Chapter 16, Problem 24P

The footprinting experiment described in Fig. 16.16 depended on having a fragment of double-stranded DNA that was labeled with radioactivity at one end of one strand.

a.	How would you make such a labeled fragment of DNA? Outline the steps you would perform, starting with two PCR primers and some genomic DNA. You also have available a kinase enzyme that adds phosphate groups to the 5′ ends of DNA strands, radioactive ATP, and a restriction enzyme of your choice. You could also use a phosphatase enzyme that removes phosphate groups from the 5′ ends of DNA strands.
b.	Why does the footprinting experiment require a fragment of DNA labeled only on one end of one strand? In other words, how would the results of the experiment differ from those shown in Fig. 16.16 if the DNA was labeled on both strands at their 5′ ends, or if the DNA was labeled with radioactive phosphate along its entire length at every phosphodiester bond?