Methods for assembling dna molecules

Abstract

The invention provides compositions and methods for assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides. The oligonucleotides of a pair have a portion of the desired sequence, and an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair, and, when arranged in order, they have at least a portion of the desired sequence. The oligonucleotides also have a 3′ or a 5′ primer binding sequence having a binding site for a primer. The oligonucleotides that correspond to the end oligonucleotides of the desired sequence also have a universal 3′ flanking sequence and a universal 5′ flanking sequence, respectively.

What is claimed is: 
     
         1 . A method of removing primer binding sequences from a dsDNA molecule comprising:
 contacting a dsDNA molecule with an enzyme mixture comprising an enzyme that specifically cleaves primer binding sequences at a non-standard base;
 wherein the dsDNA molecule comprises a 3′ and a 5′ primer binding sequence having at least one non-standard base; 
 wherein the dsDNA molecule further comprises a universal 3′ flanking sequence and a universal 5′ flanking sequence comprised to the inside of the 3′ and 5′ primer binding sequences, respectively; 
   to thereby remove the primer binding sequences from the dsDNA molecule.   
     
     
         2 . The method of  claim 1  wherein the non-standard base is deoxyuridine. 
     
     
         3 . The method of  claim 1  wherein the enzyme that specifically cleaves primer binding sequences at a non-standard base is uracil DNA-glycosylase (UDG). 
     
     
         4 . The method of  claim 3  wherein the enzyme mixture further comprises endonuclease VIII and exonuclease T. 
     
     
         5 . The method of  claim 1  wherein the primer binding sequences are 6-30 nucleotides in length. 
     
     
         6 . The method of  claim 5  wherein the 3′ and 5′ primer binding sequences are present on the 3′ and 5′ ends, respectively, of the dsDNA molecule. 
     
     
         7 . The method of  claim 1  wherein the dsDNA molecule does not comprise an expressed sequence tag. 
     
     
         8 . The method of  claim 1  wherein the non-standard base is selected from the group consisting of: 3-nitropyrrole and 5-nitroindole. 
     
     
         9 . A composition comprising:
 a DNA polymerase, dNTPs, and a plurality of oligonucleotides formed into couplets, wherein each couplet comprises an internal sequence that comprises a portion of the desired nucleic acid sequence;
 wherein when the plurality of couplets is arranged in order, adjacent to each other and according to their internal sequences they comprise at least a portion of a desired nucleic acid sequence, and each couplet further comprises a 3′ or a 5′ primer binding sequence, and each couplet contains a sequence that overlaps and is complementary to a portion of a sequence from an adjacent couplet; and
 wherein the desired nucleic acid sequence has a 3′ end and a 5′ end, and the couplets that comprise the 3′ and 5′ ends of the desired sequence further comprise a universal 3′ flanking sequence and a universal 5′ flanking sequence, respectively. 
 
   
     
     
         10 . The composition of  claim 9  comprised in a single container and further comprising an effective amount of a preservative. 
     
     
         11 . The composition of  claim 10  wherein the couplets comprise at least 50% of the oligonucleotides in the mixture, and the couplets overlap at least 33% of their sequences. 
     
     
         12 . The composition of  claim 9  wherein the universal 3′ and 5′ flanking sequences are comprised to the inside of the 3′ and 5′ primer binding sequences, respectively. 
     
     
         13 . The composition of  claim 9  wherein the primer binding sequences comprise at least one non-standard base. 
     
     
         14 . The composition of  claim 13  wherein the non-standard base is deoxyuradine. 
     
     
         15 . The composition of  claim 9  wherein the 3′ and 5′ primer binding sequences on an oligonucleotide couplet are not complementary to each other. 
     
     
         16 . The composition of  claim 9  wherein the oligonucleotides comprise from 60 to 100 nucleotides and the primer binding sequences comprise from 8 to 30 nucleotides. 
     
     
         17 . The composition of  claim 9  wherein the 3′ and 5′ primer binding sequences do not comprise a restriction site for a restriction enzyme. 
     
     
         18 . The composition of  claim 9  further comprising uracil DNA glycosylase, endonuclease VIII, and exonuclease T.