Patent US5306618A successfully refreshed from Google Patents.
US5306618A
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Method systems and reagents for DNA sequencing
Assignee: Revvity Health Sciences Inc
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Filing: 1992-01-16
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Granted: 1994-04-26
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Expires: 2012-01-16
Abstract
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.
Inventors
- 👤 James M. Prober
- 👤 Rudy J. Dam
- 👤 Charles W. Robertson, Jr.
- 👤 Frank W. Hobbs, Jr.
- 👤 George L. Trainor
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 01:14
CPC Classifications
C; C07; C07D; C07D405/00; C07D405/02; C07D405/04; C12; C12Q; C12Q1/00; C12Q1/70; C07D405/12; C07D405/14; C07D471/00; C07D471/02; C07D471/04; C07F; C07F9/00; C07F9/02; C07F9/547; C07F9/645; C07F9/6509; C07F9/6512; C07F9/6558; C07F9/65586; C07F9/6561; C07H; C07H19/00; C07H19/02; C07H19/04; C07H19/06; C07H19/10; C07H19/16; C07H19/20; C07H21/00; C12Q1/68; C12Q1/6869; G; G01; G01N; G01N27/00; G01N27/26; G01N27/416; G01N27/447; G01N27/44704; G01N27/44717; G01N27/44721; G01N27/44726; Y; Y10; Y10S; Y10S435/00; Y10S435/808; Y10S435/968; Y10S436/00; Y10S436/805; Y10T; Y10T436/00; Y10T436/13
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
130 words
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.
Claim 2
Independent Claim
130 words
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.
Claim 3
Dependent Claim
34 words
The system as set forth in claim 1 wherein the species are fragments of DNA covalently labeled with reporters capable of emitting radiant energy and which includes means adapted to separate fragments of DNA.
Claim 4
Dependent Claim
34 words
The system as set forth in claim 2 wherein the reporters have the structure ##STR22## wherein n=2 or 3, and R 1 and R 2 are H, lower alkyl, halo, lower alkoxy, or cyano.
Claim 5
Dependent Claim
28 words
The system as set forth in claim 3 wherein the DNA fragments have reporters covalently attached to their 3'-terminal residues which have been introduced via chain termination events.
Claim 6
Dependent Claim
26 words
The system as set forth in claim 4 which also includes a laser having an output wavelength lying within the excitation region of the different reporters.
Claim 7
Dependent Claim
28 words
The system as set forth in claim 2 wherein the DNA fragments have reporters covalently attached to their 3'-terminal residues which have been introduced via chain termination events.
Claim 8
Dependent Claim
87 words
The system as set forth in claim 1 wherein the means for generating first and second signals includes a means having first and second transmission characteristics, which vary as a function of wavelength, for transmitting the spectra, the transmission means and the species being relatively positionable to direct such emissions to the transmission means. first and second detectors positionable to receive respectively the first and second transmitted emissions and generating the first and second signals corresponding to the intensities of each and indicating the presence of each.
Claim 9
Dependent Claim
35 words
The system as set forth in claim 7 wherein the device characteristic has a transition from the first to the second transmission characteristic which is centered at about the middle of the reporters' emission spectra.
Claim 10
Dependent Claim
16 words
The system as set forth in claim 8 wherein the transmission means is a dichroic filter.
Claim 11
Dependent Claim
24 words
The system as set forth in claim 7 which includes means adapted to separate fragments of DNA or other molecules covalently labeled with reporters.
Claim 12
Dependent Claim
38 words
The system as set forth in claim 10 wherein the reporters have a emitting part with the structure ##STR23## wherein n=2 or 3, and R 1 and R 2 are H, lower alkyl, halo, lower alkoxy, and cyano.
Claim 13
Independent Claim
67 words
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: 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.
Claim 14
Independent Claim
67 words
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: 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.
Claim 15
Dependent Claim
18 words
The method set forth in claim 12 wherein the different senses consist of greater and lesser emission amplitudes.
Claim 16
Dependent Claim
40 words
The method as set forth in claim 13 wherein the functions are obtained by passing the emitted radiant energy through a device having a transmission/reflection characteristic which varies as a function of wavelength, and the device is a dichroic filter.
Claim 17
Dependent Claim
19 words
The method as set forth in claim 12 wherein the species are fragments of DNA covalently labeled with reporters.
Claim 18
Dependent Claim
34 words
The method as set forth in claim 15 wherein the reporters have the structure ##STR24## wherein n=2 or 3, and R 1 and R 2 are H, lower alkyl, halo, lower alkoxy, and cyano.
Claim 19
Dependent Claim
27 words
The method as set forth in claim 16 wherein the DNA fragments have reporters attached to their 3'-terminal residues which have been introduced via chain termination events.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-04-25 | 1997-10-25 | 1998-04-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-04-25 | 2001-10-25 | 2002-04-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-04-25 | 2005-10-25 | 2006-04-26 | $7700.00 | Window Closed / Lapsed |