SYNTHESIS OF FOUR-COLOR 3'-O-ALLYL MODIFIED PHOTOCLEAVABLE FLUORESCENT NUCLEOTIDES AND RELATED METHODS
First Claim
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1. A method for making a 3′
- -O-allyl modified nucleotide analogue comprising performing the steps set forth in one of FIG. 7, FIG. 8, FIG. 9, or FIG. 10.
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Accused Products
Abstract
This invention provides a process for making 3′-O-allyl-dGTP-PC-Biodopy-FL-510, 3′-O-allyl-dATP-PC-ROX, 3′-O-allyl-dCTP-PC-Bodipy-650 and 3′-O-allyl-dUTP-PC-R6G, and related methods.
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Citations
14 Claims
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1. A method for making a 3′
- -O-allyl modified nucleotide analogue comprising performing the steps set forth in one of
FIG. 7 ,FIG. 8 ,FIG. 9 , orFIG. 10 .
- -O-allyl modified nucleotide analogue comprising performing the steps set forth in one of
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2-4. -4. (canceled)
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5. A method for determining the sequence of a DNA comprising performing the following steps for each residue of the DNA to be sequenced:
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(a) contacting the DNA with a DNA polymerase in the presence of (i) a primer and (ii) four fluorescent nucleotide analogues under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein (1) the nucleotide analogues consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of dATP, (2) each nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine, or uracil and analogues thereof, (ii) a deoxyribose, (iii) a fluorophore photocleavably attached to the base, and (iv) an allyl moiety bound to the 3′
-oxygen of the deoxyribose, so that a nucleotide analogue complementary to the residue being sequenced is bound to the DNA by the DNA polymerase, and (3) each of the four analogues has a predetermined fluorescence wavelength which is different than the fluorescence wavelengths of the other three analogues;(b) removing unbound nucleotide analogues; (c) determining the identity of the bound nucleotide analogues; and (d) following step (c), except with respect to the final DNA residue to be sequenced, (i) chemically cleaving from the bound nucleotide analogue the allyl moiety bound to the 3′
-oxygen atom of the deoxyribose and (ii) photocleaving the fluorophore from the bound nucleotide analogue, wherein steps (d)(i) and (d) (ii) can be performed concurrently or in any order, and step (d)(1) is performed using a Pd catalyst at a pH of about 8.8,thereby determining the sequence of the DNA. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12)
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13. A method for removing an allyl moiety from the 3′
- -oxygen of a nucleotide analogue'"'"'s deoxyribose moiety comprising the step of contacting the nucleotide analogue with a Pd catalyst at a pH of about 8.8.
- View Dependent Claims (14)
Specification