Multiple tag labeling method for DNA sequencing
First Claim
1. A method of determining the sequence of DNA which comprises the steps ofprocessing said DNA to form four sets of DNA sequencing fragments where one set contains fragments terminating in G, a second set contains fragments terminating in A, a third set contains fragments terminating in T, and a fourth set contains fragments terminating in C, such that selected mole fractions of each one of selected sets of sequencing fragments is tagged with a first fluorophore which fluoresces at a first wavelength and such that different selected mole fractions of each one of selected sets of sequencing fragments is tagged with a second fluorophore which fluoresces at a second wavelength and in which one of said sets of sequencing fragments is comprised of a mixture of selected mole fractions of fragments where one mole fraction is labeled with the first fluorophore and the other mole fraction is labeled with the second fluorophore,separating said sets of tagged DNA sequencing fragments in a single channel or lane, anddetermining the sequence of said sequencing fragments by detecting the ratio of fluorescence intensity at the first and second wavelengths as a function of time or position.
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Abstract
A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.
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Citations
22 Claims
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1. A method of determining the sequence of DNA which comprises the steps of
processing said DNA to form four sets of DNA sequencing fragments where one set contains fragments terminating in G, a second set contains fragments terminating in A, a third set contains fragments terminating in T, and a fourth set contains fragments terminating in C, such that selected mole fractions of each one of selected sets of sequencing fragments is tagged with a first fluorophore which fluoresces at a first wavelength and such that different selected mole fractions of each one of selected sets of sequencing fragments is tagged with a second fluorophore which fluoresces at a second wavelength and in which one of said sets of sequencing fragments is comprised of a mixture of selected mole fractions of fragments where one mole fraction is labeled with the first fluorophore and the other mole fraction is labeled with the second fluorophore, separating said sets of tagged DNA sequencing fragments in a single channel or lane, and determining the sequence of said sequencing fragments by detecting the ratio of fluorescence intensity at the first and second wavelengths as a function of time or position.
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12. A method of determining the sequence of DNA which comprises the steps of
processing sets of DNA sequencing fragments such that different selected mole fractions of each one of selected sets of sequencing fragments is tagged with fluorophores which emit fluorescence at different wavelengths with one of said sets of sequencing fragments is comprised of a mixture of selected mole fractions of fragments where one mole fraction is labeled with a fluorophore which fluoresces at a first wavelength and the other mole fraction is labeled with a fluorophore which fluoresces at a second wavelength, separating said sets of tagged DNA sequencing fragments in a single channel or lane, and determining the sequence of each of said sequencing fragments by detecting the ratio of fluorescence intensity at different wavelengths as a function of time or position.
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18. A method of determining the sequence of DNA which comprises the steps of
processing sets of DNA sequencing fragments terminating in G, A, T and C, where each one of said selected sets of sequencing fragments is labeled with a selected ratio of two or more radio-isotopes having the same mobility shift and one set of sequencing fragments is comprised of a mixture of selected mole fractions of fragments, one of which is labeled with one radio-isotope and the other of which is labeled with a second radio-isotope, separating said sets of tagged DNA sequencing fragments in a single channel or lane, and determining the sequence of said sequencing fragments by detecting the ratio of isotopes as a function of position or time.
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22. A method of determining the sequence of DNA which comprises the steps of
processing sets of DNA sequencing fragments where one set contains fragments terminating in G, a second set contains fragments terminating in A, a third set contains fragments terminating in T, and a fourth set contains fragments terminating in C, tagging selected mole fractions of each one of selected sets of sequencing fragments with a first tag which provides a first signal, tagging different selected mole fractions of each one of selected sets of sequencing fragments with a second tag which provides a second signal, tagging two different mole fractions of one set of sequencing fragment with two different tags which simultaneously provide first and second signals, separating said sets of tagged DNA sequencing fragments in a single channel or lane, and determining the sequence of said sequencing fragments by detecting the ratio of intensity of the first and second signals as a function of time or position.
Specification