Methods for High Fidelity Production of Long Nucleic Acid Molecules with Error Control

Abstract

A method for synthesizing a nucleic acid having a desired sequence and length comprises providing a solid support having an immobilized nucleic acid, performing a nucleic acid addition reaction to elongate the immobilized nucleic acid by adding a nucleotide or an oligonucleotide attached to a protecting group to the nucleic acid, determining whether the nucleotide or the oligonucleotide is added to the nucleic acid, removing the protecting group, and continuing until the immobilized nucleic acid has a desired sequence and length.

1 . A method for generating a nucleic acid molecule with precise user control, the method comprising:
 a) providing a nucleic acid immobilized on a surface;   b) providing a nucleic acid molecule attached to a protecting group;   c) hybridizing said immobilized nucleic acid with said nucleic acid molecule attached to a protecting group, thereby elongating said immobilized nucleic acid;   d) dissociating said elongated immobilized nucleic acid from said protecting group; and   e) repeating steps (b) to (d) until the elongated immobilized nucleic acid has a desired sequence and length.   
     
     
         2 . The method of  claim 1 , wherein said protecting group comprises proteins, carbohydrates; diphosphates, phosphate derivatives, nucleotides, oligonucleotides, or combinations thereof. 
     
     
         3 . The method of  claim 1 , wherein the removal of said protecting group is accomplished by the application of heat, proteases, phosphotases, restriction enzymes, or combinations thereof. 
     
     
         4 . The method of  claim 1 , wherein the surface is a bead or particle and further comprising the step of:
 f) after the step of hybridizing, determining whether the nucleic acid molecule attached to a protecting group is added to the immobilized nucleic acid by detecting whether an increase in electrophoretic force applied the surface is required to keep the surface at equilibrium when a steady electric field and a magnetic field gradient are applied to the surface, wherein the required increase in electrophoretic force applied to the surface is caused by adding the nucleic acid molecule attached to a protecting group to the immobilized nucleic acid.   
     
     
         5 . The method of  claim 1 , further comprising the step of:
 before the step of disassociating, removing any mismatched base pairs by applying heat to the elongated immobilized nucleic acid in order to cause dehybridization of any mismatched base pairs from the elongated immobilized nucleic acid.   
     
     
         6 . The method of  claim 4 , further comprising the step of:
 before the step of disassociating, removing any mismatched base pairs by applying heat to the elongated immobilized nucleic acid in order to cause dehybridization of any mismatched base pairs from the elongated immobilized nucleic acid.