Strategy For Solving Restriction Digestion Problems

Strategy for solving restriction-mapping problems 1. At the outset, recognize that DNA can be circular or linear 2. Recognize that fragments can be masked; i.e., that two similar sized fragments with different sequences will appear as one band on a gel. 3. Tally the number of restriction sites for each of the enzymes by examining the number of fragments produced. With linear DNA the number of fragments is always 1 more than the number of sites; with circular DNA, the number of fragments equals the number of sites. 4. Add up the total length of the original DNA. This should help reveal if there are any “masked” fragments. 5. Look for fragments in one enzyme’s pattern that appear to be cut by the alternative enzyme. It is often good to start with the frequent cutter’s fragments and look for those that are cut by the less frequent cutter enzyme. 6. Recognize that there are several possible ends to the fragments: Free ends; E1 ends and E2 ends. Thus, fragments can be: F____________E1 F____________E2 E1___________E1 E2___________E2 E1___________E2 Of course with circular DNA there will be no free ends. 7. Try to determine what kinds of ends are on each fragment. This will tell you which ones are in the middle of a linear DNA molecule and which ones were on the ends. 8. Generally start by analyzing larger fragments and then move to smaller fragments. 9. Draw pictures to test various hypotheses.

Bio 2250 - Sample Restriction Mapping Problems In each problem, three restriction endonucleases are used to cut a piece of DNA, singly and in pairwise combination. Sizes of fragments are listed in order of size, not in linear order. Determine the correct order of restriction sites, and draw the map, with the intervals between sites labelled. Worked Example

A) 11, 6, 5 B) 14,8 C) 16,6 A x B) 8, 6, 5, 3 A x C) 11, 5, 5, 1 B x C) 8, 8, 6 D) 18, 5 E) 15, 7, 1 F) 20, 3 D x E) 10, 7, 5, 1 D x F) 15, 5, 3 E x F) 15, 4, 3, 1 G) 14, 1 H) 9, 6 I) 7, 5, 3 G x H) 9, 5, 1 G x I) 7, 5, 2, 1 H x I) 5, 4, 3, 3 Circular Restriction Mapping