? ? The long-term objective of this research is to provide a means by which to sequence 10OOs of different exons simultaneously from a genomic DNA sample with 30 to 50 times coverage of each exon. This will provide researchers with significant new information about the genetic makeup and diversity of pre- cancerous, early-cancerous, and cancerous cells. ? RainDance Technologies (Guilford, CT) has developed microfluidic technology to manipulate sub-nanoliter volumes of reagents at rates exceeding 10A9 droplets per day. We propose to use this technology to process genomic DNA for the sequencing of 1000s of exons from individual samples. We will use a single microfluidic chip for multiple processing steps. The first on-chip processing step is to combine genomic DNA on a droplet-by-droplet basis with a member of a library of 1000s of primer pairs at rates of 1000s per second. The primer pairs ensure that a single exon is amplified in a given droplet during a polymerase chain reaction (PCR) step; the product of this step is captured on a bead for sequencing. The final on-chip step is a quality control step where beads with less than 10 million copies of an exon are removed before the remaining beads are sent for sequencing on the 454 Life Sciences (Branford, CT) instrument. Successful completion of this project will allow one pooled-primer-pair-library emulsion to be used to economically sequence thousands of individual exons from an almost unlimited number of genomic, (including tumor), DNA samples. ? ? ?
| Tewhey, Ryan; Warner, Jason B; Nakano, Masakazu et al. (2009) Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27:1025-31 |
| Kiss, Margaret Macris; Ortoleva-Donnelly, Lori; Beer, N Reginald et al. (2008) High-throughput quantitative polymerase chain reaction in picoliter droplets. Anal Chem 80:8975-81 |
