Automated DNA sequencing technique
First Claim
1. A method of separating and detecting tagged polynucleotides which comprises:
- providing a plurality of polynucleotides, each tagged with a chromophore or fluorophore;
resolving to separate one of the plurality of tagged polynucleotides from other tagged polynucleotides differing in length by a single nucleotide using an electrophoretic procedure capable of resolving tagged polynucleotides differing by a single nucleotide; and
detecting the resolved tagged polynucleotides by means of the chromophore or fluorophore.
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Abstract
A process for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations wherein chromophores or fluorophores are used to tag the DNA fragments produced by the sequencing chemistry and permit the detection and characterization of the fragments as they are resolved by electrophoresis through a gel. Preferably four different fragment sets are tagged with the fluorophores fluorescein, Texas Red, tetramethyl rhodamine, and 7-nitrobenzofurazan. A system for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations comprising: a source of chromophore or fluorescent tagged DNA fragments; a zone for contacting an electrophoresis gel; means for introducing said tagged DNA fragments to said zone; and photometric means for monitoring said tagged DNA fragments as they move through said gel.
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Citations
56 Claims
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1. A method of separating and detecting tagged polynucleotides which comprises:
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providing a plurality of polynucleotides, each tagged with a chromophore or fluorophore; resolving to separate one of the plurality of tagged polynucleotides from other tagged polynucleotides differing in length by a single nucleotide using an electrophoretic procedure capable of resolving tagged polynucleotides differing by a single nucleotide; and detecting the resolved tagged polynucleotides by means of the chromophore or fluorophore. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of detecting a tagged polynucleotide, which comprises:
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providing a plurality of polynucleotides, wherein each of said plurality of polynucleotide comprises an identical primer oligonucleotide tagged with a chromophore or fluorophore; resolving the plurality of tagged polynucleotides using an electrophoretic procedure capable of resolving tagged polynucleotides differing in length by a single nucleotide; and detecting each of the plurality of resolved tagged polynucleotides by means of the chromophore or fluorophore. - View Dependent Claims (8, 9, 10, 11, 12, 13)
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14. A method of determining the sequence of a polynucleotide by analyzing polynucleotide fragments generated by a polynucleotide sequencing technique, each of said polynucleotide fragments being tagged with a chromophore or fluorophore, comprising:
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introducing the tagged polynucleotide fragments into an electrophoretic medium; separating the tagged polynucleotide fragments in said electrophoretic medium using an electrophoretic procedure capable of resolving said polynucleotide fragments differing in length by a single nucleotide; detecting the separated tagged polynucleotide fragments by means of the chromophore or fluorophore; and determining the polynucleotide sequence from the polynucleotide fragments detected. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. A method for determining the sequence of a polynucleotide which comprises:
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providing polynucleotide fragments tagged with chromophores or fluorophores, wherein the chromophores or fluorophores are distinguishable from others by their spectral characteristics; resolving the polynucleotide fragments by electrophoresis; and detecting the resolved fragments by means of the chromophores or fluorophores, and thereby determining the sequence based on the polynucleotide fragments detected. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
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41. A method for determining the sequence of a polynucleotide which comprises:
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providing polynucleotide fragments generated by a polynucleotide sequencing technique, which are tagged with chromophores or fluorophores, wherein the fragments from one or more of the four sequencing reactions A, C, G or T are distinguishable from fragments of the other reactions by their spectral characteristics; resolving the fragments by electrophoresis; detecting the fragments as they are being resolved by means of the spectral characteristics of the chromophores or fluorophores, and thereby determining the polynucleotide sequence based on the polynucleotide fragments detected. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53)
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54. A method of determining the sequence of a single-stranded polynucleotide comprising:
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obtaining a set of fragments of the complement of said single-stranded polynucleotide, wherein each fragment is tagged with a chromophore or fluorophore such that fragment differing by a single nucleotide are capable of being resolved; separating the tagged fragments according to size, using an electrophoretic procedure capable of resolving tagged fragments differing by a single nucleotide, to obtain separated tagged fragments; detecting each separated tagged fragment by means of its chromophore or fluorophore; and analyzing the sizes of the separated tagged fragments, thereby determining the sequence of the single-stranded polynucleotide. - View Dependent Claims (55, 56)
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Specification