The long term objectives of this project is to develop integrated gene synthesis systems; to bring about one to two orders of magnitude reduction in the cost of totally synthetic genes as well as reducing the time between ordering and delivery significantly. Availability of much lower cost synthetic genes has the promise to bring about revolutionary advances in combinatorial biology, gene therapy, DNA Immunization for antibody and antisera production and studies of gene expression.
The specific aims of the project are 1) to increase the purities of on chip synthesized oligonucleotides and develop protocols and tools for chip based hybridization purification of oligos; 2) develop a surface immobilized mutS based final oligo purification system for eliminating mismatched double stranded oligonucleotides; 3) Integration of the synthesis and purification chips and 4) design and fabricate chip based ligation/PCR reactors with integral heaters and temperature sensors which will convert the purified oligonucleotides into short genes or gene fragments. Given a gene or sequence we will first decompose the sense and antisense strands of the gene into a set of unique oligonucleoides of approximately 30 bases long. Each olignucleotide will be designed to hybridize with two oligonucleotides from the complimentary strand. The purity of light directed chip synthesized oligos will be improved by using a high contrast, high power projector together with ultrasonic mixing and improved synthesis chamber designs. The synthesized oligonucleotides will be purified after cleaving from the surface by hybridizing under stringent conditions to complimentary oligonucleotides synthesized on an identical chip and then passing the purified oligo pool through a chamber of bead bound mutS proteins which will retain non-perfectly hybridized oligos. After adjusting the volume and buffer etc. the highly purified oligonucleotides will be ligated in a finely temperature controlled ligation chamber to produce the desired genes. Ligation will be followed by PCR amplification using primers we will synthesize ourselves.
