Ladder assembly and system for generating diversity

A method for generating a double stranded nucleic acid molecule comprising the steps of:

• providing at least a first oligonucleotide and a second oligonucleotide, which first and second oligonucleotides are complementary to each other over at least a portion of their lengths;
• annealing said first and second oligonucleotides such that their complementary portions form a double stranded region;
• 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 annealed first and second oligonucleotides by polymerase-mediated extension of the first and second 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.

A method of generating a double-stranded nucleic acid molecule, the method comprising steps of:

• (a) providing a collection of oligonucleotides that, when hybridized with one another, form a nicked double-stranded nucleic acid molecule, which collection comprises:
• a set of top strand oligonucleotides comprising a terminal top strand oligonucleotides and at least one top strand bridging oligonucleotide;
• and
• a set of bottom strand oligonucleotides comprising a terminal bottom strand oligonucleotides and at least one bottom strand bridging oligonucleotides;
• wherein
• the terminal top strand oligonucleotide and the terminal bottom strand oligonucleotide are positioned at the opposite ends of the nicked double-stranded nucleic acid molecule;
• and wherein
• each oligonucleotide in a given strand set hybridizes with at least one oligonucleotide from the other strand set;
• and wherein
• each bridging oligonucleotide hybridizes with at least two oligonucleotides from the other strand set as binding partners, wherein the binding partners of any given bridging oligonucleotide are adjacent to one another in their strand of the nicked double-stranded nucleic acid molecule;
• (b) annealing the collection of oligonucleotides;
• (c) 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
• (d) amplifying the nicked double-stranded nucleic acid molecule 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 a first overhang.