Question

HindIII	5000, 2500
SmaI	5500, 2000
EcoRI	4000, 3500
HindIII + EcoRI	3500, 2500, 1500
HindIII + SmaI	3000, 2500, 2000
SmaI + EcoRI	4000, 2000, 1500

23. The enzyme PspN4I cleaves the sequence NNGCNN (N= any of the four bases). How many times would you expect PspN4I to digest a 10.752 kb genome?

24. Sequence A, which contains two BstBI sites (TT/CGAA), was digested with BstBI. The resulting fragment was then ligated into the unique TaqI restriction site (T/CGA) within vector B.

    Sequence A: CAG TT/CGAA TTC • • • • • • • • • • • • • • • • GGC TT/CGAA AAG Vector B: TGG T/CGA CAC

    Which enzyme (s) could be used to release the cloned DNA fragment from the recombinant vector B? BstBI, TaqI , both, or neither.

Answer 1

Please find the answers below:

Answer 1: The number of recognition sites of the restriction enzyme are 6, therefore it can recognize the DNA each 4⁶ or 4096 base pairs. Thus the total number of recognition sites in the DNA will be: 10752 / 4096 or 2.625 or nearly 3. Thus, this enzyme will digest this DNA maximum three times.

Answer 2: TaqI enzyme: Since the gene of interest has been ligated into a sequence recognized by TaqI, enzyme, its release from the site of insertion will require digestion with TaqI only since digestion with BstBI will cause breakdown of the gene of interest itself.