Patent ID: 7485423
Filing Date: 2009-02-03
Classification: C12N,C12P,C12Q

Abstract:
1. A method for generating a double stranded nucleic acid molecule comprising steps of: providing a collection of oligonucleotides comprising annealing the collection of oligonucleotides such that at least one ladder complex in generated, each such ladder complex comprising one first terminal oligonucleotide, one second terminal oligonucleotide and at least two bridging oligonucleotides such that the first terminal oligonucleotide and the second terminal oligonucleotide are connected to each other through at least two overlapping complementary bridging oligonucleotides, wherein there is no gap between oligonucleotides that are annealed to a bridging oligonucleotide; providing at least a first and a second primer, wherein at least one of the first or second primers includes at least one terminator nucleotide that does not serve as a template for at least one polymerase; and amplifying the ladder complex by polymerase-mediated extension of the first and second amplification primers such that the polymerase does not copy the terminator nucleotide and the extension reaction produces a product molecule containing at least a first overhang; wherein the step of providing bridging oligonucleotides comprises providing at least a first and a second alternate bridging oligonucleotide, which first and second alternate bridging oligonucleotides are substantially similar to each other but differ from each other in at least a first variable nucleotide, which first variable nucleotide is located at the same relative position along the first and second alternate bridging oligonucleotides;