| 摘要 |
A method is described for the simultaneous assembly of two or more primer extension generated, double stranded DNA fragments, into a construct, comprising the steps of: 1) designing the primers such that the fragments subsequently produced by PCR, have 3' termini of 15 nucleotides or more being complementary to a second and third fragment, both having 3' termini of 15 nucleotides or more, complementary to each terminal of first said fragment; 2) providing in each of said primers and subsequently in each of said fragments, at least one dU residue in place of at least one dT residue, being positioned at least 15 nucleotides from the 5' end of said primer and subsequent fragments; 3) adding to the mixture of fragments the enzyme UDG (Uracil DNA Glycosylase) and a compound such as N,N,dimethylethylenedimethylamine, to a-purinate and generate single strand nicks at said dU residues, respectively, thereby to generate a 3' single strand overhang on each fragment to be joined thereto, being complementary to the other 3' single strand overhangs of second, third or more aforementioned fragments, and 4) allowing for all fragments thus generated to anneal into a single and stable DNA construct.
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