Electrophoretic Analysis
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Electrophoretic Analysis: DNA
Fingerprinting and Evolutionary Divergence

Janet Pantella

Introduction: The early 20th century gave rise to the concept that chromosomal units called genes are responsible for transmitting heritable information from parent to offspring. The structural and functional properties unique to genes, can be evaluated in the high school laboratory. In addition the principal role of genes in the production of proteins can also be examined. The process of electrophoresis can be used to investigate various topics in biology.

Objectives: Students will examine the principles of electrophoresis. They will evaluate both the agarose and polyacrylamide system of molecular separation. In order to bring the concepts of genetics and evolution full circle from DNA to protein, students will conduct a "DNA Fingerprinting" and "Genetic Distance" laboratory.

Teacher Instructions:

1. This laboratory has been adapted from the Ward's Natural Science Establishment, Inc. that supply the electrophoretic protocol for their separation systems utilized for this laboratory. All supplies and protocols are utilized from Ward's.

2. This laboratory is appropriate for tenth through twelfth grade academic students that have a background in protein synthesis and basic genetics.

3. Background information about the concepts of electrophoresis as well as instructions on apparatus assembly and staining procedures should be outlined for students prior to the experiment.

4. After melting the stock agarose, you may find it convenient to place 15 milliliters aliquot of agarose into test tubes for students to utilize in casting their trays. This prevents you from having to remelt the stock agarose over and over and perhaps change the integrity of the gel.

5. If you believe students will have difficulty removing the well forming comb from the agarose gel, you may wish to smear a thin layer of petroleum jelly on the comb ends before immersing it into the agarose.

6. SDS Gels consist of a gel gradient and must be purchased separately for the type of separation to be conducted.

7. Prepare SDS running buffer 4X concentration according to WARD's protocol.

8. SDS or sodium dodecylsulfate (6-14%) polyacrylamide gel will be used. The SDS will give a negative charge to the protein as well as denature it. Separation is solely based on molecular weight.

Agarose Gel Electrophoresis: DNA Fingerprinting

Objectives: The use of agarose gel electrophoresis for DNA fingerprinting in this experiment is designed to allow students to examine the polymorphic units of nonsense codons unique to the individual. Students will analyze gels to determine the length of the DNA fragment by comparing its electrophoretic mobility with a DNA marker sample of known lengths. A standard curve is constructed by plotting the Rf.. value of each standard DNA fragment

Procedures:

I. Apparatus Assembly:

A. The Casting Tray:

1. Attach the well forming comb of six or twelve to the comb holder. Be sure the comb is level and has approximately a millimeters clearance from the casting tray surface.

2. Using a relatively strong tape such as duct tape to secure the ends of the casting tray. Be sure a tight seal is created between the tape and the casting tray edges.

B. Preparation of the Gel:

1. This laboratory utilizes a 0.8% agarose. Proceed to melt the agarose in a microwave or hot water bath.

2. Pour 15 milliliters of agarose into the casting tray and place the comb assembly at one end of the gel. Allow the gel to solidify and then carefully remove the well forming comb.

3. Place the casting tray with gel into the electrophoretic cell.

II. DNA Fingerprinting Procedures:

A. Transfer 10 microliters of the following into each well:

    1. Tube #1 DNA Marker - Lambda DNA/HINDIII Digest
    2. Tube #2 Unknown A - Lambda DNA
    3. Tube #3 Unknown B - Lambda DNA/ECORI

B. Be sure the loaded gel is oriented so the wells are closest to the cathode electrode.

C. Pour 200 milliliters of a 1x concentration running buffer into one compartment until the level reaches the gel. Next, fill the other compartment until the buffer covers the gel surface about 2 millimeters.

D. Run the gel at 90V for about 60 minutes. When the tracking dye that is in the samples has run halfway off the gel.

E. Place the electrophoresed gel in a staining tray and place enough DNA stain in the tray so that the gel is covered. Staining with Ward's DNA stain, takes about three hours.

F. Destaining is accomplished by letting the gel sit in distilled water until the bands are clearly observed.

SDS Polyacrylamide Gel
Electrophoresis: Genetic Distance

Objectives: This laboratory is designed to have students apply their knowledge of DNA structure and function to the concept of genetic divergence by examining the evolutionary relationships among fishes by resolving fish muscle protein (polypeptides) on polyacrylamide gel. Students will compare banding patterns between fish samples and determine the genetic distance between them. Students will create a standard curve using known high molecular weight protein maker sample. By using the standard curve and the relative mobility of the bands, students can determine the molecular weights of the unknown polypeptide.

Procedures:

I. Apparatus Assembly:

A. Add enough running buffer solution to the bottom tank so that it reaches the fill line.

B. Obtain an SDS gel cassette and insert the well forming comb in the thin opening between the 2 glass plates at the top of the gel cassette. Use the "pusher stick" to press the well 1-2 millimeters into the top surface of the gel.

C. Insert the SDS gel cassette between the gasket opening in the top tank. Assemble the buffer tanks by inserting the top tank into the cassette guides until it seats itself on the bottom of the cassette guide rails.

D. Add the running buffer solution into the top buffer tank up to its fill line.

II. Loading and Running the Gel:

A. Transfer 10 microliters of the following samples to each well:

    1. Tube #1 - Yellow Perch
    2. Tube #2 - Walleye
    3. Tube #3 - Chinook Salmon

B. Transfer 20 microliters of Tube #4, the high molecular weight protein marker into a well.

C. Set the voltage to 170 on the power supply and begin the electrophoretic process. This should continue for 60-70 minutes.

III. Staining:

A. Remove the gel cassette from the top buffer tank. Use a razor to cut the tape along one edge of the cassette and open the cassette like a book. Be sure to notch the gel with a razor so you don't lose the orientation of the gel.

B. Carefully place the gel in a staining tray and pour enough protein stain to cover the gel. Stain the gel for approximately three hours.

C. After decanting the stain, destain the gel using distilled water. This process takes about 6-8 hours.

Comments: The DNA Fingerprinting experiment allows students to determine the length of a DNA fragment by the number of base pairs present. In addition, students are introduced to the concepts of restriction enzymes and the structure of the DNA molecule. In the Genetic Distance experiment, the proteins examined are coded for by multiple genes and therefore tend to be more conserve. Most bands on the gel are composed of several polypeptides. In addition muscle tissue does not undergo much selection pressure over time. Upon analysis of their data, students should observe that the yellow perch and walleye have a similar banding pattern because of being derived from a common ancestor. The salmon will be very different from the other two. The students at the end of the laboratory are asked to make comparisons between the agarose and polyacrylamide methods of electrophoresis.


Supply Information

Source:

    Ward's
    5100 West Henrietta Rd.
    P.O. Box 92912
    Rochester, NY 14962-9012

Kit Materials:

I. DNA Fingerprinting:

DNA Fingerprinting Kit 88W8400

    (enough to run 6-gels)

Electrocell 36W5110

    Cell with cover, Gel casting tray, Comb holder, 6-well comb, 12-well comb, Color coded electrical patch cords

Power Supply:

    Two Cell Model. 36W5111
    Three Cell Model. 36W5112

II. Genetic Distance:

Protein Samples:
(yellow perch, walleye, Chinook salmon)

    1 Set of Three. 36W5269
    High Molecular Weight Protein Marker 39W2109

Reagents:

    2 - SDS Running Buffer Packs 36W45290
    2 - Bottles, WARD'S Protein Stain 38W5306
    6-14% SDS Precast Gels (pair) 36W5215

Apparatus:

    Dual Tank and Power Supply. 36W53223

On to Molecular Evolution in Plants
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