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Suppose the restriction endonuclease HindIII cuts a6.0 kb linear piece of DNA into two fragments; an 800 bp fragment and a 5200 bp fragment.....
Question:Suppose restriction endonuclease HindIII cuts a6.0 kb linear piece of DNA into two fragments; an 800 bp fragment and a 5200 bp fragment. NarI cuts DNA also into two fragments; fragments 1200 and 4800 bp long. Relative to HindIII cut site, there are two probable ways in which NarIcould have cut DNA. How can you determine correct cleavage site for NarI with respect to HindIII?
Answer
To determine this, one should subject DNA to a double digest in which both enzymes are allowed to cut DNA at the same time. When double digest is allowed to take place, if three fragments which appear upon electrophoresis of the restricted DNAare 400, 800 and 4800 bp, then the only possible way in which data can be interpreted is with 1200 bp NarI fragment containing the HindIII recognition site 800 bp from the end of linear piece of DNA. If 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.
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