Patent Document ID: 20100035768
Application ID: 12371543
Patent Flag: 0

Claim One:
1. An in vitro method of joining a set of two or more double-stranded (ds) or single-stranded (ss) DNA molecules, wherein adjacent DNA molecules to be joined contain overlapping sequences at their termini, said method comprising contacting in vitro the two or more DNA molecules in a single vessel with (a) an isolated non-thermostable 5′ to 3′ exonuclease that lacks 3′ exonuclease activity, (b) a crowding agent, (c) an isolated thermostable non-strand-displacing DNA polymerase with 3′ exonuclease activity, or a mixture of said DNA polymerase with a second DNA polymerase that lacks 3′ exonuclease activity, (d) an isolated thermostable ligase, (e) a mixture of dNTPs, and (f) a suitable buffer, under conditions that are effective for joining the two or more DNA molecules to form a first assembled dsDNA molecule in a one-step reaction.