Patent ID: 7439021
Filing Date: 2008-10-21
Classification: C12N,C12P

Abstract:
1. A method of generating a recombinant double-stranded DNA molecule, the method comprising the steps of: (a) providing a template nucleic acid molecule; (b) contacting the template nucleic acid molecule with a first primer that hybridizes thereto, the first primer including at least one termination residue characterized in that the termination residue serves as a replication template for a first DNA polymerase, but does not serve as a replication template for a second DNA polymerase that is different from the first DNA polymerase; (c) extending the first primer in a first extension reaction to generate a complementary nucleic acid strand; (d) contacting the complementary nucleic acid strand with a second primer that hybridizes thereto; (e) extending the second primer with said second DNA polymerase in a second extension reaction, so that the termination residue is not copied in the second extension reaction and the second extension reaction produces a first double-stranded DNA molecule containing a first 5′ overhang; (f) providing a second double-stranded DNA molecule containing a second overhang at least partly complementary with the first overhang; (g) forming an annealed DNA molecule by combining the first and second double-stranded DNA molecules under conditions that allow hybridization of the first and second overhangs; and (h) generating the recombinant double-stranded DNA molecule by exposing the annealed double-stranded DNA molecule to said first polymerase so that the termination residue is copied into said recombinant double-stranded DNA molecule.