Chapter 20 Branched Tutorial: Restriction Mapping

Problem Statement

Restriction sites can be mapped by comparing DNA fragments produced by digestion with restriction enzymes used alone and in various combinations.

The experiment began with one sample of a linear 13,000-bp (13-kb) DNA fragment being cut with the restriction enzyme EcoRI; a second sample of the same DNA was cut with BamHI; and a third sample was cut with both EcoRI and BamHI together.

The figure at the right represents the separation of DNA fragments by gel electrophoresis.





Question

Provide the correct restriction map of the linear DNA fragment based on the results shown on the gel image by choosing the correct enzyme names and fragment sizes from the associated dropdown menus.

A. B. C. D. E. F. G.
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Problem Strategy

Question

Determine the best approach to solve this problem by ordering the following steps:
1. Examine the results shown on the gel and compare the single digests with the double digest.
2. Identify the question that is being asked and focus on key pieces of information.
3. Examine the results shown on the gel and analyze each lane independently by constructing a restriction map for each single digest.
4. Construct a restriction map based on the double digest.

1. Which of the following is the correct order of steps that should be used to solve this problem?

A.
B.
C.
D.

2
Correct.
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Incorrect.

Instant TA

Begin with the basic information and individual analyses and then build upon this information to solve the problem. The best approach is to analyze each single digest independently and then construct the restriction map of the double digest.

Step 1: Understanding the problem

Question

1. What type of fragment is being digested?

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B.

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Correct.
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Instant TA

DNA molecules can be linear or circular. Read the question carefully to learn what type of fragment is being digested.

Step 2: Analyzing the Gel

Question

1. How many fragments result from the single digest with EcoRI?

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B.
C.
D.

2
Correct.
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Incorrect.

Instant TA

Focus on only the lane that is labeled “EcoRI alone” in order to determine your answer. A fragment is displayed as a band or single line on the gel.

Step 3: Understanding the Data

Question

1. Are there any common fragment sizes between the double digest and the EcoRI digest?

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B.

1
Correct.
Incorrect.

Instant TA

Focus on the lanes that are labeled “EcoRI alone” and “Double Digest” in order to determine your answer. A fragment is displayed as a band or single line on the gel.

Step 4: Solving the Problem

Question

1. Draw a restriction map based on the third lane of the gel. Choose from the dropdown menus below to correctly label each restriction enzyme recognition site and fragment size.

A. B. C. D. E. F. G.
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Congratulations! You have correctly determined the positions of the EcoRI and BamHI restriction sites on the original 13-kb fragment. This knowledge and approach can be used in solving similar restriction mapping problems.
At least one of your answers is incorrect. Try answering the problem again. Be sure to check your map. You should be able to see the correct size of fragments for EcoRI alone, BamHI alone, and the double digest all represented in your restriction map. The total size of the entire fragment must equal 13 kb.
Incorrect.

Instant TA

Remember that there are EcoRI fragments which are not cut by BamHI. These fragment sizes should not contain a BamHI restriction site on your map. Begin your mapping by identifying the largest EcoRI generated fragment—remember, this fragment contains a BamHI site. Be sure that you are locating the 7 kb and 1 kb fragments generated by a BamHI enzyme next to each other on the map. Keep in mind that the total size of the fragments needs to be equal to the size of the initial DNA fragment before digestion.

Notes: