Programmable oligonucleotide synthesis

Abstract

The invention relates to methods and devices for preparing synthetic nucleic acids.

What is claimed is: 
     
         1 . A method of preparation of synthetic nucleic acids, in particular of nucleic acid double strands, comprising the steps:
 (a) preparation of a multiplicity of different nucleic acid fragments by solidphase synthesis, preferably at various positions of a common solid support, and   (b) joining together of at least two nucleic acid fragments from (a) by binding to one another or by covalent linkage,   
       characterized in that the method comprises amplification steps in which the synthesized nucleic acid fragments or/and optionally double-stranded intermediates formed therefrom are submitted to amplification, e.g., PCR. 
     
     
         2 . A method for isolating an individual nucleic acid having the desired sequence from a mixture of nucleic acids, comprising:
 (a) monoclonizing a mixture of nucleic acids,   (b) parallel sequencing the monoclonized nucleic acids of step (a),   (c) localizing a nucleic acid with the desired sequence in the nucleic acids of step (b),   (d) isolating the nucleic acid from step (c), and   (e) optionally further processing of isolated nucleic acid obtained in step wherein the isolation takes place by at least one of steps (i) to (iv):
 (i) isolating one or more beads on which said nucleic acid molecule is immobilized, 
 (ii) cleaving off the nucleic acid molecule from a support on which said nucleic acid molecule is immobilized, 
 (iii) selectively amplifying the nucleic acid molecule by spatially-resolved addition of PCR reagents, and 
 (iv) eluting the nucleic acid molecule by laser capture method. 
   
     
     
         3 . A method for quality control of assembled nucleic acid molecules, said method comprising:
 (a) providing a mixture of assembled nucleic acid molecules suspected of containing sequence defects,   (b) amplifying the mixture of (a) to receive a clonal population thereof,   (c) immobilizing the clonal population of (b) on a support,   (d) parallel sequencing the clonal population immobilized according to (c),   (e) localizing at least one clonal nucleic acid molecule that meets a predefined criterion, and   (f) obtaining the at least one clonal nucleic acid molecule of (e).   
     
     
         4 . The method of  claim 3 , wherein the assembled sequences provided in step (a) are built each from one or more oligonucleotides. 
     
     
         5 . The method of  claim 3 , wherein the assembled sequences represent genes, gene clusters, chromosomes, genomes or fragments thereof 
     
     
         6 . The method of  claim 3 , wherein amplification in step (b) comprises an emulsion-based PCR. 
     
     
         7 . The method of  claim 3 , wherein immobilizing the clonal population according to step (c) comprises immobilization on beads. 
     
     
         8 . The method of  claim 3 , wherein the predetermined criterion in step (e) is the desired correct sequence. 
     
     
         9 . The method of  claim 3 , wherein the obtaining the clonal nucleic acid molecule of (f) comprises:
 (i) detachment of the clonal nucleic acid molecule from the support,   (ii) copying without disrupting the covalent linkage to the support,   (iii) laser capture, or/and   (iv) spatially resolved PCR.   
     
     
         10 . Use of a quality controlled DNA target sequence obtained by the method of  claim 1  for assembly of a gene, gene cluster, chromosome, genome or a fragment thereof 
     
     
         11 . A method of assembly of nucleic acid molecules comprising:
 (a) providing single-stranded nucleic acid molecules to be assembled into a target nucleic acid molecule,   (b) assembling by hybridization complementary regions of the single-strands nucleic acids of (a), and   (c) controlling the quality of the mixture of assembled nucleic acid molecules obtained in step (b) by the method of  claim 3 .   
     
     
         12 . The method of  claim 11 , wherein step (b) results in a gene, gene cluster, chromosome, genome or a fragment thereof.