As an alternative to fluorescence-based DNA sequencing by synthesis (SBS), we report a novel approach of using an azido moiety (N3) as label for SBS that has an intense and unique Raman shift at 2125 cm-1 where virtually all biological molecules are transparent. We demonstrated that the four 3'-azidomethyl modified nucleotides displayed surface enhanced Raman scattering at 2125 cm-1. Using these nucleotide analogues as substrates, we performed a complete 4-step SBS reaction in which each polymerase extension yielding an azido-labeled DNA product and subsequent cleavage of the azido-label was monitored by Raman spectroscopy for the appearance and disappearance of the peak at 2125 cm-1, thereby determining the DNA sequence. Due to the small size of the azido group in comparison with fluorophores, 3'-azidomethyl modified nucleotides are more efficient substrates for the polymerase. With further improvements in surface enhancement for N3, this approach can provide an attractive alternative to fluorescence-based SBS.

DNA sequencing by synthesis using surface-enhanced Raman spectroscopy

244th National Meeting (2012)

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