Chapter 12a:

Identification of Organisms Using 16S rRNA

Sequencing
Objectives
• To become familiar with the common methodology and instrumentation used in microbial
identification and taxonomy
• To be able to quickly narrow down the identity of a microorganism based on the
polymerase chain reaction (PCR) and sequences of nucleotides of a particular gene.

The Small Ribosomal Subunit

So far, we have used biochemical methods to gain information about different species of bacteria.
We will now shift to using genomic methods. The National Center for Biotechnology Information
hosts databases with genomic sequences of hundreds of thousands of organisms. As of this writing,
there were 248,492 prokaryotes in the database. We will take advantage of this in the next two
sessions.

In order to determine the relatedness of various organisms to one another, it is important to find a
trait that is present in all of them. In the 1970s the biophysicist Carl Woese used 16S ribosomal
RNA sequences to reconstruct evolutionary relationships across hundreds of prokaryotic species.
The 16S rRNA gene codes for the smaller ribosomal subunit. Woese proposed using it because
ribosomes are essential for life. While eukaryotes also have a small ribosomal subunit, it is larger
than those found in prokaryotes, and it is encoded by a different gene. The sequences of the rRNA
genes in prokaryotes contain regions that are very similar, allowing for the alignment and
comparison of these sequences. Furthermore, the gene is small enough to be easily sequenced yet
large enough to contain information for genetic comparisons. In addition, this gene contains
multiple sites with known mutation rates. Sites that mutate at a known rate, say four mutations per
site per million years, allow scientists to use them as an evolutionary clock to get an idea of how
much time has passed since two species last shared a common ancestor.

It took Woese a decade to sequence the 16S ribosomal RNA of hundreds of microbial species. His
efforts resulted in a proposal to overturn a major dogma in biology, the prokaryote- eukaryote
dichotomy. Woese proposed that there were not two, but three domains of life (Bacteria, Archaea,
and Eukaryotes). Although some specialists were quick to adopt Woese’s new scheme, the rest of
biology remained openly hostile to the idea. It wasn’t until the mid-1980s that other
microbiologists began to warm to it, but it took well over another decade for other areas of biology
to follow suit. As sequencing data poured in from around the world, it became clear that Woese’s
initial tree was, in fact, correct.
Polymerase Chain Reaction
In this session, you will take a small sample from a colony of each unknown and extract its DNA.
You will next amplify ~500 base pairs (bp) of the 1500 bp 16S gene by using PCR (polymerase
chain reaction.) PCR was developed by the Nobel Prize winner Kary Mμllis and associates in 1985
and is a method that takes a small amount of DNA and amplifies a segment of the DNA over
several orders of magnitude. With the use of PCR tests to detect SARS-CoV-2, PCR is now a
household name. In addition to its use in medicine, forensics, and paternity cases, it is widely used
by molecular biologists in research.

PCR is relatively inexpensive, and quite simple to perform. The reagents required for amplification
are:
1. DNA template
2. Two oligonucleotide primers
3. DNA polymerase
4. 4 deoxyribonucleoside triphosphates (dATP, dTTP, dCTP, dGTP)
5. Mg2+ ions.

To perform PCR, the reagents are mixed in a thin-walled tube and placed in an instrument called
a thermocycler. This machine changes the temperature very quickly according to a set of
programmed instructions. The changes in the temperature cause denaturation of the target DNA
strands, annealing (base-paring) of primers to the newly single stranded DNA, and a primer
extension (DNA polymerization). These three steps constitute a cycle. Many such cycles can be
performed quickly to obtain a large number of copies of the target DNA. It is difficult to fully
comprehend PCR with text alone.

The machine stops after the last cycle and the temperature remains at 4 ̊C until the samples are
removed. After 30 cycles, the target DNA produces 230 copies of itself, a sufficient quantity of
DNA to use for visualization of the DNA by gel electrophoresis or for sequencing. At least a single
copy of the target DNA is required for the PCR to work. PCR is an extremely sensitive test, such
that for example a single hair, or in this case, a small clump of bacteria, can provide enough
material to run the test.

Two important innovations were responsible for automating PCR. First, a heat-stable DNA
polymerase was isolated from the bacterium Thermus aquaticus, which inhabits hot springs. This
enzyme, called Taq polymerase, remains active despite repeated heating during many cycles of
amplification. Second, DNA thermal cyclers have been developed in which a computer controls
the repetitive temperature changes.

The primers are what define the portion that the researcher wishes to amplify. The ones we will
use in this lab session are indicated by the grey arrows in Figure 3. These primers were designed
to anneal to conserved regions, shown in green. This means that they will anneal to any species of
bacteria. Because of this, we call these primers universal. However, you can see that the 500 base
pair sequence in between the primers also contains regions, shown in grey, that are variable, that
is, unique to different species. By comparing the sequence of the variable regions to those
contained in the database, you will determine the identity of your unknowns.
The PCR reaction takes about 2 hours to run. Your lab instructor will store your reactions and next
week, you will check that they worked, and clean up your PCR product to submit for sequencing.
Isolating Microbial DNA for PCR Amplification of the 16s
rRNA Gene Protocol
Materials: Amount:
Sharpie marker 1 / group
Micropipettor set (p10, p20, p200, and p1000) 1 set / group
Micropipettor tips (box of red, yellow, and blue tips) 1 of each box / group
1.5 mL Eppendorf tubes 1 box / group
0.5 mL thin-walled PCR tubes 1 box / group
Microcentrifuge tube rack 1 / group
Lid locks for 1.5 mL microfuge tubes 1 / person
0.5 mL tube adaptors for microcentrifuge 3 / lab
Microcentrifuge 1 / group
Thermocycler 1 / lab
99˚C heated block 1 / lab
Inoculating loop 1 / group
Bunsen burner 1 / group
Striker 1 / group
Vortexer 1 / table

Solutions (in ice bucket): Amount:

PCRID1 primer (1.1 uM) 5.0 uL / group
PCRID2 primer (1.1 uM) 5.0 uL / group
PCR Reaction Mix 25.0 uL / group
Sterile water 1.0 mL / group

Cultures: Amount:
Unknown #1 2 broths / pair
Unknown #2 2 broths / pair

Procedure:

1. Obtain two fresh 1.5 mL Eppendorf tubes. Label them with your group number and
unknown, for example “G4U1” for Group 4, Unknown 1.

2. Vortex your broth suspension of culture and aliquot 500 uL to each of the tubes making
sure Unknown 1 and 2 go in the appropriate tubes.

3. Vortex the tube until all cells are completely dispersed.

4. Place your tube in the 99˚C heated block for 10 minutes to lyse the cells, then move the
cells to ice for 5 minutes.

5. While the tubes are incubating, two 0.5 mL thin-walled PCR tubes. Label the tubes the
same way as above and place it on ice.
6. After the samples have been on ice for 5 minutes, add the reagents as indicated in the table
below to the freshly labeled 0.5 mL tube, using a new sterile tip for each reagent.
• Add the extracted DNA to your PCR tube.
• The two primers and the PCR Reaction Mix, which contains dNTPs, Taq
polymerase, and loading dye for electrophoresis.
• Make sure to add both primers. The reaction will fail if you only add 1 primer.
• Make sure you pipet up and down each solution before adding it to ensure evenness.

Reagent Volume
Extracted DNA 2.5 uL
PCRID1 primer 2.0 uL
PCRID2 primer 2.0 uL
2X PCR Fail-Safe Pre-Mix D 10 uL
Fail-Safe Taq Polymerase 0.5 uL
Sterile DH2O 3.0 uL

Note: The PCR Rxn Mix contains MgCl2 and dNTPs. PCRID1 and PCRID2 are universal
primers, and each has a concentration of 1.1 µM. All of these reagents are located in your
ice bucket

7. Mix by pipetting for 5 seconds and microcentrifuge for 3 seconds using a special adapter
supplied for these smaller tubes.
a. Don't forget to balance the tubes

8. Once all tubes have been prepared load them into the thermocycler.

9. When all groups have their tubes in the thermocycler your instructor will start the PCRID
program. Your tubes
10. Your samples will be held at -20°C until next class.