Chapter 11, Problem 13P

You want to make a recombinant DNA in which a PCR product amplified from the human genome is inserted into a plasmid vector. The polylinker of this vector includes recognition sites for the enzymes EcoRI (5′ G^AATTC 3′) and BamHI (5′ G^GATCC 3′). (The ^ symbolizes the cut site in the DNA.) PCR primers that could amplify the fragment of human DNA are: 5′ GCTACTTCGCGTATTCCA 3′ and 5′CCCAAGTCCTAGCCGATA 3′.

a.	Describe in detail how these primers would need to be modified to create a fragment of the human genome flanked by EcoRI sticky ends so that this fragment could be cloned easily into the plasmid vector. You will need to consider the fact that most restriction enzymes, including EcoRI, cannot cut DNA if the restriction site is directly at the end of the DNA molecule; the restriction enzyme recognition site must be at least six base pairs distant from the end.
b.	Describe a potential feature of the PCR-amplified region of the human genome that could prevent you from using the strategy you described in part (a).
c.	Now describe how the primers must be modified to create a human DNA fragment with an EcoRIcompatible single-stranded overhang at one end and a BamHI-compatible overhang at the other end. (Two possibilities exist; you need to describe only one. Assume that a BamHI site also must be at least six base pairs from the end of the DNA.) Why might you want to make such a fragment?