3′-o-fluorescently modified nucleotides and uses thereof

Details 3′-o-fluorescently modified nucleotides and uses thereof 3′-o-fluorescently modified nucleotides and uses thereof

2009-10-26

2011-10-04

Riley, Jezia (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024300, C536S024330, C536S025300, C536S026600, C435S006120, C435S091100, C435S091200, C422S067000

Reexamination Certificate

active

08030466

ABSTRACT:
The present invention relates to a DNA sequencing method using a nucleoside triphosphate with a fluorescent blocking group on its 3′-OH end as a reversible terminator. Further, the present invention relates to sequencing-by-synthesis method using the mono-modified reversible terminator (MRT), the novel nucleotide monomer having a reversible fluorescent blocking group removable chemically or enzymatically at its 3′-OH end. The sequencing method of the present invention facilitates sequencing of bases inserted by terminating extension of a nucleotide chain by the nucleotide monomer and then detecting fluorescence signal from 3′-OH end. At this time, after analyzing the fluorescence signal, the blocking group conjugated to the 3′-OH end can be effectively removed, indicating that a free 3′-OH functional group can be successfully restored, so that the next monomer insertion is possible, making continuous sequencing possible.

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patent: WO 00/50642 (2000-08-01), None
Guo et al. (Jul. 8, 2008) “Four-Color DNA Sequencing with 3′-O-Modified Nucleotide Reversible Terminators and Chemically Cleavable Fluorescent Dideoxynucleotides,”Proc. Nat. Acad. Sci. USA105(27):9145-9150.
Metzker et al. (1994) “Termination of DNA Synthesis by Novel 3′-Modified Deoxyribonucleoside 5′-Triphosphates,”Nuc. Acids Res.22(20):4259-4267.
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Southworth et al. (May 1996) “Cloning of Thermostable DNA Polymerases from Hyperthermophilic Marine Archaea with Emphasis onThermococcussp. 9° N-7 and Mutations Affecting 3′-5′ Exonuclease Activity,”Proc. Nat. Acad. Sci. USA93:5282-5285.
Welch et al. (1999) “Syntheses of Nucleosides Designed for Combinatorial DNA Sequencing,”Chem. Eur. J.5(3):951-960.

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