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Chapter 12: Problem 3

A vector has a polylinker containing restriction sites in the following order: Hind III, SacI, XhoI, BglII, XbaI, and ClaI. a. Give a possible nucleotide sequence for the polylinker. b. The vector is digested with Hind III and ClaI. A DNA segment contains a Hind III restriction site fragment 650 bases upstream from a ClaI site. This DNA fragment is digested with Hind III and \(C l a I,\) and the resulting Hind III-ClaI fragment is directionally cloned into the Hind III-Clal-digested vector. Give the nucleotide sequence at each end of the vector and the insert and show that the insert can be cloned into the vector in only one orientation.

Short Answer

Expert verified
The proposed sequence for the polylinker could be 5'-AAGCTTGAGCTCCTCGAGAGATCTTCTAGAATCGAT-3'. The termini of the vector after digestion with Hind III and ClaI would be 5' AAG and 5' TCG. The insert termini after digestion with the same enzymes would be 5' TCG and 5' AAG, respectively. The insert can only be ligated into the vector in one specific orientation due to the complementary overhangs created by these restriction enzymes.

Step by step solution

01

Sequence of the polylinker

The given restriction endonucleases recognize the following sequences: Hind III: 5'-AAGCTT-3', SacI: 5'-GAGCTC-3', XhoI: 5'-CTCGAG-3', BglII: 5'-AGATCT-3', XbaI: 5'-TCTAGA-3', and ClaI: 5'-ATCGAT-3'. We can combine these sequences in the order given to generate a sequence for the polylinker, for instance: 5'-AAGCTTGAGCTCCTCGAGAGATCTTCTAGAATCGAT-3'.
02

Digestion of the vector and the insert

The vector and the DNA insert are both digested with Hind III and ClaI, which will generate complementary 'sticky' ends on both fragments. Hind III will leave a 5' overhang with the sequence AAG, while ClaI cuts to leave the sequence 5'-TCG on the 3' end of the DNA.
03

Cloning of the insert into the digested vector

The termini of the vector will be: 5' AAG and 5' TCG. The insert HindIII-ClaI fragment will have the complementary termini: 5' TCG and 5' AAG, respectively. The vector can ligate this insert at the Hind III and ClaI sites. The insert can only enter the plasmid in one orientation because Hind III and ClaI create different overhangs meaning that the Hind III cannot bind to the ClaI site and vice versa; hence the 'directional cloning'.

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