|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
24. Design and Assembly of a Turnkey, High Throughput Oligonucleotide Synthesis Facility for Use on the Human Genome Project
J. Shawn Roach and Harold R. Garner
The objective of this project was to design and assemble a highly automated, high throughput oligonucleotide synthesis facility that requires as little operator intervention as is practical. Each of the pre-processing and post-processing steps required for high throughput oligosynthesis were examined for opportunities to streamline and automate if practical. These steps include 1) loading the solid supports into the filter plates prior to synthesis, 2) cleaving the oligos from the solid supports post synthesis, 3) rapid evaporation of the residual solutions after chemical deprotection, 4) optical density evaluation of the oligo concentration after resuspension, 5) gel electrophoresis evaluation of the oligo quality, and 6) automated dilution of the oligos to a normalized concentration after resuspension.
The heart of the system is two MerMade high throughput oligonucleotide synthesizers that are each capable of synthesizing up to 192 oligos a day. The oligos are synthesized on solid supports loaded into two 96-well filter plates. Standard phosphoramidite chemistry is used to perform the synthesis. The MerMade was designed by Dr. Harold R. Garner and Dr. Simon Rayner of the University of Texas Southwestern Medical Center. The MerMades used on this project were built by Avantech Automation Corporation (New Braunfels, TX) according to the Garner-Rayner design with minor modifications. Additional devices in the facility include a Biomek 2000 Workstation (Beckman Instruments, Fullerton, CA) for performing several of the liquid transfer and filtration steps necessary, a Jetstream evaporator and Scirocco gas heater system (Helix Scientific, Warminster, PA) for rapid sample concentration, and a Spectramax 190 Plus UV-Vis spectrophotometer and 96-well plate reader (Molecular Devices, Sunnyvale, CA) for optical density analysis.
Data from oligonucleotides synthesized by the facility will be presented along with background information on the turnkey synthesis facility.
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