US5332666A Expired / Lapsed US ⏱️ 0.0 years remaining

Method, system and reagents for DNA sequencing

Assignee: Revvity Health Sciences Inc

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Filing: 1991-10-22

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Granted: 1994-07-26

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Expires: 2011-10-22

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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 2026-10-29 15:56

CPC Classifications

C; C12; C12Q; C12Q1/00; C12Q1/70; C07; C07D; C07D405/00; C07D405/02; C07D405/04; 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: 16

Claim 1

Independent Claim 87 words

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.

Claim 2

Dependent Claim 87 words

Claim 3

Dependent Claim 20 words

The process as set forth in claim 1 wherein the reporters are selected from the group comprising fluorophores and luminescers.

Claim 4

Dependent Claim 34 words

The process as set forth in claim 2 wherein the fluorophores have the structure ##STR22## wherein n=2 or 3, and R 1 and R 2 are H, lower alkyl, halo, lower alkoxy, and cyano.

Claim 5

Dependent Claim 22 words

The process as set forth in claim 1 wherein the attachment of the reporters to chain terminators is via an amide linkage.

Claim 6

Dependent Claim 32 words

The process as set forth in claim 1 wherein the reporter-labeled chain terminator includes a heterocyclic base and the attachment of the reporter to the chain terminator is through the heterocyclic base.

Claim 7

Dependent Claim 23 words

The process as set forth in claim 5 wherein a linker and optional spacer is interposed between the reporter and the heterocyclic base.

Claim 8

Dependent Claim 161 words

The process as set forth in claim 5 wherein the reporter-labeled chain terminator is a compound having the structure ##STR23## wherein X is H, NH 2 or halogen, and Y is H, halogen, OH, or NH 2 , or X=Y=OH: B is uracil, cytosine, 7-deazaadenine, 7-deazaguanine or 7-deazahypoxanthine where the pyrimidines are linked to the sugar moiety through the N 1 position, the purines and the deazapurines are linked to the sugar moiety through the N 9 position (purine numbering of the deazapurines): and A is a fluorophore having the structure ##STR24## wherein n=2 or 3. and R 1 and R 2 are H, lower alkyl, halo, lower alkoxy, and cyano: and the dotted line represents a linker and optional spacer or group joining B and A provided that if B is a pyrimidine the attachment is to the 5-position of that pyrimidine and if B is a deazapurine, the attachment is to the 7 position of that deazapurine (purine numbering).

Claim 9

Dependent Claim 146 words

The process as set forth in claim 6 wherein the reporter-labeled chain terminator is a compound having the structure ##STR25## wherein B is uracil, cytosine, 7-deazaadenine, 7-deazaguanine, or 7-deazahypoxanthine where the pyrimidines are N 1 linked and the purines are linked to the sugar moiety through the N 1 position, the pyrimidines and the deazapurines are linked to the sugar moiety through the N 9 position (purine numbering of the diazapurines), and A is a fluorophore having the structure ##STR26## wherein n=2 or 3. and R 1 and R 2 are H, lower alkyl, halo, lower alkoxy, and cyano; and the dotted line represents a linker and optional spacer joining B and A provided that if B is a pyrimidine the attachment is to the 5-position of that pyrimidine and if B is a deazapurine, the attachment is to the 7-position of that deazapurine (purine numbering).

Claim 10

Dependent Claim 30 words

The process of DNA sequence analysis according to claim 1 wherein the second mixture contains four reporter-labeled chain terminators, each carrying a different distinguishable reporter, to produce labeled DNA fragments.

Claim 11

Dependent Claim 14 words

The process as set forth in claim 9 wherein the distinguishable reporters are fluorophores.

Claim 12

Dependent Claim 18 words

The process as set forth in claim 9 including the steps of simultaneously separating the labeled DNA fragments.

Claim 13

Dependent Claim 18 words

The process as set forth in claim 11 wherein the labeled DNA fragments are combined prior to separation.

Claim 14

Dependent Claim 21 words

The process for DNA sequence analysis according to claim 9 wherein all four chain termination reactions occur in a single vessel.

Claim 15

Dependent Claim 58 words

A process for determining the base sequence of DNA comprising the steps of: 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.

Claim 16

Dependent Claim 58 words

References Cited (Backward Citations)

JPS5996454A Cited

JPS59231847A Cited

DE3446635A1 Cited

DE3501306A1 Cited

WO1986002929A1 Cited

US4855225A Cited

USPTO Annuity & Maintenance Windows

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1997-07-25	1998-01-24	1998-07-26	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2001-07-25	2002-01-24	2002-07-26	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2005-07-25	2006-01-24	2006-07-26	$7700.00	Window Closed / Lapsed

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