Methods for seamless nucleic acid assembly

Abstract

Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Such methods, systems, and compositions for seamless nucleic acid assembly include those for in vitro recombination cloning, single-stranded hierarchal DNA assembly, or overlap extension PCR without primer removal.

What we claim is: 
     
         1 . A method for nucleic acid assembly, comprising:
 (a) providing at least one double stranded nucleic acid comprising in 5′ to 3′ order: a 5′ flanking adapter sequence, a first homology sequence, an insert sequence, a second homology sequence, and a 3′ flanking adapter sequence,
 wherein the first homology sequence and the second homology sequence each comprises about 20 to about 100 base pairs in length; 
   (b) providing a vector comprising the first homology sequence and the second homology sequence; and   (c) mixing the at least one double stranded nucleic acid and the vector with a bacterial lysate.   
     
     
         2 . The method of  claim 1 , wherein the bacterial lysate comprises a nuclease or a recombinase. 
     
     
         3 . The method of  claim 1 , wherein the bacterial lysate comprises a nuclease and a recombinase. 
     
     
         4 . The method of  claim 1 , wherein the first homology sequence and the second homology sequence each comprises about 20 base pairs. 
     
     
         5 . The method of  claim 1 , wherein the first homology sequence and the second homology sequence each comprises about 41 base pairs. 
     
     
         6 . The method of  claim 1 , wherein the first homology sequence and the second homology sequence each comprises about 100 base pairs. 
     
     
         7 . The method of  claim 1 , wherein the first homology sequence or the second homology sequence is flanked by the 5′ flanking adapter sequence and the 3′ flanking adapter sequence. 
     
     
         8 . The method of  claim 1 , wherein a percentage of correct assembly is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. 
     
     
         9 . A method for nucleic acid synthesis and assembly, comprising:
 (a) de novo synthesizing a plurality of polynucleotides, wherein each polynucleotide comprises a first homology region that comprises in 5′ to 3′ order: a 5′ flanking adapter sequence, a first homology sequence, an insert sequence, a second homology sequence, and a 3′ flanking adapter sequence, wherein the first homology sequence and the second homology sequence each comprises about 20 to about 100 base pairs in length, and wherein each polynucleotide comprises a homology sequence identical to that of another polynucleotide of the plurality of polynucleotides; and   (b) mixing of the plurality of polynucleotides with a bacterial lysate to processively form nucleic acids each having a predetermined sequence.   
     
     
         10 . The method of  claim 9 , wherein the bacterial lysate comprises a nuclease or a recombinase. 
     
     
         11 . The method of  claim 9 , wherein the bacterial lysate comprises a nuclease and a recombinase. 
     
     
         12 . The method of  claim 9 , wherein the first homology sequence and the second homology sequence each comprises about 20 base pairs. 
     
     
         13 . The method of  claim 9 , wherein the first homology sequence and the second homology sequence each comprises about 41 base pairs. 
     
     
         14 . The method of  claim 9 , wherein the first homology sequence and the second homology sequence each comprises about 100 base pairs. 
     
     
         15 . The method of  claim 9 , wherein the first homology sequence or the second homology sequence is flanked by the 5′ flanking adapter sequence and the 3′ flanking adapter sequence. 
     
     
         16 . The method of  claim 9 , wherein a percentage of correct assembly is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. 
     
     
         17 . A method for nucleic acid assembly, comprising:
 (a) providing a plurality of double stranded nucleic acids;   (b) annealing a uracil at a 5′ end and a 3′ end of at least two of the double stranded nucleic acids;   (c) amplifying the double stranded nucleic acids using a uracil compatible polymerase to form amplification products;   (d) mixing the amplification products from step (c) to form a mixture; and   (e) amplifying the mixture from step (d) using a uracil incompatible polymerase to generate a single-stranded nucleic acid.   
     
     
         18 . The method of  claim 17 , wherein the uracil incompatible polymerase is a DNA polymerase. 
     
     
         19 . The method of  claim 17 , wherein the homology sequence comprises about 20 to about 100 base pairs. 
     
     
         20 . The method of  claim 17 , wherein a percentage of correct assembly is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.