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STUDENT HANDOUT:
STUDENT HANDOUT:
INTRODUCTION:
INTRODUCTION:
Bacteria are made of prokaryotic cells (Figure 1). One structure in the prokaryotic cell is the ribosome. The ribosome is the site of protein synthesis. In fact, the ribosome is also found in eukaryotic cells (the cells that you are made of!).
Figure 1. Typical Prokaryotic Cell. Image from Wikimedia Commons.
The bacterial ribosome itself comprises two subunits: the large 50S subunit and the small 30S subunit. The “S” refers to Svedberg units, a unit of measurement. The ribosome is composed of ribosomal RNA (rRNA). The 50S subunit contains 23S rRNA and the 30S subunit contains 16S rRNA (Figure 2).
Figure 2. The bacterial ribosome consists of a large 50S subunit and a small 30S subunit.
In this activity, you will learn about ribotyping (Figure 3). A ribotype is basically a DNA fingerprint of the 16S rRNA gene. Scientists can examine ribotypes to differentiate between different bacteria.
Figure 3. Overview of ribotyping procedure. Image from: Case Studies in Microscopy (Cornell University & Saint Joseph's University). Used with permission from Sue Merkel, Cornell University.
OBJECTIVES:
OBJECTIVES:
Learn how gel electrophoresis works.
Learn how to identify an unknown bacterium using ribotyping.
PART 1: EXTRACTING DNA & RESTRICTION DIGEST
PART 1: EXTRACTING DNA & RESTRICTION DIGEST
In the previous activity, you learned how to isolate DNA from cells. We can use a similar procedure to isolate the DNA of bacteria.
After the DNA is isolated, the DNA is cut with a restriction enzyme. Restriction enzymes recognize specific sequences of DNA. When they encounter that sequence of DNA, the enzyme cuts the DNA (Figure 4).
Figure 4. The restriction enzyme HindIII cutting a sequence of DNA. Image from Wikimedia Commons.
Another common restriction enzyme is HaeIII. HaeIII cuts DNA at the following:
Shown below is a sequence of DNA.
(a) Identify all the HaeIII sites.
(b) How many DNA fragments will result after incubation with HaeIII?
PART 2: GEL ELECTROPHORESIS
PART 2: GEL ELECTROPHORESIS
After digesting the DNA, scientists can visualize DNA by performing gel electrophoresis. This technique separates DNA molecules based on size and charge. The gel is made of agarose, a sugar derived from algae.
DNA is a negatively charged molecule. Hence it is loaded at the negative end of the agarose gel (Figure 5). When an electric field is applied to the gel, the DNA will migrate towards the positive end of the gel. The size of the DNA fragment will determine how fast (or slow) the DNA will move in the gel. Large molecules do not move quickly and will therefore stay at the top of the gel. Small molecules can move quickly through the gel and therefore will be found at the bottom of the gel.
To assist with determining the size of DNA in a gel, a DNA ladder is typically added to the gel. The ladder has known DNA fragment sizes (in bp, base pairs).
Figure 5. Gel electrophoresis separates molecules according to charge using an electric current.
You can watch a video on gel electrophoresis:
2) Shown below is a sample gel electrophoresis. Answer each question.
PART 3: ADDING PROBES AND VISUALIZING THE RESULT
PART 3: ADDING PROBES AND VISUALIZING THE RESULT
After the DNA is resolved on an agarose gel, the DNA is transferred to a nylon filter. Since we wish to examine only the 16S rRNA genes, a probe that hybridizes only to those genes is added.
The pattern that results is known as the "ribotype".
3. In the hypothetical situations below, review the ribotype results and determine what is the causative agent.
KEY TERMS:
KEY TERMS:
DNA
DNA ladder
eukaryotic cells
gel electrophoresis
probe
prokaryotic cells
restriction enzyme
ribosome
RNA
SUGGESTED READINGS:
SUGGESTED READINGS:
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