Chapter 9. Molecular Genetics and Mitosis

Objectives

Laboratory 9
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The purpose of this lab is to provide students the opportunity to learn some of the techniques that are central in the study of DNA. In lab you will be given DNA from suspects and crime scene evidence. You will run a gel to determine who committed the crime.

Since the labs are limited to two hours, we will provide you with DNA that has already been amplified using PCR primers. You will have six samples, and in lab you will run these samples using the electrophoresis units and determine the suspect that committed the crime.

Procedure

In this lab, you will perform gel electrophoresis on samples that were collected from the scene of an imaginary crime. These samples are purified DNA that simulate the products of PCR (polymerase chain reaction) DNA fingerprinting. Here are the facts of the imaginary crime:

A man was mugged and killed while leaving a 24-hour convenience store. There was an eyewitness to the murder, and his statement was taken by the police. The eye-witness was asked to look at mug shots and he was able to identify a suspect. However, the person accused of the murder has a brother that looks very similar to him, but they are not twins. When the eye-witness was asked to identify the suspect in a line-up he could not distinguish which of the two brothers was seen at the crime. Fortunately, there was evidence left at the scene of the crime, in the form of blood and skin collected from under the victim’s fingernails. DNA samples were then collected from the two brothers for DNA fingerprinting. The brothers were labeled Suspects X and Y.

GEL ELECTROPHORESIS

In the lab, PCR primers were used to perform PCR in the evidence DNA, Suspect X’s DNA, and Suspect Y’s DNA. The products of the PCR were labeled Evidence 1, Evidence 2, Suspect X-1, Suspect X-2, Suspect Y-1, and Suspect Y-2. Your job is to use agarose gel electrophoresis to examine the evidence as compared to the two suspects to determine who committed the crime. [Modified from Carolina Biological Supply Company “PCR Forensics Simulation Kit” (21-1210).]

You will begin class by loading the samples into the electrophoresis unit. The electrophoresis must run for at least 45 minutes.

LOAD GEL

  1. The gels will be stored in a container and will be in a buffer solution. Place the gel in the electrophoresis unit. Important: Place it such that the wells are nearest the black electrode. Handle the gel very gently, as it is very fragile.

  2. Fill the electrophoresis box with TBE buffer (a blue solution) such that it just covers the entire surface of the gel.

  3. You will use the micropipettes to load the contents of each reaction tube into a separate well in the gel. Important: Use a clean micropipette for each solution.

  4. Gently tap each container to get the DNA into the bottom of the container.

  5. Draw the sample into the micropipette. It is best to squeeze it below the bulb.

  6. Steady the micropipette with two hands.

  7. Dip the micropipette tip through the surface of the buffer, position it over the well, and slowly expel the mixture. Sucrose in the loading dye weighs down the sample, causing it to sink to the bottom of the well. Be careful not to punch the tip of the micropipette through the bottom of the gel. It is a bit like “placing” the solution directly over the well and then gently expelling the contents. The DNA should be visible in the well.

  8. Important: Carefully record what solutions are placed in each well. For this example we will suggest the order starting nearest the black electrode. Write the actual order on the diagram provided.

  9. Close the top of the electrophoresis chamber and connect the electrodes to the power supply (black to black, red to red). Make sure both electrodes are plugged into the same channel (one on top of the other). Plug in the power supply and adjust to the following settings: Set range on “low,” voltage to 155 volts.

    Time the run for 45 minutes. After a few minutes, the top of the chamber should be clouded. If it is not, call your TA to check the settings!

    After 45 minutes, unplug the power supply and disconnect the electrodes.

  10. While wearing gloves, remove the gel from the electrophoresis unit carefully as the gel will be HOT. Place the gel ONLY into the staining tray and cover the gel with stain (about 1/2 a bottle). Then, gently rock the gel for about 10 minutes. The stain will bind to the DNA and make it more visible.

  11. Carefully pour the stain in the staining tray back into the stock jar of stain and begin destaining by pouring distilled water into your staining tray, again just enough to cover the gel and resume gently rocking the gel. Destaining will take a minimum of 10 minutes.

Mitosis and the Cell Cycle

While your gels are running you will take out the compound microscope and look at mitosis. Cells normally undergo a complex series of steps that results in cells growing and dividing. The control of this process is complex and details about the control are still being discovered. Cells that grow uncontrollably are cancer cells. In lecture, we will cover this process in some detail. In lab, we want you to be able to find and recognize the different stages of mitosis.

Interphase is the time in the cell cycle between the times when the cell is dividing and encompasses the G1 (growth 1) Phase, S (DNA synthesis) Phase, and G2 (growth 2) Phase.

Mitosis is when the replicated chromosomes separate and is usually followed by cytokinesis which divides the cytoplasm into the two daughter cells. Mitosis is divided into four stages:

  • Prophase: During prophase, the chromatin condenses and become visible as chromosomes.

  • Metaphase: Chromosomes align along the equatorial plane.

  • Anaphase: The paired chromosomes separate at the kinetochores and move to opposite poles.

  • Telophase: Chromatids arrive at the opposite poles and new nuclear membranes form.

Cytokinesis: Division of the cytoplasm occurs when a protein pinches the cell into two daughter cells.

Instructions: Get a slide of the onion root tip. On the slide, find examples of each of the phases. When you have found an example, raise your hand for the TA to confirm that you have found it. Be prepared to explain why you think that cell is in that stage. On Hand-In 9, make a sketch of what each stage looks like.