Compositions and methods for synthetic gene assembly

Abstract

Methods and compositions are provided for assembly of large nucleic acids where the assembled large nucleic acids lack internal sequence modifications made during the assembly process.

What is claimed is: 
     
         1 .- 38 . (canceled) 
     
     
         39 . A method for nucleic acid assembly, the method comprising:
 a) providing a predetermined nucleic acid sequence;   b) synthesizing a plurality of single-stranded nucleic acid fragments, wherein each single-stranded nucleic acid fragment encodes for a portion of the predetermined nucleic acid sequence and comprises at least one sticky end motif, wherein the sticky end motif comprises a sequence of 5-A(N x )T-3′ (SEQ ID NO.: 1) or 5′-G(N x )C-3′ (SEQ ID NO.: 16) in the predetermined nucleic acid sequence, wherein N is a nucleotide, wherein x is the number of nucleotides between nucleotides A and T or between G and C, and wherein x is 1 to 10, and wherein no more than two single-stranded nucleic acid fragments comprise the same sticky end sequence;   c) amplifying the plurality of single-stranded nucleic acid fragments to generate a plurality of double-stranded nucleic acid fragments, wherein the plurality of double-stranded nucleic acid fragments are modified from the predetermined nucleic acid sequence to comprise (i) a non-canonical base located at a 3′ end of the sticky end motif on a first strand and (ii) a first adaptor region located 5′ of the non-canonical base on the first strand, wherein the first adaptor region comprises a nicking enzyme recognition site;   d) creating sticky ends, wherein creating sticky ends comprises:
 i) treating the plurality of double-stranded fragments with a first nicking enzyme that nicks the non-canonical base on a first strand of each double-stranded fragment, and cleaving the nicked non-canonical base; and 
 ii) treating the plurality of double-stranded fragments with a second nicking enzyme, wherein the second nicking enzyme binds to the first strand at the nicking enzyme recognition site and cleaves a second strand of each double-stranded fragment, wherein a cleavage site for the nicking enzyme is located at a junction between the sticky end motif a sequence reverse complementary to the first adaptor region of the first strand; and 
   e) annealing the double-stranded nucleic acid fragments to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site.   
     
     
         40 . The method of  claim 39 , wherein x is 1, 2, 3, 4, 5, 6, 7, 8, or 9. 
     
     
         41 . The method of  claim 39 , wherein the predetermined nucleic acid sequence is 1 kb to 100 kb in length. 
     
     
         42 .- 44 . (canceled) 
     
     
         45 . The method of  claim 39 , wherein the plurality of single-stranded nucleic acid fragments are each at least 100 bases in length. 
     
     
         46 .- 48 . (canceled) 
     
     
         49 . The method of  claim 39 , wherein the sticky ends are at least 4 bases long. 
     
     
         50 . (canceled) 
     
     
         51 . The method of  claim 39 , wherein the non-canonical base is uracil, inosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, N-6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 1-methyladenine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, N6-adenine, N6-methyladenine, N,N-dimethyladenine, 8-bromoadenine, 7-methylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-ethyluracil, 5-propyluracil, 5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, 1-methylpseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-hydroxymethyluracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, 5-(2-bromovinyl)uracil, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, or 2,6-diaminopurine. 
     
     
         52 . The method of  claim 51 , wherein the non-canonical base is incorporated into the double-stranded nucleic acid by performing a nucleic acid extension reaction from a primer comprising the non-canonical nucleotide. 
     
     
         53 . The method of  claim 51 , wherein the non-canonical base is a uracil. 
     
     
         54 . The method of  claim 53 , wherein the uracil is in a deoxyuridine-deoxyadenosine base pair. 
     
     
         55 . The method of  claim 39 , wherein the nicking recognition site is a nicking endonuclease recognition site. 
     
     
         56 . The method of  claim 39 , wherein the distance between the non-canonical base and the nicking enzyme cleavage site is less than 12 base pairs. 
     
     
         57 . The method of  claim 39 , wherein the distance between the non-canonical base and the nicking enzyme cleavage site is at least 5 base pairs. 
     
     
         58 . The method of  claim 39 , wherein the first nicking enzyme comprises a base excision activity. 
     
     
         59 . The method of  claim 58 , wherein the first nicking enzyme comprises uracil-DNA glycosylase (UDG). 
     
     
         60 . The method of  claim 59 , wherein the first nicking enzyme comprises an AP endonuclease. 
     
     
         61 . The method of  claim 60 , wherein the first nicking enzyme comprises endonuclease VIII. 
     
     
         62 . The method of  claim 39 , wherein the second nicking enzyme comprises a nicking endonuclease. 
     
     
         63 . The method of  claim 62 , wherein the nicking endonuclease is selected from the list consisting of Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII, 
     
     
         64 . The method of  claim 39 , wherein each of the plurality of double-stranded nucleic acid fragments further comprises a two sticky ends. 
     
     
         65 . The method of  claim 64 , wherein each of the two sticky ends have a different sequence from each other. 
     
     
         66 . The method of  claim 39 , wherein the sticky ends comprises a 3′ overhang. 
     
     
         67 . The method of  claim 39 , further comprising ligating the annealed double-stranded nucleic acid fragments. 
     
     
         68 . The method of  claim 39 , wherein annealing comprises: thermocycling between a maximum and a minimum temperature, thereby generating a first overhang from a first double-stranded DNA fragment and a second overhang from a second double-stranded DNA fragment, wherein the first and the second overhangs are complimentary;
 hybridizing the first and second overhangs to each other; and   ligating.   
     
     
         69 . The method of  claim 39 , wherein the annealed double-stranded nucleic acid fragments comprise a 5′ outer adaptor region and a 3′ outer adaptor region. 
     
     
         70 . The method of  claim 39 , wherein at least two non-identical single-stranded nucleic acid fragments are synthesized. 
     
     
         71 . The method of  claim 70 , wherein at least 5 non-identical single-stranded nucleic acid fragments are synthesized. 
     
     
         72 . (canceled) 
     
     
         73 . The method of  claim 39 , where a polymerase lacking 3′ to 5′ proofreading activity is added during the amplification step. 
     
     
         74 . The method of  claim 73 , wherein the polymerase is a Family A polymerase. 
     
     
         75 . (canceled) 
     
     
         76 . The method of  claim 39 , wherein the amplified plurality of single-stranded nucleic acid fragments are not naturally occurring. 
     
     
         77 . (canceled) 
     
     
         78 . A DNA library comprising:
 n DNA fragments, each comprising a first strand and a second strand, each of the n DNA fragments comprising, in order 5′ to 3′: a first nicking endonuclease recognition site, a first sticky end motif, a template region, a second sticky end motif, and a second nicking endonuclease recognition site, wherein the first sticky end motif comprises a sequence of 5′-A (N x ) i,1 U-3′ (SEQ ID NO.: 13) in the first strand; and wherein the second sticky end motif comprises a sequence of 5′-A (N x ) i,2 U-3′(SEQ ID NO.: 14) in the second strand; wherein N x  denotes x nucleosides; wherein (N x ) i,2  is reverse complementary to (N x ) i,1  and different from every other N x  found in any sticky end motif sequence within the fragment library, wherein the first nicking endonuclease recognition site in each of the DNA fragments are positioned such that there is a corresponding cleavage site immediately 3′ of the sticky end motif in the second strand, and wherein the second nicking endonuclease recognition sites are positioned such that there is a corresponding cleavage site immediately 3′ of the second sticky end motif in the first strand.   
     
     
         79 .- 89 . (canceled)