Molecular Biology

DNA Profiling

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This experiment will take four to five standard periods to complete.  Use the first period practicing with the micropipettors and labeling tubes for the upcoming restriction digest.  Use the second period setting up restriction digests and pouring agarose gels.  After a period of incubation at 37°C (can be many hours or even overnight) the digests can be transferred to a freezer.  When set, the gels can be thoroughly wrapped in saran wrap and placed in the refrigerator until needed.  The third period is for adding dye to the samples and loading the gel for electrophoresis.  Some students may be able to observe the beginning of the electrophoresis, while some will be loading their gels right to the end of the period.  Teachers can do the staining and destaining, or a few students can return to the classroom for brief periods (5 - 10 minutes each time) to do this part.  The fourth period is for photography and analysis of the gels.

A double period (without a break) for the second lab day makes for a smoother experience.  Use some of the additional time in the first period for discussing background material (in this case, leave the labeling of tubes for the second period).  Use the remaining time of the double period (after the digests are set up and the gels poured) for much needed lecture/discussion.

This lab has been written for a number of scenarios and teachers are encouraged to make up their own when they are comfortable with the lab.

Forensic DNA Amplification:  "Who Done It?"

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It has now become routine to purify DNA and isolate specific genes.  The development of the Polymerase Chain Reaction (PCR) allows incredibly small amounts of DNA to be amplified into a quantity that can be studied in the laboratory.  The technology has had a profound impact on the field of forensics.  It is now possible to isolate a minuscule sample of blood, semen, or even a hair follicle, to amplify the DNA in the sample, to analyze the amplified DNA, and to compare the DNA sample to DNA obtained from an individual suspected of committing a crime.  DNA evidence is now commonly used in criminal cases and also has widespread use in paternity investigations.

In this activity, DNA is collected from mouth epithelial cells and amplified by PCR.  The PCR-generated fragments of DNA are then separated on the basis of size using agarose gel electrophoresis.  The results of the experiment are then photographically documented.

The activity is designed to be a murder investigation.  Each one of the participants is a suspect in the crime.  Within each group of 6-8 suspects, there lies a murderer.  The bands of DNA obtained after amplification of DNA isolated from the evidence at the crime-site are compared to the amplified bands of DNA isolated from cheek cells from each suspect.  The banding pattern of the murderer will match the banding pattern of the evidence.

HIV Transmission

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This activity can be done easily in a classroom setting.  The materials are readily available and are safe for students to handle.  Role playing is involved and every student takes an active part.  It’s FUN!  ("Awesome" is the word my 10th graders used!)  This is the best way I can imagine to introduce such a sensitive/vital issue in a non-embarrassing way and still get the message across:  the HIV virus is transmitted by sharing body fluids, there are specific high risk behaviors, and what you choose to do is the greatest determining factor in whether or not you contract the disease.  There is a concern that has been expressed by some teachers that this subject needs to be dealt with in a way that all students can feel comfortable.  In some schools there may be HIV positive students and many students have personal experience with friends and relatives who are HIV positive.

How Many CAT’s?

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In this paper simulation, students will "cut" DNA samples from a mother, a baby, a husband, and a rape suspect using a restriction endonuclease.  They will then "run" the DNA fragments on a "gel" to simulate the process of electrophoresis.  A fluorescent probe is then washed over the gel.  Finally, students will analyze the gel to identify the father of the baby.

Karyotype Analysis

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In this laboratory exercise, students stain a fixed preparation of human cancer cells that were cultured in the laboratory.  The stained cells are microscopically observed; students will observe cells containing the classical "X" shaped chromatids at metaphase.  Students gain microscope practice and collect interesting data about chromosome number and chromosome type.  A karyotype of several well-spread cells is prepared and compared to a karyotype of normal human cells.  This activity can serve as a springboard to class discussions about genetic defects that can be diagnosed by the use of karyotyping.

The Properties of Enzymes:  A Study of Catalase

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The structure and function of enzymes is a central theme in cellular and molecular biology.  In this laboratory exercise, a crude cell extract is prepared from potatoes.  Activity of the enzyme, catalase [which catalyzes the reaction 2H₂O₂(l) —> 2H₂O(l) + O₂(g)], is then studied using a simple assay for O₂.  To conduct the assay, a filter is soaked in crude potato extract, then transferred to the bottom of a beaker containing hydrogen peroxide.  Catalase causes O₂ to collect in the filter which in turn causes the filter to rise.  Students are able to explore the effect of enzyme and/or substrate concentration and pH on the amount of product formed by measuring the time taken for each filter to collect enough oxygen to rise.  Students average their results, calculate the inverse of the "time to rise," and pool the data in order to plot the characteristic curve showing the dependence of enzyme activity on substrate and enzyme concentration.

Protein Gel Electrophoresis

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Students will separate a mixture of proteins from skeletal muscle using SDS-polyacrylamide gel electrophoresis (PAGE).  PAGE is a powerful analytical technique having numerous applications in modern biology.  Evidence for evolutionary relatedness amongst organisms can be determined using this technique.  Suggested organisms to compare include various fishes, mammals, poultry and/or sea foods, all of which are available from the grocery store.  During the laboratory, students will develop an experimental design, prepare samples of muscle tissue from various organisms, observe the process of electrophoresis and analyze their results.  Analysis consists of comparing protein bands according to their molecular weights on the gel.

Statistics and Probability in Evaluation of DNA Evidence

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In this lab students will apply statistical analysis to the DNA profiling results of an actual rape case.  This lab is an extension of DNA profiling labs which students have previously completed.  In part I of this exercise, students will use sets of candy to represent alleles in a particular population at a particular locus.  They will sample "alleles" from "populations" at three "loci," estimate frequencies of alleles in those samples and calculate probabilities of particular combinations of alleles.