Question

1. The restriction enzyme Sau3AI recognizes the following sequence: 5'-GATC-3'. On average, how often should this enzyme cleave DNA? In contrast, the restriction enzyme Natl recognizes the following sequence: 5'-GCGGCCGC-3'. On average, how often should this enzyme cleave DNA? Does Natl cleave DNA more frequently than Sau3AI?

2. An uncharacterized plasmid DNA was cleaved using several restriction enzymes individually and in various combinations. The DNA fragment sizes were determined by agarose gel electrophoresis and the restriction enzyme recognition sites were mapped. Subsequently, the DNA was sequenced, and an extra recognition site was found for one of the enzymes. However, all the other mapping data was consistent with sequence data. What are the simplest explanations for this discrepancy? Assume the DNA sequence had no errors.

3. A plasmid was cleaved with several restriction enzymes, individually and in combinations. The following fragment sizes (base pairs) were determined by agarose gel electrophoresis.

Note: There may be some slight discrepancy in summing up the total base pairs. Indicate the distances between sites.

Eco RI	4363
Ava I	2182
Pvu II	4363
Pstl	4363
Eco RI -Ava I	2938	1425
Eco RI - Pst I	3609	754
Ava I - Pvu II	3722	641
Ava I - Pst I	2182
Pvu II - Pst I	2820	1543
Eco RI - Pvu II	2297	2066

Make a restriction map based on this data. Hand draw on separate paper and submit the picture of your drawing map.

Note: There may be some slight discrepancy in summing up the total base pairs. Indicate the distances between sites.

4. Why is only one band detected in the Ava I - Pst I co-digest?

Answer 1

1. We consider the frequency of the given sequences on one strand of the DNA as the restriction site sequences are complimentary.

Since there are four possible bases at each of the four positions of the restriction site for Sau3AI, the frequency of the 4 base long sequence is (1/4)4 = 1/256. Therefore on an average, Sau3AI cleaves DNA after 256 bp.

Since there are four possible bases at each of the eight positions of the restriction site for Natl, the frequency of the 8 base long sequence is (1/4)8 = 1/65536. Therefore on an average Natl cleaves DNA after 65.5 kb.

Therefore Natl does not cleave DNA more frequently than Sau3AI.