Wisconsin Alumni Research Foundation

Kathryn E Richmond

Michael Richard Sussman

Peter Jeremy Belshaw

James Howard Kaysen

Brock Binkowski

Francesco Cerrina

Franco Cerrina

Matt J Rodesch

A method of rapidly producing a double-stranded target DNA is disclosed. The method includes the step of producing multiple single stranded primary DNA constructs having (a) partially overlapping and complementary internal regions that together define the target DNA, and (b) flanking regions on either side of the internal regions containing a PCR primer recognition site and a restriction enzyme recognition site. The primary DNA constructs are amplified to form a pool of double-stranded primary constructs, and a restriction enzyme is used to cleave off the flanking regions. The target double-stranded DNA is then assembled from the cleaved fragments. Hundreds of thousands of oligonucleotides can be synthesized and quickly and efficiently assembled into many different individual double-stranded DNA target sequences using this method.

Methods for rapid production of double-stranded target DNA

A method is disclosed for the direct synthesis of double stranded DNA molecules of a variety of sizes and with any desired sequence. The DNA molecule to be synthesis is logically broken up into smaller overlapping DNA segments. A maskless microarray synthesizer is used to make a DNA microarray on a substrate in which each element or feature of the array is populated by DNA of a one of the overlapping DNA segments. The complement of each segment is also made in the microarray. The DNA segments are released from the substrate and held under conditions favoring hybridization of DNA, under which conditions the segments will hybridize to form duplexes. The duplexes are then separated using a DNA binding agent which binds to improperly formed DNA helixes to remove errors from the set of DNA molecules. The segments can then be hybridized to each other to assemble the larger target DNA sequence.

Method of error reduction in nucleic acid populations

Growing community of inventors

Madison, WI, United States of America

Kathryn E Richmond