title: Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.
abstract: DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3'-OH capping group from the DNA products generated by incorporating the 3'-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3'-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3'-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3'-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.
authors.affiliation: Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Guo
authors.forename: Ning
authors.initials: N
authors.lastname: Xu
authors.forename: Zengmin
authors.initials: Z
authors.lastname: Li
authors.forename: Shenglong
authors.initials: S
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.forename: Dae Hyun
authors.initials: DH
authors.lastname: Kim
authors.forename: Mong
authors.initials: M
authors.lastname: Sano Marma
authors.forename: Qinglin
authors.initials: Q
authors.lastname: Meng
authors.forename: Huanyan
authors.initials: H
authors.lastname: Cao
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Li
authors.forename: Shundi
authors.initials: S
authors.lastname: Shi
authors.forename: Lin
authors.initials: L
authors.lastname: Yu
authors.forename: Sergey
authors.initials: S
authors.lastname: Kalachikov
authors.forename: James J
authors.initials: JJ
authors.lastname: Russo
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Turro
authors.forename: Jingyue
authors.initials: J
authors.lastname: Ju
mesh_terms: D001483:Base Sequence; D003116:Color; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054306:Dideoxynucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D004259:DNA-Directed DNA Polymerase