Directed assembly of DNA molecules via simultaneous ligation and digestion.
DNA ligation is a routine laboratory practice, yet the yield of the desired product is often very low due to competing off-pathway reactions. The sensitivity of subsequent manipulations (e.g., selection via bacterial transformation) often obviates the need for a high yield of correctly ligated products. However the ability to perform high-yield, preparative-scale DNA ligations would benefit a number of downstream applications ranging from standard molecular cloning to biophysics and DNA computing. We describe here a ligation technique that specifically converts off-pathway ligation products back into substrate. We term this second-chance strategy enzymatic ligation assisted by nucleases (ELAN) and demonstrate the ordered assembly of four DNA fragments via simultaneous ligation and digestion in the presence of eight restriction enzymes. Use of ELAN increased the yield of the desired product by more than 30-fold.
Cost
Gregory J
GJ
Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA 94720, USA. gregcost@berkeley.edu
Cozzarelli
Nicholas R
NR
D004247:DNA / Q000138:chemical synthesis*; D004262:DNA Restriction Enzymes; D003851:Deoxyribonucleases; D005821:Genetic Techniques
D004247:DNA; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes