Alternative dye-labeled ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides for automated DNA analysis
First Claim
1. A method for distinguishing polynucleotides having different 3'"'"'-terminal dideoxynucleotides in any method of chain termination DNA sequencing, said method comprising the steps of:
- forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a 3'"'"'-terminal dideoxyadenosine triphosphate, said 3'"'"'-terminal dideoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
dipyrrometheneboron difluoride linker at a 3 position of a first BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the second class having a 3'"'"'-terminal dideoxycytidine triphosphate, said 3'"'"'-terminal dideoxycytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a second BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the third class having a 3'"'"'-terminal dideoxyguanosine triphosphate, said 3'"'"'-terminal dideoxyguanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a third BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the fourth class having a 3'"'"'-terminal dideoxythymidine triphosphate, said 3'"'"'-terminal dideoxythymidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a fourth BODIPY®
fluorophore that contains at least one reactive functional group;
wherein if said first, second, third and fourth BODIPY®
fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane;
or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;
electrophoretically separating on a gel by size the polynucleotides so as to produce bands on the gel;
illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and
identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
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Accused Products
Abstract
Methods for the use of a class of dyes for improved DNA sequencing by the chain termination method of DNA sequencing, and internal labelling of polynucleotides by enzymatic incorporation of fluorescently-labeled ribonucleotides or deoxyribonucleotides are provided. A new class of dyes, BODIPY® fluorophores, has been described recently. The parent heterocyclic molecule of the BODIPY® fluorophores is a dipyrrometheneboron difluoride compound which is modified to create a broad class of spectrally-discriminating fluorophores. BODIPY® fluorophores have improved spectral characteristics compared to conventional fluorescein and rhodamine dyes. BODIPY® fluorophores have narrower band width, insensitivity to solvent or pH, and improved photostability, thus, BODIPY® fluorophores lead to improved DNA sequencing and/or detection in any method where electrophoresis and detection of DNA is required. Additionally, the spectral properties of the BODIPY® fluorophores are sufficiently similar in wavelength and intensity to be used with conventional equipment known in the art.
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Citations
17 Claims
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1. A method for distinguishing polynucleotides having different 3'"'"'-terminal dideoxynucleotides in any method of chain termination DNA sequencing, said method comprising the steps of:
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forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a 3'"'"'-terminal dideoxyadenosine triphosphate, said 3'"'"'-terminal dideoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
dipyrrometheneboron difluoride linker at a 3 position of a first BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the second class having a 3'"'"'-terminal dideoxycytidine triphosphate, said 3'"'"'-terminal dideoxycytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a second BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the third class having a 3'"'"'-terminal dideoxyguanosine triphosphate, said 3'"'"'-terminal dideoxyguanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a third BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the fourth class having a 3'"'"'-terminal dideoxythymidine triphosphate, said 3'"'"'-terminal dideoxythymidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a fourth BODIPY®
fluorophore that contains at least one reactive functional group;
wherein if said first, second, third and fourth BODIPY®
fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane;
or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;electrophoretically separating on a gel by size the polynucleotides so as to produce bands on the gel; illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method for distinguishing polynucleotides having different ribonucleotides in any method of labelling polynucleotides by enzymatic incorporation, said method comprising the steps of:
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forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having an adenosine triphosphate, said adenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a first BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the second class having a cytidine triphosphate, said cytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a second BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the third class having a guanosine triphosphate, said guanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a third BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the fourth class having a uracil triphosphate, said uracil triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a fourth BODIPY®
fluorophore that contains at least one reactive functional group;
wherein if said first, second, third and fourth BODIPY®
fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane;
or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;electrophoretically separating on a gel by size the polynucleotides; illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores. - View Dependent Claims (8, 9, 10, 11)
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12. A method for distinguishing polynucleotides having different deoxyribonucleotides in any method of labelling polynucleotides by enzymatic incorporation, said method comprising the steps of:
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forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a deoxyadenosine triphosphate, said deoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-3-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a first BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the second class having a deoxycytidine triphosphate, said deoxycytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a second BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the third class having a deoxyguanosine triphosphate, said deoxyguanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-3-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a third BODIPY®
fluorophore that contains at least one reactive functional group;
each polynucleotide in the fourth class having a deoxythymidine triphosphate, said deoxythymidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY®
linker at a 3 position of a fourth BODIPY®
fluorophore that contains at least one reactive functional group;
wherein if said first, second, third and fourth BODIPY®
fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane;
or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;electrophoretically separating on a gel by size the polynucleotides; illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores. - View Dependent Claims (13, 14, 15, 16, 17)
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Specification