Method systems and reagents for DNA sequencing
First Claim
1. A system for detecting the presence of radiant energy emitted from more than two spectrally distinct species following separation in time and/or space and the identity of such species, comprising:
- means responsive to the spectra of the species for generating a first signal that varies in amplitude in a first sense as a function of the nature of the species,means responsive to the spectra of the species for generating a second signal that varies in amplitude in a second sense as a function of the nature of the species,means responsive only to the first and second signals for obtaining a third signal corresponding to the ratio of the first and second signals, the amplitude of the third signal being indicative of the identity of each of the species.
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Abstract
A DNA sequencing system and method are described to detect the presence of radiant energy emitted from different excited reporter dye-labeled species (DNA fragments) following separation in time and/or space, and the identity of the species which emit radiant energy closely spaced in wavelength. Functions of the emitted energy are obtained which vary over the wavelengths of the closely spaced spectra in different senses and the functions ratioed, whereby the ratio is indicative of the identity of the DNA fragments.
The emitting portion of the reporter-labeled DNA fragment is preferably one of a family of fluorescent dyes based on 9-carboxyethyl-6-hydroxy-3-oxo-3H-xanthene. These xanthene dyes are covalently attached to the DNA fragments through the carboxylic acid functionality, preferably via an amide linkage. The dyes may be protected by including an alkoxy group at the 9-position. A spacer may be inserted between the dye and the amine. The fluorescent dye preferably is attached to the DNA chain terminators and provides many advantages. Thus only DNA sequencing fragments resulting from bona fide termination events will carry a reporter. The DNA sequencing may also be labeled using the xanthene dyes which have general utility as fluorescent labels. Also acyclonucleoside triphosphates are described as being useful as chain terminators in DNA sequencing using a modification of the Sanger method.
95 Citations
17 Claims
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1. A system for detecting the presence of radiant energy emitted from more than two spectrally distinct species following separation in time and/or space and the identity of such species, comprising:
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means responsive to the spectra of the species for generating a first signal that varies in amplitude in a first sense as a function of the nature of the species, means responsive to the spectra of the species for generating a second signal that varies in amplitude in a second sense as a function of the nature of the species, means responsive only to the first and second signals for obtaining a third signal corresponding to the ratio of the first and second signals, the amplitude of the third signal being indicative of the identity of each of the species. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method of detecting the presence of radiant energy emitted from different species following a separation in time and/or space and determining the identity of each such species that emit spectra comprising the steps of:
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obtaining functions of the emitted energy which vary in different senses according to the nature of the species, and ratioing such functions, the ratio being indicative of the identity of the species. - View Dependent Claims (13, 14, 15, 16, 17)
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Specification