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AssigneeGe Healthcare UK Limited
InventorsRaj Odedra, Adrian Simmonds, Lee Pickering
Estimating the term…
Nucleotides comprising a reporter moiety and a polymerase enzyme blocking moiety in which the reporter moiety does not also act as a polymerase enzyme blocking moiety are described. Also described are compounds of Formula (I): wherein W is a phosphate group, B is a base, Y is a linker comprising an enzyme-cleavable group, R2 is a reporter moiety, R3 is selected from H or OH, Z and Z′ are selected from H, OH, or a group X—R1, wherein X is a linker comprising an enzyme-cleavable group and R1 is a polymerase enzyme blocking group, provided that at least one of Z and Z′ is X—R1. 1
A nucleotide comprising a reporter moiety and a polymerase enzyme blocking moiety characterised in that the reporter moiety does not also act as a polymerase enzyme blocking moiety.
A compound of Formula I
A chemical intermediate selected from the group consisting of:
A set of nucleotides characterised in that the set contains at least one compound of Formula I.
A method for nucleic acid molecule sequencing comprising the steps of:
Dependencies are inferred from the claim text.
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Pyrimidine radicals: with the saccharide radical esterified by phosphoric or polyphosphoric acids
Purine radicals: with the saccharide radical esterified by phosphoric or polyphosphoric acids
Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate
Involving nucleic acids: nucleic acid amplification reactions
Term end not estimated
The present invention relates to nucleoside and nucleotide analogues. In particular, the invention relates to nucleotide analogues having enzyme-cleavable blocking and reporter groups positioned on separate parts of the nucleotide.
Recent improvements in DNA sequencing techniques have sought to meet the increasing demands of large scale sequencing. Increasingly, methods in which the template nucleic acid molecules are attached to a solid surface are being developed (see, for example, U.S. Pat. No. 5,302,509 and U.S. Pat. No. 5,547,839). Such methods dispense with the need for an electrophoretic separation step and, with he use of optical detection technologies (see, for example, Nie et al. Annu. Rev. Biophys. Biomol. Struct. 1997, 26: 567-96), aim to allow sequencing information at the level of a single molecule to be obtained. This has the further potential for multiple samples to be analysed simultaneously.
One example of such methods is Base Addition Sequencing Scheme (BASS) (see, for example, Metzker et al., Nucleic Acids Res 1994, Vol.22, No.20; p. 4259-4267). BASS is a method involving the incorporation of nucleotide analogues which have been modified so as to comprise a blocking group which terminates DNA synthesis. A primer is annealed to a template bound to a solid support and sequence data obtained by repetitive cycles of incorporation of modified nucleotides. At each cycle, the incorporated base is identified in situ before being deprotected to remove the blocking group and allow the next cycle of DNA synthesis.
For the purposes of clarity, certain embodiments of the present invention will now be described by way of example with reference to the following figures:
FIG. 1 shows a reaction scheme for syntesising a compound of Formula I.
FIG. 2 shows examples of compounds of Formula I.
A reaction scheme for the synthesis of an example of a compound of Formula I is set out in FIG. 1 using 5-iodo-2′-deoxyuridine (Sigma-Aldrich Chemical Co.) as the starting material and incorporating the dye, Cy3, by reaction with (N-hydroxy)succinimide Cy3 ester.
In the reaction scheme, the following abbreviations are used: TBDMS—tertbutyldimethylsilyl; DMTr—Dimethoxytrityl; TFA—trifluoroacyl; nBu—linear Butyl chain; MeCN—Acetonitrile; TCA—Trichloroacetic acid; DCM—Dichloromethane; DMF—N,N-Dimethylformamide; TEAB—Tetraethylammonium bicarbonate buffer, THF—Tetrahydrofuran; Ac—Acetyl.
Wherein TBDMS, Ac, TFA and nBu have the following structural formulae: 8