pierceessentials3e

WORKED PROBLEM

A linear piece of DNA that is 30 kb long is first cut with BamHI, then with HpaII, and finally with both BamHI and HpaII together. Fragments of the following sizes were obtained from these reactions:

BamHI: 20-kb, 6-kb, and 4-kb fragments

HpaII: 21-kb and 9-kb fragments

BamHI and HpaII: 20-kb, 5-kb, 4-kb, and 1-kb fragments

Draw a restriction map of the 30-kb piece of DNA, indicating the locations of the BamHI and HpaII restriction sites.

Solution Strategy

What information is required in your answer to the problem?

A map that includes the number and relative locations of restriction sites for BamHI and HpaII and the distances in base pairs between the sites.

What information is provided to solve the problem?

Note: This problem can be solved correctly through a variety of approaches; this solution applies one possible approach.

Hint: For linear DNA, the number of restriction sites is one less than the number of fragments produced.

The piece of DNA is 30 kb long.
The sizes of the fragments produced when the DNA is cut with BamHI, with HpaII, and with both enzymes together.

For help with this problem, review:

Physical Maps in Section 15.1.

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Solution Steps

Hint: Look for fragments in the double digest that sum to the length of a fragment present in the single digest.

When cut by BamHI alone, the linear piece of DNA is cleaved into three fragments; so there must be two BamHI restriction sites. When cut with HpaII alone, a clone of the same piece of DNA is cleaved into only two fragments; so there is a single HpaII site.

Let’s begin to determine the locations of these sites by examining the HpaII fragments. Notice that the 21-kb fragment produced when the DNA is cut by HpaII is not present in the fragments produced when the DNA is cut by BamHI and HpaII together (the double digest); this result indicates that the 21-kb HpaII fragment has within it a BamHI site. If we examine the fragments produced by the double digest, we see that the 20-kb and 1-kb fragments sum to 21 kb; so a BamHI site must be 20 kb from one end of the fragment and 1 kb from the other end.

Similarly, we see that the 9-kb HpaII fragment does not appear in the double digest and that the 5-kb and 4-kb fragments in the double digest add up to 9 kb; so another BamHI site must be 5 kb from one end of this fragment and 4 kb from the other end.

Now, let’s examine the fragments produced when the DNA is cut by BamHI alone. The 20-kb and 4-kb fragments are also present in the double digest; so neither of these fragments contains an HpaII site. The 6-kb fragment, however, is not present in the double digest, and the 5-kb and 1-kb fragments in the double digest sum to 6 kb; so this fragment contains an HpaII site that is 5 kb from one end and 1 kb from the other end.

Hint: Two fragments in the double digest that were produced by cutting a fragment in the single digest must be adjacent to one another.

We have accounted for all the restriction sites, but we must still determine the order of the sites on the original 30-kb fragment. Notice that the 5-kb fragment must be adjacent to both the 1-kb and the 4-kb fragments; so it must be in between those two fragments.

We have also established that the 1-kb and 20-kb fragments are adjacent; because the 5-kb fragment is on one side, the 20-kb fragment must be on the other, completing the restriction map: