Substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene compounds for 8-color DNA sequencing
First Claim
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1. A method of sequencing a sense strand and an antisense strand of a double-stranded polynucleotide comprising:
- i) denaturing the double-stranded polynucleotide to provide the sense strand and the antisense strand;
ii) reacting the sense strand with a first set of four differentially labeled polynucleotides;
iii) reacting the antisense strand with a second set of four differentially labeled polynucleotides;
iv) identifying each of the eight polynucleotides by a fluorescence or an absorption spectrum of the fluorophore; and
v) determining the sequence of the sense strand from the polynucleotides differentially labeled with the first set of fluorophores and the sequence of the antisense strand from the polynucleotides differentially labeled with the second set of fluorophores.
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Abstract
The present invention describes a method of 8-color sequencing. Specifically, the present invention is directed to sequencing a sense strand and an antisense strand of a double-stranded polynucleotide comprising forming eight polynucleotide products differentially labeled with eight characteristic fluorophores, wherein said eight fluorophores comprise a set; and identifying each of the eight polynucleotide products by a fluorescence or an absorption spectrum of the characteristic fluorophores.
70 Citations
33 Claims
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1. A method of sequencing a sense strand and an antisense strand of a double-stranded polynucleotide comprising:
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i) denaturing the double-stranded polynucleotide to provide the sense strand and the antisense strand;
ii) reacting the sense strand with a first set of four differentially labeled polynucleotides;
iii) reacting the antisense strand with a second set of four differentially labeled polynucleotides;
iv) identifying each of the eight polynucleotides by a fluorescence or an absorption spectrum of the fluorophore; and
v) determining the sequence of the sense strand from the polynucleotides differentially labeled with the first set of fluorophores and the sequence of the antisense strand from the polynucleotides differentially labeled with the second set of fluorophores. - View Dependent Claims (2, 3, 4, 5)
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6. A method of 8-color sequencing of a polynucleotide comprising the steps of:
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i) forming eight classes of polynucleotides wherein each class of polynucleotides is labeled with a fluorophore and each fluorophore is different;
ii) electrophoretically separating the classes of polynucleotides;
iii) illuminating the separated polynucleotides with a wavelength capable of causing the fluorophores to fluoresce; and
iv) identifying the classes of polynucleotides by the fluorescence or absorption spectrum of the fluorophores. - View Dependent Claims (7, 8, 9, 10, 11, 12)
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13. A method of distinguishing polynucleotides having different 3′
- -terminal dideoxynucleotides in a chain termination method of DNA sequencing, the method comprising the steps of;
i) forming eight classes of polynucleotides by extending from primers a plurality of polynucleotides by means of a DNA polymerase or a reverse transcriptase in the presence of a dideoxyadenosine triphosphate, a dideoxycytosine triphosphate, a dideoxyguanosine triphosphate, and a dideoxythymidine triphosphate, and wherein the eight classes of polynucleotides are labeled at a 5′
position with a different fluorophore;
ii) electrophoretically separating the classes of polynucleotides;
iii) illuminating the separated polynucleotides with a wavelength capable of causing the fluorophores to fluoresce; and
iv) identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22)
- -terminal dideoxynucleotides in a chain termination method of DNA sequencing, the method comprising the steps of;
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23. A method for distinguishing polynucleotides having different ribonucleotides in a method of labeling polynucleotides by enzymatic incorporation, said method comprising the steps of:
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i) forming a mixture of four classes of polynucleotides, the four classes comprising polynucleotides having different terminal nucleotide triphosphates, wherein said triphosphates are linked to a BODIPY fluorophore that contains at least one reactive functional group; and
wherein said BODIPY fluorophores comprise a first set and all are different;
ii) forming a second mixture of four classes of polynucleotides, the four classes comprising polynucleotides having different terminal nucleotide triphosphates;
wherein said triphosphates are linked to a BODIPY fluorophore that contains at least one reactive functional group; and
wherein said BODIPY fluorophores comprise a second set and all are different, and are different than the first set;
iii) electrophoretically separating the polynucleotides in the first mixture and the second mixture;
iv) illuminating the first and second mixtures with a wavelength capable of causing the fluorophores to fluoresce; and
v) identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores. - View Dependent Claims (24, 25, 26, 27)
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28. A method of labeling a nucleic acid for 8-color sequencing comprising the steps of:
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i) forming a plurality of oligonucleotides substituted with at least two fluorophores comprising a donor and an acceptor;
wherein said oligonucleotides are separated into eight classes, wherein said eight donor fluorophores comprise a donor set and are the same or different; and
wherein eight acceptor fluorophores comprise an acceptor set and are all different;
ii) annealing said oligonucleotide classes to a strand of a polymerase chain reaction product to generate a substrate for a 5′
to 3′
exonuclease activity;
iii) amplifying said oligonucleotide classes, wherein said exonuclease activity degrades said oligonucleotides, wherein said donor is released; and
iv) detecting said oligonucleotide classes. - View Dependent Claims (29, 30)
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31. As a composition of matter, a 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-styryloxyacetate.
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32. As a composition of matter, a 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-styryloxyacetate.
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33. As a composition of matter, a 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid.
Specification