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DNA Fingerprinting for High School Biology

By Richard Myers



Type of entry:

  • lesson/class activity

Type of activity:

  • hands-on

Target audience:

  • Biology
  • Genetics, Biotechnology


Abstract of Activity

In this laboratory investigation students work with several of the tools and techniques used in genetic engineering. Students are provided with an unknown sample of DNA. The students dissect this sample into fragments of DNA using restriction enzymes, sometimes called "molecular scissors". They then sort these fragments, measure and analyze them according to size. The students then identify and match their unknown DNA sample with known samples. (The matching of unknown samples of DNA with known samples is often referred to as "DNA fingerprinting" and is an important tool used in police work).

This laboratory activity is performed in a high school general biology laboratory with class periods of 50 minutes. The unit takes about 7 to 10 days. Materials needed to perform this laboratory activity are available commercially.


Background information

Notes to teachers:

  • This is a laboratory activity I use in my high school general biology program. The class is made up of students in grades 9 through 12 and the class period is 50 minutes.
  • All materials are available commercially except for plasmids "A and B". To use this procedure you will need to replace or delete the use of these plasmids.
  • Unknown plasmid C is pBR322; D is pBR325 and E is pUC 19. I use about O.2 to 0.4 Ál per unknown.
  • Restriction enzymes used are EcoRI and BamHI.
  • The DNA ladder is a 1kb ladder from Bethesda Research Laboratories (BRL). You will need to transfer the information on base pair sizes of the ladder to Chart 1, page three before running the lab.
  • For general information see a standard standard molecular cloning manuals such as Maniatus, Fritsch, Sambrook.
  • Be aware of safety concerns such as electric currents, DNA stains and ultra-violet light.


Systems, Molecular basis of heredity, Science and technology:

Laboratory Instruction: DNA Fingerprinting for High School Biology
(per pair, two pairs per table)

Day 1: INTRODUCTION AND USE OF EQUIPMENT

Day 2: INTRODUCTION TO GEL ELECTROPHORESIS

Day 3: RESTRICTION PROCEDURE

1. Obtain a 1.5 ml eppendorf tube containing an unknown DNA sample. Record the unknown # in your notebook.

2. Add to this tube 5 Ál of restriction enzyme mixture.

3. Mix; spin briefly.

4. Incubate 37°C for 1 hour. (Step 5 will be completed for you).

5. Store overnight in refrigerator.

Day 4: GEL ELECTROPHORESIS:

1. Set out restricted (unknown) tube. Spin gently.

2. Each member of a pair obtain a tube. Label as A or B (to distinguish each pair member). Also label with your table number and mark in a way to distinguish your tube from the other tubes at your table.

3. Each individual add 5 Ál of sample buffer (blue) to your tube (A or B).

4. Transfer 5 Ál from your unknown tube to your tube (A or B).

5. Mix; spin gently.

6. Load 10 Ál of your tube (A or B) to a Gel. Tube A to Gel "A" and tube B to Gel "B". (Record the well #, your name, and your unknown sample # on the chart by the Gel). Note: One well in each Gel has to be reserved for DNA ladder.

7. Store tubes A and B in assigned location. Clean up.

Day 5/6: MEASUREMENT, DATA ORGANIZATION, GRAPHING AND ANALYSIS OF RESULTS.

Questions: (You can answer these questions using diagrams; also explain your answers fully; answer on another labeled page).

(1) EXPLAIN WHAT RESTRICTION ENZYMES DO?

(2) WHAT IS A DNA LADDER?


Observing your results: Procedure

I. GEL ELECTROPHORESIS RESULTS. (Xerox copies of photos)

Whole (uncut) plasmid DNA will appear as a smear and compared to DNA fragments will not move very far into the gel. Cut (restricted) plasmid DNA will show one or more fragments and will move further into the gel than the whole uncut plasmid.

(3) DO THE RESULTS INDICATE THAT YOUR PLASMID WAS "CUT"? EXPLAIN.

Systems, Molecular basis of heredity, Science and technology:

The DNA ladder is a set of various DNA fragments of known length in base pairs (bp) (See chart I, page 3). It is added to the gel in order to provide a comparison for the unknown DNA sample. Thus, when you observe a gel photo of a plasmid DNA sample that has been "cut" with restriction enzymes, you should be able to see fragments of various sizes and be able to compare these to the known length fragments of the DNA ladder. To make the comparison, you can first observe, for an estimate, which fragment sizes your unknown falls between.

DNA samples move through the gel in a logarithmic fashion, relative to the # of base pairs; eg. the larger the fragment, the slower it moves. Using semi-logarithmic paper, you can plot the DNA ladder and then compare your unknown sample to this.

A. RECORDING THE RESULTS: (Obtain a copy of the photo from your gel)
1. DNA ladder: Measure, in centimeters, the distance the DNA ladder moved in the gel (from the leading edge of the well to the leading edge of the fragment image). Record this information in column A of Chart 1, page 3. Place the data in relation to ladder fragments shown on the data sheet.

2. Unknown DNA fragments: Measure, in cm., the distance the restricted DNA sample fragments moved in the gel. Record this information in column B of Chart 1. (Place the data in an approximate location to the size fragments of the DNA ladder).

B. PREPARING THE SEMI-LOGARITHMIC GRAPH: (Note: Make it neat for display. First make the graph lightly in pencil. When satisfied that it is accurate, then darken with ink. Be sure to title the page, title the graph, label each axis, make a key, keep it neat and accurate, highlight or note the results of the unknown clearly on the graph.)
1. Divide the horizontal axis into cm units. Between 1 - 7 cm.

2. Divide the logarithmic scale (vertical axis) into 100's, 1000's, and 10,000's, as instructed.

3. Plot the data for the DNA ladder on the graph. Make a small circle around each point and indicate the exact size of that fragment.

4. Connect, as best possible, the points with a straight line. (use the points between 300 to 5000 base pairs).

5. Plot the results of the restricted fragments on this graph. Do this by finding the appropriate value on the graph (in cm.) for each DNA fragment. Once this value is located, then move up to where this value intersects the plotted line for the known DNA ladder. Place an "X" at that point where they cross. Label this point clearly.

6. List the values for the base pairs that each of these points represent in column C of Chart 1.

Now observe the following (Diagram A):

Diagram A This is a set of fragment patterns from five different DNA plasmid samples that were cut with the same restriction enzymes you used to prepare your DNA.

Systems, Molecular basis of heredity, Science and technology:

(4) WITH WHICH SET OF FRAGMENT PATTERNS, (A, B, C, D, or E), DOES YOUR SAMPLE BEST COMPARE? EXPLAIN YOUR CHOICE.

Observe Charts of possible plasmids (From supplier of plasmids) which show the structure of five different plasmids (Plasmids A, B, C, D, and E). Note various fragment lengths formed by the restriction enzymes.

Consider the fragment lengths obtained from the results of the restriction procedure you performed on your unknown DNA sample. (See chart I, column C).

(5) DOES THE CHOICE FOR YOUR UNKNOWN PLASMID (A, B, C, D, or E) COMPARE FAVORABLY WITH THE STRUCTURE OF THE PLASMID ON THE CHART? WHAT INFORMATION DO THE RESTRICTION RESULTS GIVE YOU ABOUT THIS PLASMID? EXPLAIN FULLY.


Chart I: DNA Ladder/Unknown Fragment Size Comparison

Unknown DNADNA Ladder
C
Estimated
Fragment
Size (bp)
B
Migration
Distance(cm)
A
Migration
Distance(cm)
DNA Ladder Fragment
Size (bp)
    
    
    
     
     
    
     


What question does this activity help students to answer?

In this laboratory activity students digest (cut) a plasmid DNA sample with two different restriction enzymes, load samples of the restricted (cut) samples of DNA on a gel-electrophoresis apparatus set-up and photo document the results for later analysis and the identify an unknown DNA sample by analysis of the photo document. By identifying the sizes of the various DNA fragments, the students are able to identify their particular plasmid sample.


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