You want to insert your favorite gene, which has been digested with Ncol endonuclease on both ends, into a vector. Ncol recognizes the DNA sequence C*CATGG. The vector you want to use contains many endonuclease recognition sites, but does not include an Ncol site. Which restriction endonuclease would be the most suitable replacement to digest the vector?

Question

A.  Sphl - G*CATGC
B.  Sunl - C*GTACG
C.  Kpnl - GGTAC*C
D.  CViAll - C*ATG

Answer 1

A.  Sphl - G*CATGC

Clarify Question  
· What is the key concept addressed by the question?
        o This question addresses restriction enymes.
· What type of thinking is required?
        o This question is asking you to analyze the information given, using logic, to dissect the problem and determine the answer.
· What key words does the question contain and what do they mean?
        o Digesting with an endonuclease means to treat the DNA with a restriction enzyme that cleaves within the DNA, only at particular sequences.
        o Those sequences are called endonuclease recognition sites.
        o The vector is the plasmid used for cloning the gene.
 
Gather Content
· What do you already know about restriction enzymes?
        o Restriction enzymes recognize a specific DNA sequence, ranging from 4 bases to 12 bases, and cleave the DNA at a specific base within this sequence.
        o The recognition sites for most type II enzymes are palindromes. The palindromic DNA sequence reads the same from 5¢ to 3¢ on one strand as it does on the complementary strand.
        o Given this kind of sequence, cutting the DNA at the same base on either strand produces staggered cuts that leaves “sticky ends,” or overhangs.
 
Consider Possibilities
· What other information is related to the question? Which information is most useful?
        o What is the goal of digesting both the gene and the vector with restriction enzymes? The goal is to allow the insert ends to stick to the ends of the vector, so that it can be ligated in.
        o What must be true of the DNA ends for the gene to be able to insert and ligate into the vector successfully? The sticky ends of the gene and vector must complement each other – they must be compatible.
        o Can you draw out the results of each enzyme to help answer this question?
        o On the vector, Ncol - C*CATGG would create 5’ sticky ends of CATG
        o Sunl - C*GTACG would create 5’ sticky ends of GTAC
        o Kpnl - GGTAC*C would create 3’ sticky ends of GTAC
        o CViAll - C*ATG would create 5’ sticky ends of ATG
        o Sphl - G*CATGC would create 5’ sticky ends of CATG
   Choose Answer
· Given what you now know, what information and/or problem solving approach is most likely to produce the correct answer?
        o For digesting the gene to be inserted, only Sphl - G*CATGC would create the samecompatible sticky ends as NcoI.
        o Both create 5’ ends of CATG.
        o Remember, since the enzyme cleaves on both strands, the other strand is cleaved by SphI like 3’ CGTAC*G 5’. So the overhang with both enzymes is four nucleotides, 5’ CATG 3’. They are compatible ends even though the recognition sites are slightly different.
 
Reflect on Process
· Did your problem-solving process lead you to the correct answer? If not, where did the process break down or lead you astray? How can you revise your approach to produce a more desirable result?
        o This question asked you to choose the best restriction enzyme.
        o The question required you to analyze the information given, using logic, to dissect the problem and determine the answer.
        o Did you recognize that the overhangs needed to complement – that is, they needed to be the same?
        o Did you recognize that a 5’ overhang cannot bind with a 3’ overhang?
        o Did you try writing out the results of each enzyme?