Method, system and reagents for DNA sequencing
First Claim
1. A process of DNA sequence analysis by chain elongation comprising reacting a template of DNA contained in an appropriate sequencing vector, a primer, a polymerase, a first mixture of DNA nucleotides or their analogs, and a second mixture of radiation omitting reporter-labeled chain terminators corresponding to the DNA nucleotides or their analogs to produce fragments of DNA having reporters covalently attached to their 3'"'"' terminal residues introduced by chain termination events, and analyzing the fragments so produced for the presence of reporters, thereby identifying the DNA sequence.
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Accused Products
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.
262 Citations
14 Claims
- 1. A process of DNA sequence analysis by chain elongation comprising reacting a template of DNA contained in an appropriate sequencing vector, a primer, a polymerase, a first mixture of DNA nucleotides or their analogs, and a second mixture of radiation omitting reporter-labeled chain terminators corresponding to the DNA nucleotides or their analogs to produce fragments of DNA having reporters covalently attached to their 3'"'"' terminal residues introduced by chain termination events, and analyzing the fragments so produced for the presence of reporters, thereby identifying the DNA sequence.
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14. A process for determining the base sequence of DNA comprising the steps of:
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attaching a different radiation emitting reporter to a chain terminator for each of the bases, preparing, labeled DNA fragments by a modified chain termination method, separating the DNA fragments according to size, and detecting the reporter for each DNA fragment thereby identifying the 3'"'"'-terminal base.
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Specification