to conserved regions and amplify variable regions. Bacteria can be identified based on the sequence information found on the Ribosomal Database Project website (www.cme.msu.edu/RDP/html/index.html) or NCBI (www.ncbi.nlm.nih.gov).

    In this laboratory, each lab section will amplify a different region of the 16s rDNA gene (based on the E. coli 16s rDNA sequence) using Polymerase Chain Reaction (PCR). The DNA will be sequenced using the capillary DNA sequencer at the Grice Marine Lab. We will then compare the results obtained with the different primers and determine which primer set was most effective in identifying unknown bacteria.
 

EXPERIMENTAL PROTOCOL:

1. Each student will obtain one liquid culture of bacteria. Note the code number. Also note the characteristics of the liquid culture such as color or consistency. 

2. Every unknown culture should be treated as though it were a human pathogen. Wear gloves while handling the bacteria. Be careful not to contaminate anything, especially yourself.

3. Carefully resuspend the culture in the test tube. Remove 500 ul of culture into a special locking  eppendorf tube. Pellet in the microfuge for 2 minutes at 10,000 x g. Carefully draw off the medium using a P200. Some pellets are not firm and are easily disturbed.

4. Thoroughly resuspend cells in 400  ul dH₂O. Freeze cells in a dry ice bath for 2 minutes. Place in the heating block (70 - 80' C) for 7-10 minutes. Open the cap carefully to release pressure. Add 100 ul of resuspended Chelex to the bacterial cells. Resuspend the Chelex-100 solution each time before use. Mix thoroughly using a vortex or pipettor.

5. Again freeze cells in the dry ice bath for 2 minutes, followed by 7-10 minutes in the heat block. Open cap carefully to release any built-up pressure. Then close cap tightly and spin in the microfuge for 2 minutes at 10,000 x g. Remove 200 ul of the clear supernatant to a new microfuge tube and keep the tube on ice.

6. Set up PCR reactions. Each student will add 8 ul of dH₂O and 2 ul of the DNA supernatant to the small (0.5 ml) PCR tube. You should have a total of 10 ul in the tube. Write your initials on the TOP of the tube. 

7. The instructor will prepare Master Mix plus primers, as shown in the table below. The Master Mix contains all the common components for a set of reactions. It improves consistency among the reactions and reduces pipetting error.

                                               PCR Parameters:

Adapted from:
"Identification of bacteria using two degenerate 16s rDNA sequencing primers" by Boye, et al., the "Microbiology Laboratory Manual" by J. D. Newman, Lycoming College (srv2.lycoming.edu/~newman), and Dr Dag Harmstad, University of Muenster, Germany (personal communication).