Ladder assembly and system for generating diversity

Abstract

The present invention provides novel methods of generating a nucleic acid molecule. In certain embodiments, a double stranded nucleic acid chunk is generated from a ladder complex comprising partially complementary oligonucleotides, which chunk is combined with a nucleic acid acceptor molecule. In certain embodiments, the assembled chunk/nucleic acid acceptor molecule complex may be propagated in vivo or in vitro. The present invention also provides improved systems for generating a plurality of nucleic acid molecules that differ at one or more nucleotide positions. In certain embodiments, the plurality of nucleic acid molecules encodes a polypeptide or portion of a polypeptide.

1 . A method for generating a double stranded nucleic acid molecule comprising steps of:
 providing a collection of oligonucleotides comprising
 at least one first terminal oligonucleotide, 
 at least one second terminal oligonucleotide, and 
 at least two bridging oligonucleotides; 
 wherein the first and second terminal oligonucleotides are characterized in that each terminal oligonucleotide anneals with only one other oligonucleotide in the collection and each bridging oligonucleotide is characterized in that each bridging oligonucleotide anneals with at least two other oligonucleotides in the collection; 
   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;   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.   
   
   
       2 . The method of  claim 1  wherein in the step of annealing, there is no gap between the oligonucleotides that are annealed to a bridging oligonucleotide. 
   
   
       3 . The method of  claim 1  wherein in the step of annealing, at least one gap is created between the two oligonucleotides that are annealed to a given bridging oligonucleotide. 
   
   
       4 . (canceled) 
   
   
       5 . (canceled) 
   
   
       6 . (canceled) 
   
   
       7 . The method of  claim 1 , wherein the first overhang of each nucleic acid molecule of the collection is substantially similar;
 the method further comprising providing at least one second double stranded DNA molecule containing a second overhang, which second overhang is at least partly complementary to the first overhang; and   combining the collection of double stranded DNA molecules and the second DNA molecules under conditions that allow hybridization of the first and second overhangs.   
   
   
       8 . The method of  claim 7 , wherein the step of combining comprises combining individual double stranded molecules of the collection with the second double stranded DNA molecule in separate combination reactions. 
   
   
       9 . The method of  claim 7 , wherein the step of combining comprises combining more than one double stranded molecule of the collection with the second double stranded DNA molecule simultaneously in a single combination reaction, such that only one double stranded molecule of the collection is combined with a single double stranded DNA molecule. 
   
   
       10 . The method of  claim 1 , wherein a portion of each bridging oligonucleotide is exactly complementary to either the portion of the terminal oligonucleotide or to the portion of the other bridging oligonucleotide to which it anneals. 
   
   
       11 . The method of  claim 1 , wherein a portion of each bridging oligonucleotide is exactly complementary to the portion of at least one other bridging oligonucleotide to which it anneals. 
   
   
       12 . The method of  claim 2 , 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;
 and wherein the step of providing bridging oligonucleotides further comprises providing at least a third and a fourth alternate bridging oligonucleotide, which third and fourth alternate bridging oligonucleotides are substantially similar to each other but differ from each other in at least a second variable nucleotide, which second variable nucleotide is located at the same relative position along the third and fourth alternate bridging oligonucleotides;   wherein the differing first variable nucleotide of the first alternate bridging oligonucleotide is complementary to the differing second variable nucleotide of the third alternate bridging oligonucleotide;   and wherein the differing first variable nucleotide of the second alternate bridging oligonucleotide is complementary to the differing second variable nucleotide of the fourth alternate bridging oligonucleotide.   
   
   
       13 . (canceled) 
   
   
       14 . (canceled) 
   
   
       15 . (canceled) 
   
   
       16 . (canceled) 
   
   
       17 . The method of  claim 3 , wherein the step of providing bridging oligonucleotides comprises providing at least two alternate bridging oligonucleotides, each alternate bridging oligonucleotide being substantially similar but differing from each other in at least one nucleotide located at the same relative position along the alternate bridging oligonucleotides, which position is located such that neither of the two oligonucleotides that anneal to the alternate bridging oligonucleotide and form said gap anneal with the alternate bridging oligonucleotide at said position. 
   
   
       18 . (canceled) 
   
   
       19 . (canceled) 
   
   
       20 . (canceled) 
   
   
       21 . The method of  claim 1  wherein the generated nucleic acid molecule encodes a polypeptide comprising a functional domain of a protein. 
   
   
       22 . (canceled) 
   
   
       23 . 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.   
   
   
       24 . The method of  claim 23  further comprising:
 providing at least a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide, wherein the first and second oligonucleotides are complementary to each other over at least a portion of their lengths, and wherein the third oligonucleotide is at least partly complementary to at least a portion of the second oligonucleotide that is different from the portion of the second oligonucleotide that is complementary to the first oligonucleotide;   annealing the first, second and third oligonucleotides such that the first and third oligonucleotides hybridize with the second 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 annealed oligonucleotide triplex 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.   
   
   
       25 . The method of  claim 24 , wherein in the step of annealing, there is no gap between the first and third oligonucleotides that are annealed to the second oligonucleotide. 
   
   
       26 . The method of  claim 24 , wherein in the step of annealing, there is a gap between the first and third oligonucleotides that are annealed to the second oligonucleotide. 
   
   
       27 . 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.   
   
   
       28 . The method of  claim 27 , wherein at least one oligonucleotide comprises a plurality of oligonucleotides of different sequence. 
   
   
       29 . The method of  claim 27 , wherein at least one bridging oligonucleotide comprises a plurality of oligonucleotides of different sequence. 
   
   
       30 . The method of  claim 29  wherein at least one binding partner of the bridging oligonucleotide also comprises a plurality of oligonucleotides of different sequence that are complementary with different members of the bridging oligonucleotide plurality. 
   
   
       31 . The method of  claim 29  wherein each member of the plurality of bridging oligonucleotides has complementary binding partners.