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  3. DNA sequencing by synthesis using 3'- O- azidomethyl nucleotide reversible terminators and surface-enhanced Raman spectroscopic detection

DNA sequencing by synthesis using 3'- O- azidomethyl nucleotide reversible terminators and surface-enhanced Raman spectroscopic detection

  • RSC Adv. 2014 Jan 1;4(90):49342-49346. doi: 10.1039/C4RA08398A.
Mirkó Palla 1,  Wenjing Guo 2,  Shundi Shi 2,  Zengmin Li 2,  Jian Wu 3,  Steffen Jockusch 4,  Cheng Guo 2,  James J Russo 2,  Nicholas J Turro 4,  Jingyue Ju 5
Affiliations

Affiliations

  • 1 Center for Genome Technology and Biomolecular Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA ; Department of Mechanical Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA.
  • 2 Center for Genome Technology and Biomolecular Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA ; Department of Chemical Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA.
  • 3 Center for Genome Technology and Biomolecular Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA.
  • 4 Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA.
  • 5 Center for Genome Technology and Biomolecular Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA ; Department of Chemical Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA ; Department of Pharmacology, Columbia University, 3000 Broadway, New York, NY 10027, USA.
PMID: 25396047 DOI: 10.1039/C4RA08398A
Abstract

As an alternative to fluorescence-based DNA Sequencing by synthesis (SBS), we report here an approach using an azido moiety (N3) that has an intense, narrow and unique Raman shift at 2125 cm-1, where virtually all biological molecules are transparent, as a label for SBS. We first demonstrated that the four 3'-O-azidomethyl nucleotide reversible terminators (3'-O-azidomethyl-dNTPs) displayed surface enhanced Raman scattering (SERS) at 2125 cm-1. Using these 4 nucleotide analogues as substrates, we then performed a complete 4-step SBS reaction. We used SERS to monitor the appearance of the azide-specific Raman peak at 2125 cm-1 as a result of polymerase extension by a single 3'-O-azidomethyl-dNTP into the growing DNA strand and disappearance of this Raman peak with cleavage of the azido label to permit the next nucleotide incorporation, thereby continuously determining the DNA sequence. Due to the small size of the azido label, the 3'-O-azidomethyl-dNTPs are efficient substrates for the DNA Polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for the azido moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS.

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