Restriction endonucleases are DNA-cutting enzymes found originally in bacteria and put to use by scientists. These enzymes are unique in that they identify specific palindromic gene sequences and splice the DNA in a patterned form, shown below. Some enzymes, such as AluI, cut straight across. However, it is more common to see enzymes create “”sticky ends”” that result in some overhanging DNA. These sequences are able to bind with a complimentary overhanging sequence. Ever since their discovery, it has been possible to manipulate genetic sequences of bacteria in order to understand more about genes and their transfer. Experiments are carried out using bacterial plasmids, circular molecules of DNA found outside of the bacterial chromosome. Plasmids are particularly easy to work with because they are relatively short, only containing a few genes. However, they must contain only one origin of replication. Otherwise, different DNA sequences will be polymerized than originally intended, skewing the experimental results.
A biology student was studying two populations of antibiotic resistant bacteria. After performing various genetic studies, the student discovered a plasmid in each bacterium that conferred its resistance. One was ampicillin-resistant (pAMP), and the other was kanamycin-resistant (pKAN), and their plasmids are shown below. The student also researched some common restriction enzymes and compared them to the DNA sequences on pAMP and pKAN to determine where restriction enzymes might cut the DNA and potentially come up with a way to remove their resistances. However, the student also discovered a tumor suppressor gene (tsg1 and tsg2) on each plasmid that might be useful to save. Not entirely familiar with restriction enzymes, the student first treated one plasmid at a time with a two restriction enzymes and ran a gel electrophoresis to measure the results. Although the data were sound, the student unfortunately forgot to label which restriction enzymes were used.
DNA samples from pAMP and pKAN were used as controls on a gel electrophoresis. Lanes 1 and 2 were two trials using different restriction enzymes on pAMP, while 3 and 4 were two different restriction enzymes on pKAN.
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