EP0252683A2 — Process and reagents for DNA sequence analysis

Claim 1

Independent Claim 134 words

A system for detecting the presence of radiant energy emitted from different 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 different than the first sense as a function of the nature of the species, and means responsive to the first and second signals for obtaining a third signal corresponding to the ratio of a function 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 134 words

A system for detecting the presence of radiant energy emitted from different 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 different than the first sense as a function of the nature of the species, and means responsive to the first and second signals for obtaining a third signal corresponding to the ratio of a function 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 31 words

The system as set forth in claim 2 wherein the reporters have the structure wherein n=2 or 3, and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and 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 3ʹ-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 3ʹ-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 35 words

The system as set forth in claim 10 wherein the reporters have a emitting part with the structure wherein n=2 or 3, and R₁ and R₂ 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 31 words

The method as set forth in claim 15 wherein the reporters have the structure wherein n=2 or 3, and R₁ and R₂ 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 3ʹ-terminal residues which have been introduced via chain termination events.

Claim 20

Dependent Claim 85 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 reporter-labeled chain terminators corresponding to the DNA nucleotides or their analogs to produce fragments of DNA having reporters covalently attached to their 3ʹ terminal residues introduced by chain termination events, and analyzing the fragments so produced for the presence of reporters, thereby identifying the DNA sequence.

Claim 21

Dependent Claim 85 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 reporter-labeled chain terminators corresponding to the DNA nucleotides or their analogs to produce fragments of DNA having reporters covalently attached to their 3ʹ terminal residues introduced by chain termination events, and analyzing the fragments so produced for the presence of reporters, thereby identifying the DNA sequence.

Claim 22

Dependent Claim 26 words

The process as set forth in claim 18 wherein the reporters are selected from the group comprising chromophores, fluorophores, luminescers, spin labels, and electron dense materials.

Claim 23

Dependent Claim 31 words

The process as set forth in claim 19 wherein the fluorophores have the structure wherein n=2 or 3, and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano.

Claim 24

Dependent Claim 22 words

The process as set forth in claim 18 wherein the attachment of the reporters to chain terminators is via an amide linkage.

Claim 25

Dependent Claim 32 words

The process as set forth in claim 18 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 26

Dependent Claim 23 words

The process as set forth in claim 22 wherein a linker and optional spacer is interposed between the reporter and the heterocyclic base.

Claim 27

Dependent Claim 156 words

The process as set forth in claim 22 wherein the reporter-labeled chain terminator is a compound having the structure wherein X is H, NH₂ or halogen, and Y is H, halogen, OH, or NH₂, 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 N₁ position, the purines and the deazapurines are linked to the sugar moiety through the N₉ position (purine numbering of the deazapurines); and A is a fluorophore having the structure wherein n=2 or 3, and R₁ and R₂ 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 28

Dependent Claim 139 words

The process as set forth in claim 23 wherein the reporter-labeled chain terminator is a compound having the structure wherein B is uracil, cytosine, 7-deazaadenine, 7-deazaguanine, or 7-deazahypoxanthine where the pyrimidines are N₁ linked and the purines are linked to the sugar moiety through the N₁ position, the pyrimidines and the deazapurines are linked to the sugar moiety through the N₉ position (purine numbering of the diazapurines), and A is a fluorophore having the structure wherein n=2 or 3, and R₁ and R₂ 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 29

Dependent Claim 30 words

The process of DNA sequence analysis according to claim 18 wherein the second mixture contains four reporter-labeled chain terminators, each carrying a different distinguishable reporter, to produce labeled DNA fragments.

Claim 30

Dependent Claim 14 words

The process as set forth in claim 26 wherein the distinguishable reporters are fluorophores.

Claim 31

Dependent Claim 18 words

The process as set forth in claim 26 including the steps of simultaneously separating the labeled DNA fragments.

Claim 32

Dependent Claim 18 words

The process as set forth in claim 28 wherein the labeled DNA fragments are combined prior to separation.

Claim 33

Dependent Claim 21 words

The process for DNA sequence analysis according to claim 26 wherein all four chain termination reactions occur in a single vessel.

Claim 34

Dependent Claim 56 words

A process for determining the base sequence of DNA comprising the steps of: attaching a different 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 3ʹ-terminal base.

Claim 35

Dependent Claim 56 words

A process for determining the base sequence of DNA comprising the steps of: attaching a different 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 3ʹ-terminal base.

Claim 36

Dependent Claim 106 words

A compound having the structure wherein (a) n is 2 or 3, R₁ is H, lower alkyl, fluoro, chloro, iodo, lower alkoxy, or cyano, and R₂ is halo, lower alkoxy, or cyano, or (b) n is 2 or 3, R₁ is lower alkyl, fluoro, iodo, lower alkoxy, or cyano, and R₂ is H or lower alkyl, or (c) n is 2 or 3, R₁ is bromo, and R₂ is lower alkyl, fluoro, chloro, iodo, lower alkoxy, or cyano, (d) n is 2, R₁ is chloro, and R₂ is H or lower alkyl, or (e) n is 3, R₁ is bromo, and R₂ is H or bromo.

Claim 37

Dependent Claim 106 words

A compound having the structure wherein (a) n is 2 or 3, R₁ is H, lower alkyl, fluoro, chloro, iodo, lower alkoxy, or cyano, and R₂ is halo, lower alkoxy, or cyano, or (b) n is 2 or 3, R₁ is lower alkyl, fluoro, iodo, lower alkoxy, or cyano, and R₂ is H or lower alkyl, or (c) n is 2 or 3, R₁ is bromo, and R₂ is lower alkyl, fluoro, chloro, iodo, lower alkoxy, or cyano, (d) n is 2, R₁ is chloro, and R₂ is H or lower alkyl, or (e) n is 3, R₁ is bromo, and R₂ is H or bromo.

Claim 38

Dependent Claim 36 words

A compound having the structure wherein n=2 or 3 and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano and Rʹ is an alkyl or aryl group and Rʺ is an alkyl group.

Claim 39

Dependent Claim 36 words

A compound having the structure wherein n=2 or 3 and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano and Rʹ is an alkyl or aryl group and Rʺ is an alkyl group.

Claim 40

Dependent Claim 28 words

A method of preparing the compound of claim 33 comprising the steps of treating 3-hydroxy-6-oxo-9-carboxyalkyl-6H-xanthenes with a carboxylic acid anhydride in pyridine followed by treatment with an alcohol.

Claim 41

Dependent Claim 42 words

A compound having the structure wherein n=2 or 3 and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano, Rʹ is an alkyl or aryl group, and Rʺ is an alkyl group and X is a good leaving group.

Claim 42

Dependent Claim 42 words

A compound having the structure wherein n=2 or 3 and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano, Rʹ is an alkyl or aryl group, and Rʺ is an alkyl group and X is a good leaving group.

Claim 43

Dependent Claim 42 words

A compound having the structure wherein n=2 or 3 and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano, Rʹ is an alkyl or aryl group, and Rʺ is an alkyl group and X is a good leaving group.

Claim 44

Dependent Claim 42 words

A compound having the structure wherein n=2 or 3 and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano, Rʹ is an alkyl or aryl group, and Rʺ is an alkyl group and X is a good leaving group.

Claim 45

Dependent Claim 136 words

A compound having the structure wherein X is H, NH₂ or halogen, and Y is H, halogen, OH, or NH₂, 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 N₁ position, the purines and the deazapurines are linked to the sugar moiety through the N₉ position (purine numbering); and A is a fluorophore having the structure wherein n=2 or 3, and R₁ and R₂ 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 46

Dependent Claim 136 words

A compound having the structure wherein X is H, NH₂ or halogen, and Y is H, halogen, OH, or NH₂, 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 N₁ position, the purines and the deazapurines are linked to the sugar moiety through the N₉ position (purine numbering); and A is a fluorophore having the structure wherein n=2 or 3, and R₁ and R₂ 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 47

Dependent Claim 99 words

A compond having the structure wherein B is uracil, cytosine, 7-deazaadenine, 7-deazaguanine or 7-deazahypoxanthine here the pyrimidines are N₁-linked and the deazapurines) are N₉-linked (purine numbering); and A is a fluorophore having the structure wherein n=2 or 3, and R₁ and R₂ 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 48

Dependent Claim 99 words

A compond having the structure wherein B is uracil, cytosine, 7-deazaadenine, 7-deazaguanine or 7-deazahypoxanthine here the pyrimidines are N₁-linked and the deazapurines) are N₉-linked (purine numbering); and A is a fluorophore having the structure wherein n=2 or 3, and R₁ and R₂ 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 49

Dependent Claim 51 words

A compound having the structure wherein n=2 or 3 and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano and Rʹ is an alkyl or aryl group and Rʺ is an alkyl group and R₄ is H or a trivalent phosphorus moiety and B is a heterocyclic base.

Claim 50

Dependent Claim 51 words

A compound having the structure wherein n=2 or 3 and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano and Rʹ is an alkyl or aryl group and Rʺ is an alkyl group and R₄ is H or a trivalent phosphorus moiety and B is a heterocyclic base.

Claim 51

Independent Claim 44 words

A method of DNA sequence analysis according to the chain terminator method using dideoxy chain terminating nucleotides which includes the step of substituting a compound having the structure wherein B is 1-uracil, 1-cytosine, 1-thymine, 9-adenine, 9-guanine, or 9-hypoxanthine for the dideoxy chain terminating nucleotides.

Claim 52

Independent Claim 44 words

A method of DNA sequence analysis according to the chain terminator method using dideoxy chain terminating nucleotides which includes the step of substituting a compound having the structure wherein B is 1-uracil, 1-cytosine, 1-thymine, 9-adenine, 9-guanine, or 9-hypoxanthine for the dideoxy chain terminating nucleotides.

Claim 53

Dependent Claim 13 words