Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

• This invention relates generally to fluorescent dye compounds useful as molecular probes. More specifically, this invention relates to 4,7-dichlororhodamine dyes useful as fluorescent labeling reagents.
• non-radioactive detection of biological analytes is an important technology in modern analytical biotechnology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact of reagent disposal is greatly reduced, resulting in decreased costs for analysis.
• methods utilizing such non-radioactive detection methods include DNA sequencing, oligonucleotide probe methods, detection of polymerase- chain-reaction products, immunoassays, and the like.
• the independent detection of multiple spatially overlapping analytes in a mixture is required, e.g., single-tube multiplex DNA probe assays, irnmuno assays, multicolor DNA sequencing methods, and the like.
• multicolor labeling allows for the analysis of all four bases in a single lane thereby increasing throughput over single-color methods and eliminating uncertainties associated with inter-lane electrophoretic mobility variations.
• Multiplex detection imposes a number of severe constraints on the selection of dye labels, particularly for analyses requiring an electrophoretic separation and treatment with enzymes, e.g., DNA sequencing.
• the set may still not be suitable if the respective fluorescent efficiencies are too low. For example , in the case of DNA sequencing, increased sample loading cannot compensate for low fluorescence efficiencies (Pringle).
• the fluorescent dyes must be compatible with the chemistry used to create or manipulate the fragments, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like.
• rhodamine dyes particularly useful in multicolor applications are the rhodamine dyes, e.g., tetramethylrhodamine (TAMRA), rhodamine X (ROX), rhodamine 6G (R6G), rhodamine 110 (RI 10), and the like (Bergot).
• TAMRA tetramethylrhodamine
• ROX rhodamine X
• R6G rhodamine 6G
• RI 10 rhodamine 110
• Rhodamine dyes are particularly attractive relative to fluorescein dyes because (1) rhodamines are typically more photostable than fluoresceins, (2) rhodamine-labeled dideoxynucleotides are better substrates for thermostable polymerase enzymes, and (3) the emission spectra of rhodamine dyes is significantly to the red (higher wavelength) of fluoresceins.
• rhodamine dyes in the context of multiplex detection methods are relatively broad emission spectrum of such dyes. This broad emission spectrum results in poor spectral resolution between spectrally neighboring dyes thereby making the multicomponent analysis of such dye combinations difficult.
• the fluorescence emission spectra shown in FIG. 7A demonstrate this high degree of spectral overlap.
• a second drawback of currently available rhodamine dyes is that their absorption spectrum does not match the wavelength of currently available solid state frequency-doubled green diode lasers, e.g., neodymium solid-state YAG lasers, which have an emission line at approximately 532 nm. It is highly advantageous to use such lasers because of their compact size, long useful life, and efficient use of power.
• the present invention is directed towards our discovery of a class of 4,7- dichlororhodamine dyes useful as molecular probes.
• the invention includes a compound having the formula:
• the invention includes a labeled nucleotide having the formula:
• the invention includes a labeled polynucleotide containing a nucleotide having the formula:
• the linkage linking B and D is attached to D at one of positions R 1 -R 6 or X ⁇ -X 3 .
• the linkage linking B and D is attached to D at one of positions X 2 or X 3 .
• the linkage is
• B is a purine
• the linkage is attached to the 8-position of the purine.
• B is 7-deazapurine
• the linkage is attached to the 7-position of the 7-deazapurine.
• B is pyrimidine
• the linkage is attached to the 5 -position of the pyrimidine.
• the present invention includes a method of polynucleotide sequencing, such method including the following steps. Forming a mixture of a first, a second, a third, and a fourth class of polynucleotides such that each polynucleotide in the first class includes a 3'- terminal dideoxyadenosine and is labeled with a first dye, each polynucleotide in the second class includes a 3 '-terminal dideoxycytidine and is labeled with a second dye, each polynucleotide in the third class includes a 3 '-terminal dideoxyguanosine and is labeled with a third dye, and, each polynucleotide in the fourth class includes a 3 '-terminal dideoxythymidine and is labeled with a fourth dye.
• the dyes are selected such that one of the first, second, third, or fourth dyes is a 4,7-dichlororhodamine dye of the invention, the other of the dyes being spectrally resolvable from the 4,7-dichlororhodamine dye and from each other. Electrophoretically separating the polynucleotides thereby forming bands of similarly sized polynucleotides, illuminating the bands with an illumination beam capable of causing the dyes to fluoresce, and, identifying the classes of the polynucleotides in the bands by the fluorescence spectrum of the dyes.
• the present invention comprises a novel class of 4,7-dichlororhodamine compounds useful as fluorescent dyes, reagents employing such dyes as molecular labels, and methods utilizing such dyes and reagents in the area of analytical biotechnology.
• the compounds of the present invention find particular application in the area of multicolor fluorescent DNA sequencing and fragment analysis.
• the invention is based in part on the discovery that the fluorescent properties of 4,7- dichlororhodamines and related dyes are highly favorable for use as molecular probes.
• Their emission band widths are generally 20-30 percent narrower than analogs lacking the 4,7- dichloro derivatives, and, their emission and absorption maxima are at wavelengths generally about 10-30 nm higher than analogs lacking the 4,7-dichloro derivatives.
• Linking group refers to a functionality capable of reacting with a "complementary functionality” attached to a reagent, such reaction forming a "linkage” connecting a dye to a reagent.
• the particular linking group used depends on the nature of the complementary functionality and the type of linkage desired. In some cases, the linking group must be activated prior to reaction with a complementary functionality, e.g., the activation of a carboxylate linking group with dicyclohexylcarbodiimide and N-hydroxysuccinimide to form a N- hydroxysuccinimide (NHS) ester.
• the linking group of the invention is isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde or glyoxal, epoxide, carbonate, aryl halide, imidoester, carbodiimide, anhydride, 4,6-dichlorotriazinylamine, or other active carboxylate.
• the linking group is haloacetyl, alkyl halide, maleimide, halo acetyl, aziridine, acryloyl, arylating agent, e.g., fluorobenzene, and the like.
• the linking group is preferably diazoalane, diazoacetyl, carbonyldiimidazole, and carbodiimide (Hermanson).
• the linking group is an activated NHS ester which reacts with an amine complementary functionality, where to form the activated NHS ester, a dye of the invention including a carboxylate linking group is reacted with dicyclohexylcarbodiimide and N- hydroxysuccinimide to form the NHS ester (Khanna; Kasai).
• Table 1 shows a sampling of representative linking groups along with compatible complementary functionalities and resulting linkages.
• lower alkyl denotes straight-chain and branched hydrocarbon moieties containing from 1 to 8 carbon atoms, i.e., methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, and the like.
• propano in particular refers to the moiety — CH 2 CH 2 CH 2 — .
• Cycloalkyl refers to hydrocarbon moieties that form rings, e.g. cyclohexyl and cyclopentyl. Nitrogen atoms with cycloalkyl substituents may form aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, larger rings, and substituted forms thereof.
• “Lower substituted alkyl” denotes a lower alkyl including electron-withdrawing substituents, such as halo, cyano, nitro, sulfo, and the like.
• “Lower haloalkyl” denotes a lower substituted alkyl with one or more halogen atom substituents, usually fluoro, chloro, bromo, or iodo.
• “Lower alkene” denotes a lower alkyl or lower substituted alkyl wherein one or more of the carbon-carbon bonds is a double bond.
• “Lower alkyne” denotes a lower alkyl or lower substituted alkyl wherein one or more of the carbon-carbon bonds is a triple bond.
• Lower Alkoxy refers to a moiety including lower alkyl single bonded to an oxygen atom.
• Aryl refers to single or multiple phenyl or substituted phenyl, e.g., benzene, naphthalene, anthracene, biphenyl, and the like.
• nucleoside refers to a compound consisting of a purine, deazapurine, or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like, linked to a pentose at the 1 ' position, including 2'-deoxy and 2'- hydroxyl forms (Stryer).
• nucleotide refers to a phosphate ester of a nucleoside, e.g., triphosphate esters, wherein the most common site of esterification is the hydroxyl group attached at the C-5 position of the pentose.
• nucleoside will be intended to include both nucleosides and nucleotides.
• nucleosides in reference to nucleosides include synthetic analogs having modified base moieties, modified sugar moieties, and/or modified phosphate ester moieties, e.g., as described elsewhere (Scheit; Eckstein).
• labeled nucleoside refers to nucleosides which are covalently attached to the dye compounds of Formula I.
• polynucleotide or “oligonucleotide” refer to linear polymers of natural nucleotide monomers or analogs thereof, including double and single stranded deoxyribonucleotides, ribonucleotides, -anomeric forms thereof, and the like.
• nucleoside monomers are linked by phosphodiester linkages, where as used herein, the term “phosphodiester linkage” refers to phosphodiester bonds or analogs thereof including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like, including associated counterions, e.g., H + , NH , Na + , and the like if such counterions are present.
• Polynucleotides typically range in size from a few monomeric units, e.g. 8-40, to several thousands of monomeric units.
• a polynucleotide is represented by a sequence of letters, such as "ATGCCTG,” it will be understood that the nucleotides are in 5'— 3' order from left to right and that "A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted.
• spectral resolution in reference to a set of dyes means that the fluorescent emission spectra of the dyes are sufficiently distinct, i.e., sufficiently non- overlapping, that reagents to which the respective dyes are attached, e.g., polynucleotides, can be distinguished on the basis of the fluorescent signal generated by the respective dyes using standard photodetection systems, e.g., employing a system of band pass filters and photomultiplier tubes, a charged-coupled device in conjunction with a spectrograph, or the like, as exemplified by systems described elsewhere (Hunkapiller; Wheeless).
• substituted refers to a molecule wherein one or more hydrogen atoms are replaced with one or more non-hydrogen atoms, functional groups or moieties.
• an unsubstituted nitrogen is — NH
• a substituted nitrogen is — NHCH 3
• substituents include but are not limited to halo, e.g., fluorine and chlorine, lower alkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic, heterocycle, and linking group.
• the present invention comprises a novel class of 4,7- dichlororhodamine dye compounds having the general structure shown immediately below as Formula I.
• Formula I 4,7- dichlororhodamine dye compounds having the general structure shown immediately below as Formula I.
• variable substituents are defined as follows. taken separately are hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group, or combinations thereof, or, when taken together, Ri and R ⁇ is benzo, or, when taken together, Rj and R 5 is benzo.
• Ri-R ⁇ are hydrogen, methyl, or ethyl.
• Yj-Y 4 taken separately are hydrogen, lower alkyl, alkyl sulfonate, alkyl carboxylate, or cycloalkyl.
• Y ⁇ and R 2 , Y 2 and R 1 ⁇ Y 3 and R 3 , and/or Y 4 and R» are propano, ethano, or substituted forms thereof to form fused rings.
• X]-X 3 taken separately are hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonic acid (sulfonate), hydroxymethyl (-CH OH), and linking groups.
• Xi is carboxylate and X and X 3 taken separately are hydrogen and linking group.
• R is hydrogen (dJON) or methyl (DMDJ), shown below as FORMULA II.
• FORMULA II Another particularly preferred compound of the present invention is referred to as dR650, shown below as FORMULA HI.
• a third particularly preferred set of compounds are where R is 6-hexanoic acid (dJODA) or methyl-/?-benzoic acid (dR134), as shown below in FORMULA IV.
• dJODA 6-hexanoic acid
• dR134 methyl-/?-benzoic acid
• a fourth particularly preferred compound of the present invention is referred to herein as dR139, where Y ⁇ and Y 2 , and Y 3 and Y 4 , form pyrrolidinyl rings as nitrogen substituents, R ⁇ _6 are hydrogen, X] is carboxyl, and X 2 and X 3 are carboxyl and hydrogen.
• the structure of dR139 is shown below as Formula V.
• the present invention comprises a novel class of 4,7- dichlororhodamine dye compounds having the general structure shown below as FORMULA VI.
• R 7 -R ⁇ o , R] -R ⁇ 6 , and R ]8 may be hydrogen, fluorine, chlorine, methyl, ethyl, lower alkyl, lower alkene, lower alkyne, cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino, amido, -nitrile, lower alkoxy, linking group, or combinations thereof.
• R 7 -R ⁇ 0 and R ⁇ 3 -Ri 6 are hydrogen, methyl, or ethyl.
• Ri i and R ]7 may be hydrogen, lower alkyl, alkyl sulfonate, alkyl carboxylate, lower alkene, lower alkyne, cycloalkyl, phenyl, aryl, linking group, or combinations thereof.
• R] i and R 17 are methyl or phenyl.
• X ⁇ -X 3 taken separately are hydrogen, chlorine, fluorine, lower alkyl, amine, amide, carboxylate, sulfonic acid (sulfonate), hydroxymethyl (-CH 2 OH), and linking groups.
• X] is carboxylate and X 2 and X 3 taken separately are hydrogen or linking group.
• Particular preferred embodiments are where R 7 , R 8 , R 10 R ⁇ 3 , R ⁇ 4 , and R ]7 are hydrogen or methyl, R 9 and R] 5 are hydrogen, Ri and R] 5 are methyl or phenyl, and R )2 and R ] are hydrogen.
• the present invention comprises reagents labeled with the 4,7- dichlororhodamine dye compounds of Formulas I- VI.
• Reagents of the invention can be virtually anything to which the dyes of the invention can be attached.
• the dyes are covalently attached to the reagent directly or through a linkage.
• Exemplary reagents include proteins, polypeptides, polysaccharides, nucleotides, nucleosides, polynucleotides, lipids, solid supports, organic and inorganic polymers, and combinations and assemblages thereof, such as chromosomes, nuclei, living cells, such as bacteria, other microorganisms, mammalian cells, tissues, glycoproteins, and the like.
• chromosomes such as bacteria, other microorganisms, mammalian cells, tissues, glycoproteins, and the like.
• a preferred class of reagents of the present invention comprise nucleotides and nucleosides which incorporate the asymmetric benzoxanthene dyes of the invention.
• Such nucleotide/side reagents are particularly useful in the context of labeling polynucleotides formed by enzymatic synthesis, e.g., nucleotide triphosphates used in the context of PCR amplification, Sanger-type polynucleotide sequencing, and nick-translation reactions.
• nucleotide/side reagents of the present invention are shown below in Formula VII wherein
• B is a nucleoside base; 7-deazapurine, purine, or pyrimidine nucleotide base, analogs thereof, and preferably uracil, cytosine, deazaadenine, or deazaguanosine.
• D is the 4,7- dichlororhodamine dye compound of the Formulas I- VI of the invention. Wi and W 2 taken separately are H or OH.
• W 3 is OH, OPO 3 , OP 2 O 6 , OP 3 O 9 , including analogs thereof, e.g., phosphorothioate, phosphoroanilidate, phosphoroamlothioate, phosphoramidiate, and other like phosphate analogs, including associated counterions if present, e.g., H + , Na + , NH , and the like.
• Wi is H
• W is OH
• W 3 is OP 3 O 9
• Wi and W 2 are H and W 3 is OP 3 O 9 .
• the linkage linking B and D may be attached to D at any one of positions R]-R ⁇ 8 or Xi-
• the linkage is attached at one of X 2 or X 3 .
• the linkage linking B and D is attached to the 8-position of the purine
• B is 7-deazapurine
• the linkage is attached to the 7-position of the 7-deazapurine
• B is pyrimidine
• the linkage is attached to the 5-position of the pyrimidine.
• the nucleotides of the present invention are dideoxynucleotide triphosphates having the structure shown below in Formula VIII, including associated counterions if present.
• Labeled dideoxy nucleotides such as that shown in Formula VIII find particular application as chain terminating agents, or "terminators", in Sanger-type DNA sequencing methods (Sanger).
• nucleotides of the present invention are deoxynucleotide triphosphates having the structure shown in Formula IX below, including associated counterions if present.
• FORMULA IX Labeled deoxynucleotides such as that shown in Formula IX find particular application as means for labeling polymerase extension products, e.g., in the polymerase chain reaction (Mullis).
• Nucleotide/side labeling can be accomplished using any of a large number of known nucleoside/tide labeling techniques using known linking groups, and associated complementary functionalities to form linkages. See above for a discussion of preferred linking groups.
• the linkage linking the dye and nucleoside should (i) not interfere with oligonucleotide-target hybridization, (ii) be compatible with relevant enzymes, e.g., polymerases, li gases, and the like, and (iii) not quench the fluorescence of the dye.
• the dyes of the invention are covalently linked to the 5- carbon of pyrimidine bases or to the 7-carbon of 7-deazapurine bases.
• Several suitable base labeling procedures have been reported that can be used with the invention. (Gibson; Gebeyehu; Haralambidis; Nelson 1992; Bergstrom; Fung 1988; Ward; Woo.)
• the linkages are acetylenic amido or alkenic amido linkages, the linkage between the dye and the nucleotide base being formed by reacting an activated NHS ester of the dye with an alkynylamino- or alkenylamino-derivatized base of a nucleotide.
• the resulting linkage is 3-(carboxy)amino-l-propynyl or 3-amino-l-propyn-l-yl (Formula X.l).
• Formulas X.l -6 Khan.
• alkynylamino-derivatized nucleosides The synthesis of alkynylamino-derivatized nucleosides is described by (Hobbs 1989, 1992). Briefly, the alkynylamino-derivatized nucleotides are formed by placing the appropriate halodideoxynucleoside (usually 5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides) and Cu(I) in a flask, flushing with argon to remove air, adding dry DMF, followed by addition of an alkynylamine, triethylamine and Pd°. The reaction mixture is stirred for several hours, or until thin layer chromatography indicates consumption of the halodideoxynucleoside.
• halodideoxynucleoside usually 5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides
• Cu(I)
• the alkynylamino- nucleoside can be isolated by concentrating the reaction mixture and chromatographing on silica gel using an eluting solvent which contains ammonium hydroxide to neutralize the hydrohalide generated in the coupling reaction.
• methanol/methylene chloride can be added to the reaction mixture, followed by the bicarbonate form of a strongly basic anion exchange resin. The slurry can then be stirred for about 45 minutes, filtered, and the resin rinsed with additional methanol/methylene chloride.
• the combined filtrates can be concentrated and purified by flash-chromatography on silica gel using a methanol-methylene chloride gradient. The 5 '-triphosphates are obtained by standard techniques.
• Yet another preferred class of reagents of the present invention comprise polynucleotides labeled with the 4,7-dichlororhodamine dyes of the invention.
• labeled polynucleotides are useful in a number of important contexts including as DNA sequencing primers, PCR primers, oligonucleotide hybridization probes, and the like.
• polynucleotides of the invention include a nucleotide having the formula:
• D is a 4,7- dichlororhodamine dye compound of the invention.
• B is a 7-deazapurine, purine, or pyrimidine nucleotide base, preferably uracil, cytosine, deazaadenine, or deazaguanosine.
• Zi is H, OH, or OCH 3 .
• Z 2 is H, OH, OPO 3 , OP 2 O 6 , OP 3 O 9 , or Nuc, a neighboring nucleotide, wherein Nuc and the nucleoside are linked by a phosphodiester linkage or analog thereof, e.g., phosphorothioate, phosphoroanilidate, phosphoroanilothioate, phosphoramidiate, and other like phosphate analogs, including associated counterions if present, e.g., H + , Na + , NH 4 + , the linkage being attached to the 5'-position of Nuc.
• a phosphodiester linkage or analog thereof e.g., phosphorothioate, phosphoroanilidate, phosphoroanilothioate, phosphoramidiate, and other like phosphate analogs, including associated counterions if present, e.g., H + , Na + , NH 4 + , the linkage being attached to
• Z 3 is H, OPO 2 , including phosphate analogs, or Nuc, wherein Nuc and the nucleoside are linked by a phosphodiester linkage or analog thereof, the linkage being attached to the 3 '-position of Nuc wherein Nuc refers to a nucleoside, nucleotide, or polynucleotide.
• B is purine or 7-deazapurine
• the sugar moiety is attached at the N 9 -position of the purine or deazapurine
• B is pyrimidine
• the sugar moiety is attached at the N'-position of the pyrimidine.
• B is attached to the sugar moiety and to the dye compound as described above for the nucleotide reagent of the invention.
• the labeled nucleotide of Formula XI can be the 5'-terminal nucleotide, the 3'- terminal nucleotide, or any internal nucleotide of the polynucleotide.
• the polynucleotide of the present invention includes multiple dyes, at least one of which is a dye compound of the invention, located such that fluorescence energy transfer takes place between a donor dye and an acceptor dye.
• a dye compound of the invention located such that fluorescence energy transfer takes place between a donor dye and an acceptor dye.
• Such multi- dye polynucleotides find application as spectrally-tunable probes or DNA sequencing primers (Ju; Lee).
• Labeled polynucleotides may be synthesized either enzymatically, e.g., using a DNA polymerase or ligase (Stryer), or by chemical synthesis, e.g., by the phosphoramidite method, the phosphite-triester method, and the like (Gait). Labels may be introduced during enzymatic synthesis utilizing labeled nucleotide triphosphate monomers as described above or may be introduced subsequent to synthesis.
• the labeled polynucleotide is made by enzymatic synthesis, the following procedure may be used.
• a template DNA is denatured and an oligonucleotide primer is annealed to the template DNA.
• a mixture of deoxynucleotide triphosphates and/or dideoxynucleotide triphosphates is added to the reaction including dGTP, dATP, dCTP, ddTTP, ddGTP, ddATP, ddCTP, and ddTTP, where at least a fraction of one of at least one the deoxynucleotides and/or dideoxynucleotides is labeled with a dye compound of the invention as described above.
• a polymerase enzyme is added under conditions where its polymerase activity is operative.
• a labeled polynucleotide is formed by the incorporation of the labeled deoxynucleotides and/or dideoxynucleotides during polymerase strand synthesis.
• two primers are used instead of one, one primer complementary to the + (plus) strand and the other complementary to the - (minus) strand of the target, the polymerase is a thermostable polymerase, and the reaction temperature is cycled between a denaturation temperature and an extension temperature, thereby exponentially synthesizing a labeled complement to the target sequence by PCR (Mullis; Innis).
• the polynucleotide may be labeled at a number of positions including the 5 '-terminus (Eckstein; Orgel; Smith); the phosphodiester backbone (Eckstein); or at the 3 '-terminus (Nelson 1992a; Nelson 1992b; Nelson 1995).
• Edstein the 5 '-terminus
• Eckstein the phosphodiester backbone
• 3 '-terminus the 3 '-terminus
• an oligonucleotide is labeled as follows.
• a dye including a carboxylate linking group is converted to the NHS ester by reacting with approximately 1 equivalent of 1,3-dicyclohexylcarbodiimide and approximately 3 equivalents of N-hydroxysuccinimide in dry ethyl acetate for 3 hours at room temperature.
• the reaction mixture is washed with 5% HC1, dried over magnesium sulfate, filtered, and concentrated to a solid which is resuspended in DMSO.
• the DMSO dye stock is then added in excess (10-20 x) to an aminohexyl derivatized oligonucleotide in 0.25 M bicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours (Fung 1988).
• the dye labeled oligonucleotide is separated from unreacted dye by passage through a size-exclusion chromatography column eluting with buffer, e.g., 0.1 molar triethylammonium acetate (TEAA).
• buffer e.g., 0.1 molar triethylammonium acetate (TEAA).
• TEAA triethylammonium acetate
• the fraction containing the crude labeled oligonucleotide is further purified by reverse phase HPLC employing gradient elution.
• the dyes and reagents of the present invention are well suited to methods utilizing fluorescent detection, particularly methods requiring the simultaneous detection of multiple spatially-overlapping analytes.
• Dyes and reagents of the invention are particularly well suited for identifying classes of polynucleotides that have been subjected to a biochemical separation procedure, such as electrophoresis, where a series of bands or spots of target substances having similar physiochemical properties, e.g. size, conformation, charge, hydrophobicity, or the like, are present in a linear or planar arrangement.
• bands includes any spatial grouping or aggregation of analytes on the basis of similar or identical physiochemical properties. Usually bands arise in the separation of dye-polynucleotide conjugates by electrophoresis.
• Classes of polynucleotides can arise in a variety of contexts.
• fragment analysis or “genetic analysis” methods
• labeled polynucleotide fragments are generated through template-directed enzymatic synthesis using labeled primers or nucleotides, e.g., by ligation or polymerase-directed primer extension; the fragments are subjected to a size-dependent separation process, e.g., electrophoresis or chromatography; and, the separated fragments are detected subsequent to the separation, e.g., by laser-induced fluorescence.
• a size-dependent separation process e.g., electrophoresis or chromatography
• the separated fragments are detected subsequent to the separation, e.g., by laser-induced fluorescence.
• multiple classes of polynucleotides are separated simultaneously and the different classes are distinguished by spectrally resolvable labels.
• amplified fragment length polymorphism detection is based on amplified fragment length polymo ⁇ hisms, i.e., restriction fragment length polymo ⁇ hisms that are amplified by PCR (Vos). These amplified fragments of varying size serve as linked markers for following mutant genes through families. The closer the amplified fragment is to the mutant gene on the chromosome, the higher the linkage correlation. Because genes for many genetic disorders have not been identified, these linkage markers serve to help evaluate disease risk or paternity.
• the polynucleotides may be labeled by using a labeled polynucleotide PCR primer, or by utilizing labeled nucleotide triphosphates in the PCR.
• VNTRs variable number of tandem repeats
• VNTRs are regions of double-stranded DNA that contain adjacent multiple copies of a particular sequence, with the number of repeating units being variable. Examples of VNTR loci are pYNZ22, pMCTl 18, and Apo B.
• a subset of VNTR methods are those methods based on the detection of microsatellite repeats, or short tandem repeats (STRs), i.e., tandem repeats of DNA characterized by a short (2-4 bases) repeated sequence.
• STRs short tandem repeats
• classes identified in accordance with the invention are defined in terms of terminal nucleotides so that a correspondence is established between the four possible terminal bases and the members of a set of spectrally resolvable dyes (Fung 1989). Such sets are readily assembled from the dyes of the invention by measuring emission and abso ⁇ tion bandwidths with commercially available spectrophotometers. More preferably, the classes arise in the context of the chemical or chain termination methods of DNA sequencing, and most preferably the classes arise in the context of the chain termination method, i.e., dideoxy DNA sequencing, or Sanger sequencing.
• This method involves the synthesis of a DNA strand by a DNA polymerase in vitro using a single- stranded or double-stranded DNA template whose sequence is to be determined. Synthesis is initiated at only the one site where an oligonucleotide primer anneals to the template. The synthesis reaction is terminated by inco ⁇ oration of a nucleotide analog that will not support continued DNA elongation.
• the chain-terminating nucleotide analogs are typically 2',3'- dideoxynucleoside 5'-triphosphates (ddNTPs) which lack the 3 -OH group necessary for 3' to 5' DNA chain elongation.
• dNTPs 2'-deoxynucleoside 5'- triphosphates
• enzyme-catalyzed polymerization will be terminated in a fraction of the population of chains at each site where the ddNTP can be inco ⁇ orated.
• labeled primers or labeled ddNTPs are used for each reaction, the sequence information can be detected by fluorescence after separation by high-resolution electrophoresis.
• dyes of the invention can be attached to either sequencing primers or dideoxynucleotides.
• polynucleotides are preferably separated by electrophoretic procedures (Rickwood and Hames; Osterman)
• the type of electrophoretic matrix is crosslinked or uncrosslinked polyacrylamide having a concentration (weight to volume) of between about 2-20 weight percent. More preferably, the polyacrylamide concentration is between about 4-8 percent.
• the electrophoresis matrix includes a strand separating, or denaturing, agent, e.g., urea, formamide, and the like. Detailed procedures for constructing such matrices are given by (Maniatis 1980; ABI PRISMTM 377 DNA Sequencer User 's Manual ).
• the optimal polymer concentration, pH, temperature, concentration of denaturing agent, etc. employed in a particular separation depends on many factors, including the size range of the nucleic acids to be separated, their base compositions, whether they are single stranded or double stranded, and the nature of the classes for which information is sought by electrophoresis. Accordingly application of the invention may require standard preliminary testing to optimize conditions for particular separations.
• the dye-polynucleotide conjugates are detected by measuring the fluorescence emission from the dye labeled polynucleotides.
• the labeled polynucleotides are illuminated by standard means, e.g. high intensity mercury vapor lamps, lasers, or the like.
• the illumination means is a laser having an illumination beam at a wavelength between 488 and 550 nm.
• the dye- polynucleotides are illuminated by laser light generated by an argon ion laser, particularly the 488 and 514 nm emission lines of an argon ion laser, or the 532 emission line of a neodymium solid-state YAG laser.
• argon ion lasers are available commercially which lase simultaneously at these lines, e.g., the Model 2001 from Cyonics, Ltd. (Sunnyvale, Calif).
• the fluorescence is then detected by a light-sensitive detector, e.g., a photomultiplier tube, a charged coupled device, or the like.
• the crude product was purified by silica gel chromatography to give N-methyl, N-(/n-hydroxyphenyl)-O-fluoro-benzamide as a white foam (1.2 gm, 5 mmole, 50%).
• Sodium hydride 200 mg, 60% dispersion in oil, 5 mmol
• the mixture was then refluxed for 2 hours and cooled to room temperature.
• Solvent was evaporated under vacuum and 5 ml hydrochloric acid (2M) was added.
• the mixture was extracted with ethyl acetate twice. The combined ethyl acetate extracts were washed with water and sat.
• Dimethyl sulfide/borane complex (3.75 ml, 7.5 mmole, 2M in THF) was added dropwise to the tricyclic amide (0.56 gm, 2.5 mmole) in 10 ml dry tetrahydrofuran cooled in an ice bath. The mixture was refluxed for one hour, cooled in an ice bath, and 10 ml methanol was added slowly. Solvent was evaporated under vacuum, more methanol was added, and evaporation was repeated thoroughly under vacuum. The crude product was purified by silica gel chromatography to give the tricyclic amine as a white solid (400 mg, 1.9 mmol, 76%).
• the fraction containing dye-labeled nucleotide and unlabeled nucleotide was concentrated in a vacuum centrifuge and subjected to a second HPLC using a reverse-phase column.
• the unlabeled nucleotide and each dye isomer of dye-labeled nucleotide were separated using an elution gradient of 15% acetonitrile/85% 0.1 M triethylammonium acetate to 35% acetonitrile / 65% 0.1 M triethylammonium acetate.
• the solutions containing dye- labeled nucleotide were concentrated in a vacuum centrifuge, redissolved in 10 mM carbonate/bicarbonate buffer, pH 9, and quantified by measuring the absorbance of the solution in a UV/Visible spectrophotometer. Yields were approximately 1%.
• the fraction containing dye-labeled oligonucleotide and unlabeled oligonucleotide was collected and subjected to HPLC purification on a reverse-phase column.
• the unlabeled oligonucleotide and each dye isomer of dye-labeled oligonucleotide were separated using an elution gradient of 10% acetonitrile/85% 0.1 M triethylammonium acetate to 30% acetonitrile/65% 0.1 M triethylammonium acetate.
• the solutions containing dye- labeled oligonucleotide were concentrated in a vacuum centrifuge, and redissolved in a buffer containing 10 mM Tris, 1 mM EDTA, pH 7.0 (TE).
• Reactions were set up in 0.5 ml tubes for the Perkin-Elmer 480 DNA Thermal Cycler (PE Biosystems). Total reaction volumes were 20 ⁇ l, including 15 ⁇ l of the above reaction premix, an appropriate amount of dye labeled terminator, and water.
• Dye terminator reactions were set up with either 1 pmole of dye terminator for A and G terminators or with 15 pmole of dye terminator for C and T terminators, with dyes of the present invention. In a few cases, the dye terminators for C or T were at too low a concentration, such that 5 ⁇ L resulted in less than 15 pmole of dye terminator. In these cases, 5 ⁇ l of dye terminator was used and no water was added to the reaction.
• the dried samples were resuspended in 25 ⁇ l of Template Suppression Reagent (PE Biosystems), vortexed, heated to 95 °C for 2 minutes, cooled on ice, vortexed again, and centrifuged (13,000 x g). 10 ⁇ l of the purified sample was aliquoted into sample vials adapted for use with the PE ABI PRISMTM 310 Genetic Analyzer (PE Biosystems). Electrophoresis on the Model 310 used a 61 cm long , 50 ⁇ m ID uncoated fused silica capillary having a length to the detector of 50 cm.
• PE Biosystems Template Suppression Reagent
• the capillary was filled with a solution of a linear dimethylpolyacrylamide (DMA) sieving polymer (Madabhushi), buffer, and containing nucleic acid denaturants (PE Biosystems). Samples were electrokinetically injected for 30 sec at 2.5 kV. Electrophoresis was performed for 2 hr at 12.2 kV with the capillary temperature maintained at 42 °C.
• DMA linear dimethylpolyacrylamide

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