A new family of nucleotides has been designed, synthesised and evaluated for DNA sequencing by synthesis. Each of the nucleotide analogues had a free 3’-OH group and a fluorophore attached to the base through a small peptide linkage that was designed to stop multiple base additions. Analysis by HPLC and MALDI-TOF proved the modified nucleotides were incorporated into a growing DNA strand by a DNA polymerase and that the peptide linker was cleaved with high efficiency after incubation of the extended primer with a protease. Enzymatically cleavable nucleotides were successfully applied to chip-based sequencing by synthesis cycles. The fluorescent emission revealed the identity of the incorporated nucleotide and the removal of the fluorophore by a protease ensured the detection of the next base in the following cycle. Additionally, a practical microwave-mediated solid-phase protocol has been developed for the synthesis of cyanine dyes spanning the visible and the near infrared spectrum to allow the preparation of a series of fluorescent nucleotides for the design of a four colour DNA sequencing technology.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:654026 |
| Date | January 2009 |
| Creators | Lopalco, Maria |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15233 |
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