Chapter 17, Problem 20P

To further analyze the CRABS CLAW gene (see Problems $19$ and $\text{20}$), you create a map of the genomic clone. The $\text{11-}$ kbEcoRI fragment is cloned into the EcoRI site of the MCS of the vector shown in Problem $18$. You digest the double $\text{-}$ stranded form of the genome with several restriction enzymes and obtain the following results. Draw, as far as possible, a map of the genomic clone of CRABS CLAW.

What restriction digest would help resolve any ambiguity in the map?

You have isolated a genomic clone with an EcoRI fragment of $\text{11}$ kb that encompasses the CRABS CLAW gene (see Problem $\text{18}$). You digest the genomic clone with HindIII and note that the 11-kb EcoRI fragment is split into three fragments of $\text{9}$ kb, 1.5 kb, and 0.5 kb.

Does this tell you anything about where the CRABS CLAW gene is located within the 11-kb genomic clone?

Restriction enzyme sites within a cDNA clone are often also found in the genomic sequence. Can you think of a reason why occasionally this is not the case? What about the converse: Are restriction enzyme sites in a genomic clone always in a cDNA clone of the same gene?

You have identified a $0.80\text{-}kb$ cDNA clone that contains the entire coding sequence of the Arabidopsis gene CRABS CLAW. In the construction of the cDNA library, linkers with EcoRI sites were added to each end of the cDNA, and the cDNA was cloned into the EcoRI site of the MCS of the vector show in the accompanying figure. You perform digests on the CRABS CLAW cDNA clone with restriction enzymes and obtain the following results. Can you determine the orientation of the cDNA clone with respect to the restriction enzyme sites in the vector? The enzymes listed in the dark blue region are found only in the MCS of the vector.