Outbreak Lab: In this lab, biotech procedures will be used to see if a sample of viral DNA is the deadly Alabama virus. The specific technique that you will perform is a gel electrophoresis of digested viral DNA At the end of the lab investigation, you will have created a DNA profile. A DNA profile is an analysis of DNA fragments to determine if it comes from a particular individual or organism or virus. You are creating a DNA fingerprint.

How can you create a DNA Profile? DNA Extraction – remove DNA from virus Polymerase Chain Reaction (PCR) – make copies of the viral DNA in questions DNA Restriction Digest – use restriction enzymes to cut DNA at given sites Gel Electrophoresis – separate the fragments of viral DNA to identify it

Here are the three samples of digested viral DNA Here are the three samples of digested viral DNA. The red capped vial has the Alabama strain, the yellow capped vial has the Missouri strain and the green capped vial contains the Pennsylvania strain. Note the blue liquid in the red capped vial. There is 20 ul of DNA and loading dye.

Prepare the gel for the electrophoresis The bottle with the blue gel will be melted in a microwave. The blue liquid gel will be poured into the gel rig in front of the bottle. Notice a plastic white comb in the gel rig. This comb will form all the gel to form wells for the samples of DNA in the previous slide.

Why is the gel blue? The gel is blue because a stain called methylene blue was added to the gel when it was made. The small bottle to the left of the large gel bottle contains the stain. This stain will be used to stain the DNA fragments during and after the electrophoresis. Be careful: it will stain hands and clothes.

This is the work area for the gel electrophoresis This is the work area for the gel electrophoresis. Look at the gel box that contains the blue liquid. The blue liquid is TAE buffer. It is blue because methylene blue stain was added. The box is hooked up to the electrophoresis electric supply which will supply the current to move the DNA fragments down the gel. A micropipette set to 20 ul and yellow tips are ready for loading.

Gel in the gel box filled with TAE buffer Note the comb is still in the gel. This must be removed before loading with DNA.

Loading the DNA Sample The gel is being loaded with the Missouri strain virus DNA. Other wells will be used for the Alabama and Pennsylvania strains.

Below is a gel that has run Below is a gel that has run. Note the blue line at the bottom of the gel. This is the loading dye that was mixed in with the viral DNA. It runs faster than the fragments. The fragments are hard to see. To visualize the fragments, the gel will be stained.

Visualizing the Restriction Fragments Staining the gels De-staining the gels

The DNA Fingerprint Red Yellow Green Examine the three lanes. Do any of the lanes exhibit the same pattern of DNA fragments? Lane Yellow and Green have the same restriction pattern. This means that the DNA from these two states are the same which means that the virus infecting these two states are the same strain. Red Lane was Alabama strain. Yellow Lane was the Missouri strain. Green Lane was the Pennsylvania strain. The DNA Fingerprint

12.11 The analysis of genetic markers can produce a DNA profile DNA profiling is the analysis of DNA fragments to determine whether they come from a particular individual Compares genetic markers from noncoding regions that show variation between individuals Involves amplification (copying) of markers for analysis Sizes of amplified fragments are compared For BLAST Animation DNA Fingerprinting, go to Animation and Video Files. Student Misconceptions and Concerns 1. Television programs might lead some students to expect DNA profiling to be quick and easy. Ask students to consider why DNA profiling actually takes many days or weeks to complete. Teaching Tips 1. Figure 12.11 describes the general steps of DNA profiling. This overview is a useful reference to employ while the details of each step are discussed. Copyright © 2009 Pearson Education, Inc.

Crime scene Suspect 1 Suspect 2 DNA isolated DNA of selected Crime scene Suspect 1 Suspect 2 1 DNA isolated 2 DNA of selected markers amplified Figure 12.11 An overview of DNA fingerprinting. This figure shows the process of DNA profiling (also called DNA fingerprinting). DNA is isolated. DNA is amplified by polymerase chain reaction (see Module 12.12). The amplified fragments are compared by agarose gel electrophoresis (see Module 12.13). 3 Amplified DNA compared