Ligation blunt ends

FIGURE 13.4 Blunt-end ligation using phage T4 DNA ligase, which catalyzes the ATP-dependent ligation of DNA molecules. AMP and PP are by-products. 

Another method is to enzymatically add suitable restriction sites to the blunt ends of the DNA. For this, a specially designed blunt-ended synthetic oligonucleotide species, known as a linker, is added and enzymatically ligated to the larger DNA. 

An important extension of this method is the procedure of blunt-end ligation high concentrations of the T4 ligase will catalyze the ligation of DNA fragments containing flush ends (equation 14.4). Flush ends may be generated by a number... 

Before blunt-end ligation of an insert, the vector needs to be dephosphorylated because resealing of the two ends of the linear vector to form a circular plasmid representing an intramolecular reaction is entropically favored, and thus faster (Chapter 2, Section 2.2.3), than the intermolecular insertion of the blunt-end frag-... 

Digest 1 pg of the pLXSN retroviral plasmid with 1 pi of the restriction enzyme Hpal in 10 pi of 1 x NEBuffer 4 in a 1.5 ml Eppendorf tube at 37°C overnight. This produces a linear 5.9 kb backbone fragment with blunt ends. Add 1 pi of calf intestinal alkaline phosphatase to the tube which removes 5 and 3 phosphoryl groups. This prevents the plasmid from self ligation. 

Restriction enzymes recognize specific recognition sequences and cut the DNA to leave cohesive ends or blunt ends. The ends of restricted DNA molecules can be joined together by ligation to create new recombinant DNA molecules. 

Alternatively, if the phosphatase treatment is to be used, the Eco Rl-cut vector can be used for blunt-ended ligation after repair of the EcoRI sticky ends. In practice, this latter procedure appears... 

If the enzyme gives 3 - or 5 -sticky ends which cannot be ligated directly into a site in M13mp7 (e.g. Hhal, Hinfl), the fragment termini can be converted into blunt ends ... 

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