Four-color DNA sequencing by synthesis on a chip using cleavable fluorescent nucleotide reversible terminators

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a new paradigm to decipher DNA sequences. This thesis focuses on the construction of such a novel DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. First, we used a 2-nitrobenzyl based photocleavable (PC) linker to attach a fluorophore to 3'-O-allyl-modified nucleotides, forming photocleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-PC-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-aallyl-dNTPs-PC-fluorophore in a polymerase reaction, are removed by photocleavage (irradiation at 355 nm) and Pd-catalyzed deallylation reaction, respectively. This allows the re-initiation of the polymerase reaction and continuation of SBS. We then found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-ally1 group on a DNA extension product were now able to be removed simultaneously in 30 seconds by Pd-catalyzed deallylation reaction in an aqueous buffer solution. This one-step dual-deallylation reaction thus allowed the re-initiation of the polymerase reaction and increased the SBS efficiency. We also found an alternative sequencing method that is a hybrid between Sanger dideoxy chain terminating reaction and sequencing by synthesis (SBS) and discuss the advantages that come with this hybrid sequencing approach. In this approach, four nucleotides, modified as reversible terminators (3'-O-PC-dNTPs) by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are used in combination with four photocleavable fluorophore labeled dideoxynucleotides (ddNTPs-PC-fluorophore) to generate Sanger sequencing fragments during SBS. DNA sequences were determined by the unique fluorescence emission of each fluorophore on the ddNTPs. Upon removing the 3'-OH blocking group on the dNTPs and the fluorophore from the ddNTPs, the polymerase reaction can reinitiate and the DNA sequences can be continuously determined. Four-color DNA sequencing was performed using these novel fluorescent nucleotide analogues and a four-color fluorescent scanner to identify sequences of DNA template immobilized on a chip. The DNA chip was constructed by covalently attaching alkyne-modified self-priming DNA template onto an azido-PEG functionalized glass slide by using 1,3-dipolar cycloaddition chemistry. DNA sequences were obtained for various different DNA templates, including DNA templates containing homopolymeric regions in its sequence.