Question

A directional (forced) cloning experiment is planned using the restriction enzymes PstI and SphI to generate incompatible termini in the vector pUC18. A student performing this experiment notes that the enzymes have different buffer requirements and must be digested sequentially rather than simultaneously. The plasmid is first digested with SphI, purified on a column, eluted and resuspended in PstI buffer and digested with PstI. However, when this plasmid was used in the cloning experiment with linear inserts having SphI and PstI ends, no recombinant plasmid clones were obtained. Explain? How could the problem be corrected? [2 marks][Clue: ‘Who’s on first’.]

Answer 1

Pst1 restriction enzyme recognizes the site 5'-CTGCAG-3' and optimally digests the DNA fragments at 37^oC in Buffer O system from the Thermofischer company. Tango buffer is efficient if multiple compatible enzymes are used for digesting the plasmid for cloning the insert. Sph1 restriction enzyme recognizes the 5'-GCATGC-3' and digests the DNA fragments optimally under standard temperature conditions of 37^oC in Thermofischer buffer B.NEB buffers can also be used for digesting the plasmid cloning the insert.

The restriction enzymes Pst1 and Sph1 sites are located in the multiple cloning site of the pUC 18 vector but the sites are located close to each other. This will hinder the effective digestion of the sequences due to close vicinity of the recognition sequences in the vector pUC 18 vector. Therefore sequential cloning is not leading to the generation of clones in the plasmid vector. The digestion sites of the two restriction enzymes used for cloning should be located at a proper distance in the multiple cloning site.

Different enzymes which have restriction sequences recognition sites far away in the multiple cloning sites should be used for cloning in the vector. The restriction ends of the insert to be ligated should be chosen accordingly. This will ensure the ligation of the insert in the pUC18 vector with efficient cloning of the vector. The sites which can be chosen for digestion which are far in the multiple cloning sites in the pUC 18 vector are HindIII, BamH1, Sal1, Kpn1 and EcoR1.

Various adapters and linkers with multiple cloning sites can be used for the ligation of the insert containing restriction digestion ends which are compatible with the enzyme sites in the multiple cloning site of the restriction vector pUC 18. The adaptor or linkers can be annealed to the 5' and 3 ' ends of the restriction digestion fragments which can effectively used for cloning.