Genetics/Biotechnology, Advanced/AP Biology, Biology, Life Science.
Students will analyze the results of a hypothetical cloning experiment using a restriction endonuclease map obtained from an on-line data source.
The question they will answer is: "How can I tell if the DNA I have obtained from this hypothetical cloning experiment is the DNA I am expecting to get?"
Notes for the teacher: This activity requires access to the World Wide Web or a DNA sequence analysis computer program such as DNA Strider.
Required of students: Students must have a fundamental understanding of DNA structure and a rudimentary knowledge of what restriction endonucleases do. They must also have basic computer skills.
Preparation time needed: Minimal. Photocopies of the DNA sequences provided must be made available to students. Class time will be required to provide students with necessary background information.
Class time needed: Entering the DNA sequence into the computer will take about 10 minutes. On-line manipulation of the data will require less than 15 minutes and the results can be printed out for subsequent analysis by the students. Time required for student analysis will vary.
One of the most precise techniques a researcher uses to determine the order of nucleotides, and thus the identity of the product of a cloning experiment, is DNA sequence analysis. However this procedure is quite complicated, and frequently uses radioactive isotopes, making it unsuitable for the average high school student. Another less precise method used to identify cloning products involves the use of restriction endonucleases.
In this activity, students will utilize an on-line data source provided by the Baylor College of Medicine, to analyze DNA sequence data. Students will develop a strategy for determining if a DNA sequence provided by the instructor could match an expected DNA sequence.
To make this determination, the students will enter the DNA sequence provided by the instructor into the computer, create a "restriction endonuclease map" of the sequence using the on-line data source, and describe how they could develop a series of experiments using restriction enzymes which could provide evidence of a match with an expected DNA sequence.
Restriction endonucleases are widely used to cut the DNA molecule at various points along its length. These enzymes are highly selective, only acting on a very specific sequence of nucleotides and are routinely used in conjunction with agarose gel electrophoresis to help determine the identity of DNA samples.
Powerful computer software as well as on-line databases are available to help the research scientist determine which restriction enzymes will act on a given DNA sequence. The researcher must then, in effect, assemble a puzzle from the results of restriction enzyme reactions, to help ascertain the identity of unknown DNA.
To allow students to use problem solving skills in conjunction with some of the same experimental techniques research scientists use to identify an unknown DNA sequence.
Computer with modem, access to the world wide web and a DNA sequence. (The DNA sequence provided is 300 base pairs long. A larger number of nucleotides may also be used.)
Present the following scenario to the students: " We have a sample of DNA which is the result of a cloning experiment. We believe this DNA has the sequence (provide the sample sequence). Your task is to design a series of experiments using restriction enzymes which would help to verify that the results of the cloning experiment are, in fact, the DNA we expect. To do this, follow the instructions listed below."
Enter the DNA sequence into a word processing program and copy it using the Edit/Copy command.
Login on your computer to the world wide web and go to the following address:
You are now at the Baylor College of Medicine Search Launcher home page.
Under "Current Launch Pages:" click on Sequence Utilities
Place the cursor in the box at the top of the page and using the Edit/Paste command, paste in the sequence previously typed.
Click on the circle in front of the words "WebCutter" and then click on Submit. The restriction map will appear on your screen.
Select the File/Print command and your restriction map will be printed out for you for later analysis.
Students must now use the restriction maps to determine which enzymes they would use to cut the DNA sample in a series of diagnostic restriction digests. Since several of the enzymes produce fragments of similar size, the students will have to suggest a series of experiments using a number of the enzymes. Their goal is to design experiments which will support or reject the hypothesis that the sample DNA they are analyzing matches the expected DNA.
They should be advised that in real applications of this technique, the size of the fragments produced from the restriction enzyme reactions are determined by using agarose gel electrophoresis and comparing the results of the experiment to a size marker. The resolution is such that they would have difficulty verifying the size of very small fragments or distinguishing between fragments of very similar sizes. In short, they must find enzymes which would provide significantly different results from each other.
A single restriction digest which yields fragments of the expected sizes is not definitive proof that their sample is a match of the expected. Results of several different digests which produce fragments of the expected lengths increases the likelihood that the sample DNA is identical to the expected DNA sequence.
Students should prepare either a written or oral presentation for the teacher explaining how their proposed experiments might help to identify the sequence of the sample DNA. Their reports should specify the exact sizes of the fragments they would expect from each of the reactions they propose.
If actual samples of DNA are available, students could attempt to verify their proposed results by actually performing the digests and viewing the results using agarose gel electrophoresis. To perform this experiment, one would require a DNA sample with a known sequence, a supply of restriction endonucleases and agarose gel electrophoresis apparatus.