DNA sequencing and DNA synthesis are major, complementary capabilities that enable exquisite control and new understanding of biological systems. The cost of sequencing/synthesis, per base pair, continues to drop at a remarkable rate. The new discipline of synthetic biology intends to exploit both DNA synthesis and DNA sequencing toward practical ends; simultaneously, the success of synthetic biology will accelerate the deployment of both DNA synthesis and DNA sequencing.
DNA sequencing with polonies
We adapt a conventional epifluorescence microscope to function as a non-electrophoretic DNA sequencing instrument. To validate our approach, we resequence an engineered, evolved strain of E.coli. A clone-free in vitro protocol yields a complex mate-paired library from which single DNA molecules are amplified in parallel to one-micron beads by emulsion PCR. Millions of DNA-bearing beads are immobilized in an acrylamide gel and subjected to automated cycles of sequencing-by-ligation and four-color imaging to query 26 bases per amplicon. Our best instrument-run to date yielded 30 megabases of sequence. Multiplex technologies, in which complex mixtures are manipulated within a shared reagent volume, have strong potential to significantly impact our approach to DNA sequencing.
- Shendure J, Mitra R, Varma C, Church GM (2004) Advanced Sequencing Technologies: Methods and Goals. Nature Reviews of Genetics May;5(5):335-44. Image:2004shendure.pdf