Suppose the restriction endonuclease HindIII cuts a 6.0 kb linear piece of DNAinto two fragments; an 800 bp fragment and a 5200 bp fragment.....

QuestionSuppose the restriction endonuclease HindIII cuts a 6.0 kb linear piece of DNAinto two fragments; an 800 bp fragment and a 5200 bp fragment. NarI cuts the DNA also into two fragments; fragments 1200 and 4800 bp long. Relative to the HindIII cut site, there are two possible ways in which NarI could have cut the DNA. How can you determine the correct cleavage site for NarI with respect to HindIII?

Answer

To determine this, one must subject the DNA to a double digest in which both the enzymes are allowed to cut the DNA at the same time. When the double digest is allowed to take place, if the three fragments that appear upon electrophoresis of the restricted DNAare 400, 800, and 4800 bp, then the only possible way in which the data can be interpreted is with the 1200 bp NarI fragment containing the HindIII recognition site 800 bp from the end of the linear piece of DNA. If the 4800 bp NarI fragment
contained the cut site, you would visualize fragments of sizes 800, 1200, and 4000 bp after electrophoresing the doubly digested DNA.