Methods for rapid production of target double-stranded DNA sequences

Abstract

It has been previously disclosed that DNA segments can be made in massively parallel chemical synthesis operations on a common substrate followed by release of the segments from the substrate and assembly of the segments into target DNA molecules. Here it is taught that if the DNA primary constructs are sufficiently long and properly designed, that the copy numbers of the primary constructs can be multiplied as needed by a PCR process using as a template regions at the ends of the primary constructs. The end regions, called flanking regions, can also be designed so that they may be cleaved easily from the amplification products. The target double-stranded DNA can then be assembled from the cleaved fragments. Hundreds of thousands of oligonucleotides can be synthesized and assembled into many different individual genes by this process in a relatively quick and efficient process.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority from U.S. provisional patent application Ser. No. 60 / 578,195 filed Jun. 9, 2004.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with United States government support awarded by the following agency: DOD ARPA DAAD 19-02-2-0026. The United States has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]The invention pertains to the field of molecular biology and techniques and apparatus for the manufacture of DNA molecules of defined or desired sequences. The ex vivo manufacture of DNA molecules makes possible the use of those DNA molecules in vivo to synthesize any desired peptides, proteins or assemblies of proteins or combinations of nucleic acids, as may be desired, and to perform a large variety of genetic experiments in living organisms.[0004]In modern biotechnology it is common to create DNA sequences chemically, that is to say apart from any l...

AI Technical Summary

Benefits of technology

[0008]It is an object of the present invention to improve the process of the synthesis of DNA sequences specifically by massively parallel chemical synthesis of small segments followed by assembly of the small segments into larger DNA sequences.

[0009]It is an advantage of the method of the present invention in that it permits amplification of the number of copies of DNA synthesized to increase the amount of DNA available for DNA assemble procedures.

Problems solved by technology

While these methods permit the synthesis of long DNA segments, using the massively parallel synthesis capabilities of the MAS style of instrument, there is one drawback to the methodology described in the aforementioned published PCT applications.

While the amount of DNA is so small as to be difficult to measure physically, it is believed that approximately 20 picomoles of DNA are synthesized on a typical DNA microarray made by an MAS instrument.

Conversely, if the solution is diluted to a reasonable volume, then the DNA molecules are so dilute in the solution that the normal enzymes and other agents used for altering and manipulating DNA are difficult to use because of the dilution of the DNA.

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