Blunt-End Ligation

 Blunt-end ligation is covalently joining two or more double-stranded DNA fragments with flush ends by the enzyme DNA Ligase (1). Many restriction enzymes produce blunt ends, when they cleave both polynucleotide chains at the same site, and some cohesive ends are converted to blunt ends by the fill-in reaction. Blunt end ligation is a much less efficient reaction than ligation of DNA fragments with cohesive ends. One technique for overcoming the low efficiency of blunt-end ligation is ligating a short oligonucleotide linker fragment to the DNA ends that are to be joined (2.( Because a large molar concentration of the linker can be added to the reaction, its ligation to the ends of the DNA occurs more readily than joining of the ends themselves. Linkers are designed to contain a unique restriction site so that the ligation mixture is digested with the corresponding enzyme to create cohesive ends, which can be joined by conventional ligation.

 The efficiency of blunt-end ligation is greatly increased by carrying out the reaction in the presence of 15% polyethylene glycol, 6,000 MW (PEG), although only linear molecules are produced in this reaction. Circular plasmids are required for efficient transformation of Escherichia coli. An efficient way of producing circular molecules from the linear product of blunt-end ligation is including a bacteriophage P1 lox recombination site in one of the molecules being ligated (3). After ligation, the DNA is treated with Cre recombinase, which by recombination between the lox sites produces a circular molecule from a linear molecule that contains two lox sites. This type of molecule is created when the molecule without a lox site is ligated to two molecules with a lox site, one at each end.

It is possible to amplify the products of a low-efficiency ligation containing the desired insert by PCR, using primers that bind to vector DNA on each side of the cloning site. The PCR product of the expected size is purified and recloned. This results in transformants from ligations that give no positive colonies on direct transformation (4).

References

1.T. Maniatis, E. F. Fritsch, and J. Sambrook (1982) Molecular Cloning; a Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY. 

2.G. J. Bhat, M. J. Lodeg, P. J. Myler, and K. D. Stuart (1991) Nucleic Acids Res. 19, 398. 

3.A. C. Boyd (1993) Nucleic Acids Res. 21, 817–821. 

4. H. W. Son and E. Lolis (1995) BioTechniques 18, 644–650.