Worked Problem

One sample of a linear 13,000-bp (13-kb) DNA fragment is cut with the restriction enzyme EcoRI; a second sample of the same DNA is cut with BamHI; and a third sample is cut with both EcoRI and BamHI together. The resulting fragments are separated and sized by gel electrophoresis (I Figure 19.4). Determine the positions of the EcoRI and BamHI restriction sites on the original 13-kb fragment.

Using the sizes of the fragments produced from the three digests in Figure 19.4, we can order the positions of the restriction sites on the original 13-kb piece of DNA. First, note that digestion with EcoRI alone produced 8-kb, 3-kb, and 2-kb fragments, indicating that there are two EcoRI restriction sites in the original linear piece of DNA. Digestion with BamHI produced 9-kb and 4-kb fragments, indicating that there is only one BamHI site. The BamHI restriction site must be 9 kb from one end and 4 kb from the other end.

The double digest produced four pieces of DNA: 7-kb, 3-kb, 2-kb, and 1-kb fragments. Neither of the fragments generated by BamHI alone is present in the double digest, and so EcoRI must have cut both of the BamHI fragments. Consider the 9-kb fragment. How could this fragment be cut by EcoRI to produce the fragments found in the double digest? Two of the fragments produced by the double digest, the 7-kb and 2-kb fragments, add up to 9 kb, the length of one fragment produced by digestion by BamHI alone. Similarly, the 3-kb fragment and the 1-kb fragment of the double digest add up to 4 kb, the length of the other fragment produced by BamHI alone. Therefore, EcoRI cut the first BamHI fragment into 7-kb and 2-kb fragments and cut the second BamHI fragment into 3-kb and 1-kb fragments:

9 kb

9 kb

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