High-fidelity Production of Long Nucleic Acid Molecules

Applications

Applications for this technology include vaccine trials, gene therapy, and bacterial engineering.

Problem Addressed

Presently, there are two major issues with the current technology: Scaling and Errors. 

Firstly, scaling. As the size of the desired sequence grows, the production time and costs increase. Ideally, smaller amounts of reagents, shorter cycle times for oligonucleotide synthesis, a greatly improved parallelization of the synthesis process used to provide the oligonucleotide, and an improved process for the assembly of the oligonucleotides into larger molecules would 

Considering errors, the technology available today produces many errors in DNA synthesis.  This is primarily the result of producing larger DNA sequences.  The expected per base error rates (1-2% per coupling step) essentially guarantee that conventional methods will result in sequences with many errors. Thus to produce larger DNA sequences, the molecule is not synthesized as a single long piece. Instead, current methods involve combining many shorter oligonucleotides to build the larger desired sequence.

Technology

This invention provides methods for the error-free production of long nucleic acid molecules with precise user control over sequence content. Long error-free nucleic acid molecules can be generated in parallel from oligonucleotides immobilized on a surface, such as on an oligonucleotide microarray. The movement of the growing nucleic acid can be controlled through the stepwise repositioning of the growing molecule. Stepwise repositioning refers to the position of the growing molecules as it interacts with the oligonucleotides immobilized on the surface.

Detecting and correcting errors that arise in the process of constructing long nucleic acid molecules are also covered in this invention. A force-feedback system using magnetic or optical tweezers is implemented, either separately or in combination. Using this system, double or single-stranded DNA is grown off a solid-phase support. The solid-phase support is magnetic in nature and is held in a fixed equilibrium position by applying an electric field and magnetic field  gradient created by the magnetic tweezers that oppose the electrophoretic force. As oligonucleotides are annealed to the growing strand, the negatively charged phosphate backbone adds charge to the bead-strand complex. However, the added oligonucleotide adds essentially no mass or surface area to the complex.

Advantages

  • Creation of long error-free nucleic acid molecules
  • Low production time and cost