| US 7,579,146 B2 | ||
| Nucleic acid cloning | ||
| Kevin A. Jarrell, Lincoln, Mass. (US); Vincent W. Coljee, Gentofte (Denmark); William Donahue, Quincy, Mass. (US); and Svetlana Mikheeva, Allston, Mass. (US) | ||
| Assigned to Trustees of Boston University, Boston, Mass. (US) | ||
| Filed on Jun. 29, 2001, as Appl. No. 9/897,712. | ||
| Application 09/897712 is a continuation in part of application No. PCT/US00/00189, filed on Jan. 05, 2000. | ||
| Application PCT/US00/00189 is a continuation in part of application No. 09/225990, filed on Jan. 05, 1999, granted, now 6,358,712. | ||
| Claims priority of provisional application 60/114909, filed on Jan. 05, 1999. | ||
| Prior Publication US 2005/0191623 A1, Sep. 01, 2005 | ||
| This patent is subject to a terminal disclaimer. | ||
| Int. Cl. C12Q 1/68 (2006.01); C07H 21/04 (2006.01); C12N 15/00 (2006.01) | ||
| U.S. Cl. 435—6 [435/320.1; 435/254.2; 536/24.3; 536/24.33; 536/24.5] | 47 Claims |
| 1. A method of generating a nucleic acid molecule, the method comprising steps of:
providing a first precursor nucleic acid molecule including a first double stranded portion flanked by first and second single-stranded
overhangs;
providing a second precursor nucleic acid molecule including a second double-stranded portion flanked by third and fourth
single-stranded overhangs, the third single-stranded overhang being complementary with the second overhang of the first precursor
nucleic acid molecule;
providing a third precursor nucleic acid molecule including a third double-stranded portion flanked by fifth and sixth single-stranded
overhangs, the fifth overhang being complementary with the fourth overhang of the second precursor nucleic acid molecule and
the sixth overhang being complementary with the first overhang of the first nucleic acid molecule; and
combining the first, second, and third precursor nucleic acid molecules together in a single reaction under conditions that
allow for hybridization of the complementary overhangs, so that a new nucleic acid molecule is generated;
wherein at least one of the precursor nucleic acid molecules is generated by extending first and second primers in a polymerase-mediated
extension reaction, at least one of the first and second primers including at least one base-containing residue, which base-containing
residue is not used as a template by the polymerase employed in the polymerase-mediated extension reaction, but which is capable
of being used as a template by a different polymerase.
|