The goal of this project is to achieve a five- to ten-fold cost reduction relative to standard """"""""1/8x"""""""" ABI sequencing reactions in an easily automatable and exportable way, by completing the development and testing of novel microtiter plate-based consumables. These consumables consist of a 384 well thermocycling lid that allows sequencing reactions as small as I microliter to be performed in conventional 384 well PCR plates, and a reusable 96 well ultrafiltration (UF) plate that enables cleanup of the resulting samples, driven by a commercially available pipetting device. Current production prototypes have generated sequence data with quality metrics equivalent to conventional methods, and have demonstrated the feasibility of their use in high-throughput sequencing automation. During Year-1 the development of the 384 well thermocycling lid will be completed (with appropriate modifications for automated use), a small production run of lids will be performed, and an automated sequencing reaction setup platform will be configured to use the lids. The UF plate will be upgraded from 96 to 384 wells, and a reusable disposable UF plate will be designed in preparation for a production run in year 2. Thermocycling and cleanup protocols will be optimized, and alternate ultrafiltration membrane materials will be explored to reduce variability, increase the average sequencing pass rates and quality metrics, and increase reusability of the UF plates. All design and manufacture tasks will be done in collaboration with vendors who plan to manufacture the thermocycling lids and UF plates for sale. During Year-2 a small production run of the disposable 384 well UF plates will be performed and the previously-developed semi-automated clean up platform will be upgraded to use these plates in a fully automated mode. Rigorous testing and qualification of the plates on these automation platforms in GTC's high-throughput sequencing center will demonstrate achievement of the goal.
| Murakami, T; Reiter, L T; Lupski, J R (1997) Genomic structure and expression of the human heme A:farnesyltransferase (COX10) gene. Genomics 42:161-4 |
| Murakami, T; Sun, Z S; Lee, C C et al. (1997) Isolation of novel genes from the CMT1A duplication/HNPP deletion critical region in 17p11.2-p12. Genomics 39:99-103 |
