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Nucleic Acids Research, 1979, Vol. 6, No. 3 1013-1024
© 1979

Articles

Addition of oligonucleotides to the 5'-terminus of DNA by T4 RNA ligase

N.Patrick Higgins1,2, Adam P. Geballe1,3 and Nicholas R. Cozzarelli1,4

1Departments of Biochemistry, University of Chicago Chicago, IL 60637 USA 4Biophysics and Theoretical Biology, University of Chicago Chicago, IL 60637 USA

Received December 28, 1978.

Bacteriophage T4-induced RNA ligase catalyzes the controlled template-independent addition of RNA to the 5'-phosphoryl end of large DNA molecules. Restriction enzyme-generated fragments of ColEl DNA with completely base-paired or cohesive ends and linear single-stranded øX174 viral DNA were all good substrates. DNA molecules from 10 to 6000 nucleotides long were quantitatively joined in an hour to a number of different RNA homopolyers. The most efficient of these was A(pA)5; I(pI)5 and C(pC)5 were also utilized while U(pU)5 was not. The optimum ribohomopolymer length was six nucleotides. Joining of ribohomopolymers between 10 and 20 nucleotides long occurred at approximately 1/2 the maximal rate and a trimer was the shortest substrate. Thus RNA ligase provides a method for generating extensions of predetermined length and base composition at the 5'-end of large DNA molecules that complements the available procedures for extending the 3'-hydroxyl terminus of DNA.

2Present address: Department of Biochemistry, University of Wyoming, Laramie, WY 82071

3Present address: Department of Medicine, University of Chicago, Chicago, IL 60637, USA.


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O. R. Faridani, G. M. McInerney, K. Gradin, and L. Good
Specific ligation to double-stranded RNA for analysis of cellular RNA::RNA interactions
Nucleic Acids Res., September 1, 2008; 36(16): e99 - e99.
[Abstract] [Full Text] [PDF]


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