Patent ID: 6143527
Filing Date: 2000-11-07
Classification: A61K,C07K,C12N

Abstract:
A method of synthesizing a composite nucleic acid molecule comprising the steps of:a) heating a first double-stranded nucleic acid molecule and a second double-stranded nucleic acid molecule at a denaturing temperature;b) forming a mixture comprising said first denatured nucleic acid molecule, said second denatured nucleic acid molecule, a bridging oligonucleotide and a thermostable ligase,wherein said denatured nucleic acid molecules each consist of a first strand and a second strand complementary thereto, andwherein said bridging oligonucleotide comprises, from 5' to 3', a 10-40 nucleotide sequence which is complementary to a nucleotide sequence on the 5' end of the first strand of said first nucleic acid molecule and a 10-40 nucleotide sequence which is complementary to a nucleotide sequence on the 3' end of the first strand of said second nucleic acid molecule;c) maintaining said mixture at an annealing temperature, wherein said bridging oligonucleotide hybridizes to sequences on the first strands of both said first and second nucleic acid molecule, thereby orienting said first and second molecules adjacent to each other, and wherein said thermostable ligase ligates either said 3' end of said first nucleic acid molecule to said 5' end of said second nucleic acid molecule or said 5' end of said first nucleic acid molecule to said 3' end of said second nucleic acid molecule to form a first strand of said composite nucleic acid molecule hybridized to said bridging oligonucleotide;d) maintaining said mixture at a denaturing temperature, wherein said bridging oligonucleotide dissociates from said first strand of said composite nucleic acid molecule; ande) maintaining said mixture at an annealing temperature, wherein said first strand of said composite nucleic acid molecule hybridizes to complementary strands of said first nucleic acid molecules and said second nucleic acid molecule, wherein said thermostable ligase ligates the end of said complementary strand of said first nucleic acid molecules to the end of said complementary strand of second nucleic acid molecule to form a second strand of said composite nucleic acid molecule.