CA1338597C - Reagents for the preparation of 5'-tagged oligonucleotides - Google Patents

Reagents for the preparation of 5'-tagged oligonucleotides

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Publication number
CA1338597C
CA1338597C CA000611327A CA611327A CA1338597C CA 1338597 C CA1338597 C CA 1338597C CA 000611327 A CA000611327 A CA 000611327A CA 611327 A CA611327 A CA 611327A CA 1338597 C CA1338597 C CA 1338597C
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group
compound according
reporter
alkyl
dna
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French (fr)
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Frank Worden Hobbs Jr.
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PerkinElmer Health Sciences Inc
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EI Du Pont de Nemours and Co
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07FACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02Phosphorus compounds
    • C07F9/547Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Abstract

Reagents useful in the preparation of 5'-fluorescence-tagged oligonucleotides are disclosed. A class of oligonucleotides is also disclosed.

Description

1338~97 TITLE

Reagents For The Preparation of S'-Tagged Oligonucleotides BACKGROUND OF THE I NVENTION

Field of the Invention This invention relates to nonnucleoside-containing, fluorescence-tagged, phosphorus reagents, which are useful in the preparation of 5'-tagged oligonucleotides. A
class of 5'-fluorescence-tagged oligonucleotides is also disclosed as part of this invention.

Summary of the Background Deoxyribonucleic acid (DNA) is the molecule that stores the information needed to direct all processes in living systems. It i~ a polymer composed of four mononucleotide ~ubunits linked by phosphodiester bonds. Naturally occurring DNA is usually found in a double-stranded form with two complimentary linear polymers held together by hydrogen bonds.
Double-stranded DNA can be dissociated into single strands of DNA and, conversely, complimentary single-stranded DNA will associate to form double-stranded DNA.
Although the terms "DNA~ and "oligonucleotide" are often used interchangeably, "DNA" is used herein to refer to large ( ~ 100 nucleotides long) or naturally occurring molecules, especially those being subjected to CR-8663 35 various assays. "Oligonucleotide" is used herein to refer to pieces of single-stranded DNA that ,~.
2 133~97 are small enough to be made by current, practical chemical synthesis ( < 100 nucleotides long).
The distinction between the terms ~DNA" and "oligonucleotide, n however, $s recognized to be somewhat artificial. DNA can be broken into well-defined pieces that are ~mall enough to be considered as oligonucleotides, and chemically synthesized oligonucleotides can be joined enzymatically to make double-stranded polymer6 large enough to be called DNA and to direct life processes.
The ability to introduce reporter~ into oligonucleotides and DNA is, in part, responsible for the recent explosive growth in the field of molecular biology. A reporter can be defined as a chemical group that has a physical or chemical characteristic readily measurable or detectable by appropriate physical or chemical detector systems or procedures. Ready detectability can be provided by ~uch characteri~tics as color change, luminescence, fluore~cence, and radioactivity or it may be provided by~the ability of the reporter to serve as a ligand recognition site to form specific ligand-ligand complexes in which the second ligand contains a group detectable by conventional (e.g., colorimetric, ~pectrophotometric, fluorometric or radioactive) detection procedures. The ligand-ligand complexes can be in the form of protein-ligand, enzyme-substrate, antibody-antigen, carbohydrate-lectin, protein-cofactor, protein-effector, nucleic acid-nucleic acid, or nucleic acid-ligand complexes. The complex formed between biotin and avidin is an example of such a ligand-ligand complex.
Althouqh high specific activity 32p has generally been used to tag oligonucleotides as well as DNA for a variety of applications, the use of this radioisotope is problematic from both a logistical and a health standpoint. The short half-life of 3 2 p necessitates the anticipation of reagent requirements several days in advance and prompt use of such a reagent. Once 3 2 P-taqged DNA sequencing fragments have been qenerated, they are prone to self-destruction and must be immediately subjected to electrophoretic analysis. Subsequent autoradiography required for visualization of the labeled DNA fragments in the electrophoretic gel is a slow process (overnight exposures are common). Finally, possible health risks are associated with the use and disposal of such potent radioisotopes.
To address these problems, replacement of 3 2 P/autoradiography with alternative, nonradioisotopic reporter/detection systems has been considered. New reporter/detection systems must be exceptionally sensitive to replace 3 2 p.
In one sense, DNA can be its own ~reporter"
because it can be detected by ultraviolet ( W) light absorption. Many important assays, however, require that DNA be detected at concentrations many orders of magnitude below concentrations at which DNA can be detected by W
absorbance. DNA sequencing, for example, requires reporter/detection systems that can detect 10-16 mole (or about 10C molecules) of DNA. Therefore, practical non-isotopic reporter/detection systems must offer sensitivity at least comparable to that of 32p. Hereafter, the term "reporter" shall refer only to chemical 4 133~97 groups capable of replacing high specific activity 3 2 p.
Prober, et al., Science 238, 336-41 (1987) and Smith, et al., Nature 321, 674-79 (1986), have shown that, in conjuction with an argon laser and filtered photomultiplier tube detection system, certain fluorescent dyes can replace 3 2 p as reporters for DNA sequencing. To achieve the re~uired sensitivity, these dyes were carefully selected for their strong absorption at the wavelength of the argon la~er, their high quantum efficiency of fluorescent emission, and the ability to distinguish their fluore~cent emission from background signals.
The ability to introduce readily detected reporters at a specific site in DNA is ab601utely critical to many methods of analyzing DNA. For example, all currently known method~
for sequencing DNA require that several hundred different oligonucleotides be separated by gel electrophoresi6. (About 10-l5 to 10-16 mole of each oligonucleotide is generally prebent.) Therefore, it is critical that the reporter does not complicate separation by gel electrophorecis.
3 2 p is an ideal reporter in this respect because substituting 3 2 p for nonradioactive 31 p has no effect on gel electrophoresis. When a nonradioactive reporter, such as a fluorescent dye, ifi attached to an oligonucleotide, the electrophoretic mobility of that oligonucleotide changes. If only a single reporter i~ attached to the oligonucleotide at a preci~ely defined location, such changes are uniform and tolerable.
If, however, a variable number of reporterc are attached or if a single reporter ifi attached to a s 1338~97 variety of positions, electrophoretic analysis becomes impossible.
DNA amplification by the polymerase chain reaction (PCR) is another technique for analyzing DNA that requires separation by gel electrophoresis. Preferably, oligonucleotides used in this method will al60 have a non-isotopic reporter at a single location.
Although several methods of non-site-specific enzymatic tagging of DNA are known, only one type of site-specific tagging with non-isotopic reporters i5 known. The enzyme, terminal transferase, i6 capable of adding a variety of modified and/or tagged nucleotides to the 3'-end of an oligonucleotide.
This enzyme affects single-site tagging only when the 3'-hydroxyl group of the modified and/or tagged nucleotide is removed or changed.
Unfortunately, DNA tagged by this method cannot be used in many enzymatic assays. DNA sequencing and amplification, for example, require that the tagged oligonucleotides used in these assays have a normal hydroxyl group at the 3'-end.
Many chemical methods for tagging DNA
have been developed, but most of these involve non-site-specific reactions, thereby producing tagged DNA that is not suitable for analysis by gel electrophoresis. Site-specific tagged oligonucleotides have been prepared by total, i.e., chemical, synthesis. With one exception, this has always been done by synthesizing an oligonucleotide possessing an added group with unusual reactivity, e.g., an aliphatic amino group or a thiol. In this approach, the added 3S amino or thiol groups have either replaced the 5'-hydroxyl group or have been added to the 6 1338~97 5'-hydroxyl group by means of a linker or have been added to the base by means of a linker.
This site-specific tagging process comprises:
(i) preparation of a monomeric nucleotide reagent that contains a protected form of the unusually reactive group; (ii) chemical synthesis and purification of the desired oligonucleotides with the unusually reactive group, usually with conco~itant deprotection of t~.e unusually reactive group; and (iii) selective attachment of a fluorescent dye (or other reporter) to the unusually reactive group.
Examples of this and related approaches have been disclosed by Draper, et al., Biochemistry l9, 1774-81 (1980); Smith, DE
3,446,635 Al (19B5); Smith, et al., Nucleic Acids Res. 13, 2399-2412 (1985); Coull, et al., ~etrahedron Lett. 27, 3991-94 (1986); Sproat, et al., Nucleic Acids Res. 15, 4837-48 (1987);
~-0 Sproat, et al., Nucleic Acids Res. 15, 6181-96 (1987); Tanaka, et al., Tetrahedron Lett. 28, 2611-14 (1987); Tanaka, et al., Nucleic Acids Res. 15, 6209-24 (1987); Agrawal, et al., Nucleic Acids Res. 14, 6227-45 (1986); Connolly, Nucleic Acids Res. 15, 3131-39 (1987); Connolly, et al., Nucleic Acids Res. 13, 4485-4502 (1985); and Sinha, et al., Nucleic Acids Res. 16, 2659-69 (1988).
~otally synthetic site-specific tagging approaches present several problems in the synthesis of tagged oligonucleotides.
First, it is a multi-step procedure involving synthesis and purification of a modified oligonucleotide, addition of the reporter to the reactive group of this modified oligonucleotide, and a final purification.

13~8~97 Second, both DNA sequencing and DNA
amplification require that the tagged oligonucleotide be a substrate for a DNA
polymerase. secauSe these polymerases catalyze reactions at the 3'-end of the oligonucleotide, the 5'-end of the oligonucleotide i6 the preferred site for attaching non-isotopic reporters. When the unusually reactive group is attached to or incorporated within a nucleotide, thiC approach lacks versatility. The 5~-nucleotide can be dA, dT, dC or dG; therefore, four appropriate reagents are needed for incorporating an unusually reactive group along with the desired 5'-nucleotide. Because these reagent6 are typically air- and moisture-sensitive and have a limited shelf-life, the need to stock a set of at least four reagents is burdensome.
Third, if a linking group is used to introduce the unusually reactive functional group onto the 5'-position, additional problems ari6e.
It is frequently difficult to determine whether the unusually reactive group has been successfully linked to a synthetic oligonucleotide. Because the reagents used to attach the unusually reactive group to the oligonucleotide have a limited shelf-life, failure to incorporate the desired reporter is common.
Fourth, when problems are encountered, it is usually difficult to determine which step failed.
Fifth, large excesses of the reporter are generally used for successful coupling to the unusually reactive group. This both wastes the 8 1338~97 reporter and complicates the purification of the oligonucleotide.
In the one exception to the totally synthetic site-specific tagging approaches described above, Prober, et al., EP-~ 252,683 (1988), have outlined a more direct and reliable method for synthesizing fluorescence-taqged oligonucleotides for DNA sequencing. An unusually reactive functional group was not used in this approach. Instead, a fluorescent reporter was attached directly to a nucleotide before the nucleotide was incorporated into the desired oligonucleotide.
The principal disadvantage of this method i5 that it relies on attachment of the reporter to a specific nucleotide and therefore lacks versatility. The resulting fluorescence-tagged oligonucleotidc was used in a DNA sequencing system that calls for four distinguishable fluorescent dyes. Complete versatility would require a set of 16 different fluorescence-tagged nucleotide reagent~ suitable for the synthesis of oligonucleotides. The reagents are also air- and moisture-sensitive and have a limited shelf-life.
The purpose of the present invention is to overcome the problems encounterea in the prior art by providing nonnucleoside-containing, fluorescence-tagged, phosphorus reagents to ~roduce 5'-tagged oligonucleotides. The reagents disclosed in the present invention are easier to prepare and are more versatile than are the compounds found in Prober, et al., ~P-A 252,683.
The presence of the reporters of the present invention in the resulting oligonucleotides does not interfere with analysis by gel 9 1338~97 electrophoresis or with use in DNA sequencinq or DNA amplificaton. Fewer steps are required and the chances for error or confusion have been reduced when these reporters are used.
The 5'-tagged oligonucleotide~ of the present invention can be used as labeled primer~
for automated DNA sequencing and for DNA
amplification by the polymerase chain reaction (PCR).
SUMMARY OF THE INVENTION

The present invention provides chemical reagents of the formula reporter-A-Q

wherein the reporter is selected from the group consisting of protected fluorescent reporters and ~0 unprotected fluorescent reporters; A is selected from the group consisting of -O-, -S-, -NR, and -CRRl, wherein R and Rl are independently selected from the group consisting ~5 of H, C3-C10 branched alkyl, Cl-Cl0 unbranched kyl~ C6-Clo aryl~ C~-Cl 2 alkaryl, and C -C
aralkyl; and Q is selected from the group consisting of OH O O

-P-NR R3, -P-N R , -P-OH, -P-OH, and -P-X

Y Y H Y

and salts thereof, wherein R2 and R3 are lo 1338~97 independently selected from the group consistinq of C3 -C10 branched alkyl and Cl-C10 unbranched alkyl; R~ is selected from the group consisting of -(CRR ).(A) n ( CRRl ) - ~

C~( C R R ~ C 2~ c H 2-wherein R, Rl, and A are as defined above, ~ 6, n ~ 0-1, p - 1-10, and q - 0-10, provided that 2m+n < 12 and further provided that 2 ~ n+p+q < 13; X is selected from the group consisting of -F, -Cl, -Br, -I, imidazol-l-yl, 1,2,4-triazol-1-yl, tetrazol-1-yl, and lS 1-hydroxybenzotriazol-O-yl; and Y is any phosphate-protecting group.
Additionally, the present invention includes a class of 5'-fluorescence-tagged oligonucleotides. These oligonucleotides are conveniently prepared using the reagents of the present invention. The general structure of this class of oligonucleotides is R~ Rs 0~0~ 0' 25p6 ~ p~

o~N~ o IJ~ ,, s ~

o- ~ -o--j 0 ~
--n HO R~
wherein Rs and R6 are independently selected from the group consisting of -H, Cl-C~ alkyl, -F, -Cl, -sr, -I, Cl-C~ alkoxy, and -CN; B is selected from the group consisting of 1-thyminyl, 1-cytosinyl, l-uracilyl, 9-adeninyl, and 9-guaninyl; ~9 is selected from the group consisting of -H and -OH; and n ~ O to about 100.

LRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a plot of fluorescent emission against time observed during electrophoresis for the four s~mples prepared in Example 7. The fi~ure demonstrates that primer extension products derived from 5'-fluorescence-tagged oligonucleotides can be detected by their fluorescent emission. The figure al60 demonstrates that the sequence of bases in the DNA template can be deduced from this plot.
Figure 2 shows a plot of fluorescent emission against time o~served during electrophoresis of the amplified sample of DNA
produced in Example 8 and a similar plot of size markers. This figure demonstrates that amplification products derived from 5'-fluoresence-tagged oligonucleotide primers can be detected by their fluorescent emis6ion. The figure also demonstrates that the major product of the amplification process is, as expected, a fluoresence-tagged oligonucleotide of approximately 337 base pairs in size.

DETAILED DESCRIPTION OF THE INVENTION

Nonnucleoside-containing, fluorescence-tagged phosphorus reagents of the present invention are useful in the preparation of 5'-tagged oligonucleotides. These reagents are of the formula 12 1338a97 reporter-A-Q

wherein the reporter is ~elected from the group consisting of protected fluorescent reporters and unprotected fluorescent reporters; A is selected from the group consisting of -O-, -S-, . I I
-NR, and -CRRl, wherein R and Rl are independently selected from the group consi~ting of H, C3 -Cl0 branched alkyl, Cl-Cl0 unbranched alkyl~ C6-Clo aryl~ C~-Cl 2 alkaryl, and C~-C
aralkyl; and Q is selected from the group consistinq of OH O O

-P-NR2R , -P-N~ R , -P-OH, -P-OH, and -P-X

Y Y H Y

(1) (2) (3) (4) (S) and salts thereof, wherein R2 and R3 are independently selected from the group consisting of C3 -C10 branched alkyl and Cl-Cl0 unbranched alkyl; R' is selected from the group consisting of -(CRR )~(A) (CRRl) -, (A)n-(CRR')p-3 0 ~ ~ a r~ H ."Lr\ ~ H
~CRRl)q- -CH2 \J~CH2 wherein R, Rl, and A are as defined above, m - 1-6, n - 0-1, p - 1-10, and q ~ 0-10, provided that 2m+n < 12 and further provided that 2 < n+p+q < 13; X is selected from the group consisting of -F, -Cl, -Br, -I, imidazol-1-yl, 1,2,4-triazol-l-yl, tetrazol-1-yl, and 1-hydroxybenzotriazol-O-yl; and Y is any phosphate-protecting group. The reporter ~s covalently linked to the activatable phosphorus group, Q.
Preferably, A is -O-, in which case these reagents are commonly known (with reference to the activatable phosphorus group, Q) as phosphoramidites (1 and 2), phosphorous acids (3), H-phosphonates (4), and activated phosphodiesters (5). Structures (3) and (4) represent ~oderately strong acids, and the reagents represented by these structures are generally isolated and used as their organically soluble salts.
The H-phosphonate forms ( 4 ) of these reagents are generally in equilibrium with the phosphorous acid forms (3), with the H-phosphonates strongly favored. Analogous equilibria are established when A is -S-, - NR, or - CRR .
The phosphorus group, Q, includes Y, which can be any phosphate-protecting group.
Preferably, Y is selected from the group consisting of 4-Cl-C6H~-O-, 2-Cl-C6H~-O-, NO2 C6 H4--O--~ 4--~02--C6 H~ CH2 CH2--O--, 2, 4-NO2--C6 H3 CH2 CH2--O- ~ 2 ~ 4--C12--C6 H3--O- ~
2,3--Cl2--C6 H3--O-, NCCH2 CH2 O-, NCCH2 C ( CH3 ) 2 ~~ ' CH 0--, (Z)3CCH2--O-, (Z)3CC(CH3 )2 , R
R ' SCH2 CH2 -O-, R ' SO2 CH2 CH2--O-, and R ' NH--, wherein Z is selected from the group consisting of Cl, Br, and I, and R' is selected from the group consisting of H, C3-Cl o branched alkyl, Cl-ClO
unbranched alkyl, C6-C1o aryl, C7-Cl 2 alkaryl, 14 1338~97 and C~-Cl 2 aralkyl. The most preferred Y groups are NCCH2CH2O-, CH~O-, and 2-cl-c6H~-o-.
A suitable fluorescent reporter is one that can be detected in its unprotected form at or below the level of detection that can be quickly achieved with 32p, i.e., about 10-1~
moles. Specific desirable characteristics may include a large coefficient of extinction in the region of excitation, a high quantum yield, an optimal excitation or emission wavelenqth (preferably above 350 nm), and photostability.
For example, fluorescent dyes that are efficiently excited by an argon laser are desirable because of the low cost of this laser.
Preferably, in its unprotected form, the reporter is a fluorescent dye chosen from the group consisting of xanthenes (e.g., fluoresceins, eosins, erythrosins), rhodamines (e.g., tetramethylrhodamine, Texas RedQ), benzamidizoles, ethidiums, propidiums, anthracyclines, mithramycins, acridines, actinomycins, merocyanines, coumarins (e.g., 4-methyl-7-methoxycoumarin), pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes (e.g., 2~ dansyl, 5-dimethylamino-1-naphthalenesulfonyl), salicylic acids, benz-2-oxa-1-diazoles (also known as benzofurazans) (e.g., 4-amino-7-nitrobenz-2-oxa-1,3-diazole), and fluorescamine. Useful forms of many of these dyes are commercially available. For a review of fluorescent dyes used in tagging DNA, see A. S.
Waggoner, Chapter 1, Applications of Fluorescence in the siomedical Sciences, ed. by D. L. Taylor, et al., Alan R. Liss, New York (1986).
Most preferably, the reporter is a xanthene, especially a xanthene dye represented by the structure ~'C(.O~OX~OC(=O)~

O N~

wherein Rs and R6 are independently selected from the group consisting of -H, Cl-C~ alkyl, -F, -Cl, -Br, -I, C1-C~ alkoxy, and -CN; R is Cl-C~
alkyl; and ~ is selected from the group consisting of alkyl or aryl.
Using methods known in the art, a covalent link can be made between these dyes and the activatable phosphorus group, Q. For reasons of synthetic ease and stability, Q is usually attached to an oxygen, which was formerly part of a hydroxyl group in the reporter. In some cases, the activatable phosphorus group, Q, can be attached directly to the fluorescent dye at a site that does not interfere with its utility as a reporter. In other cases, a covalent linkage can be formed by selectively attaching one of these dyes to one end of a small, difunctional molecule and the activatable phosphorus to the other end of this molecule. Most of the fluorescent reporters listed above are available commercially in a form suitable for attachment to a small, difunctional molecule.
In some cases, it may be necessary to protect sensitive functional groups on the reporter during the attachment of the activatable phosphorus group, activation of the phosphorus 16 1338~97 qroup, or attachment of the phosphorus group to the 5'-hydroxyl qroup of the oligonucleotide.
The nature of the protecting group(s), if present, will depend on the sensitive functional groups on the reporter. The preferred xanthene dyes of this invention have nucleophilic hydroxyl groups that need protection. Methods for protecting and deprotecting a wide variety of functional groups are known in the art and have been reviewed in J. F. W. McOmie (ed.), Protective Groups in Organic Chemistry, Plenum Press, New York (1973).
~ecause automated DNA synthesizers generally use the phosphoramidite approach to oligonucleotide synthesis, the preferred phosphorus reagents of this invention are phosphoramidites, i.e., when A is -O- and Q is selected from the group consisting of -P-NR R and -P-N _R

Y y (1) (2) employed with xanthene dyes, preferably of the 6tructure specified above. A preferred embodiment is wherein R2 and R3 of the activatable phosphorus group, Q, are -CH(CH3) 2 .
The reagents discussed above are useful in the preparation of 5'-fluorescence-tagged oligonucleotides. Appropriate processes for using these reagents to form a covalent bond between the activatable phosphorus group, Q, and the 5'-hydroxyl group of an oligonucleotide are known. These processes can be combined with 17 1338~97 other known methods of synthesizing oligonucleotides to prepare 5'-fluorescence-taqged oligonucleotides. For a general review of the field of oligonucleotide synthesis, see M.J.
Gait (ed.), Oligonucleotide Synthesis, A
Practical Approach, IRL Press, Oxford (1984). In cases wherein the fluorescent reporter is used in a protected form, an additional deprotection step may be required.
In addition to the disclosed reagents, - the present invention includes a class of 5'-fluorescence-tagged oligonucleotides. These oligonucleotides are conveniently prepared using the reagents of the present invention and processes known in the art of oligonucleotide synthesis. The general structure of this class of oligonucleotides is ~'R~

~0--1' 0 ~

O' o--j 0 ~
--n HO Rs wherein R5 and R6 are independently selected from the group consisting of -H, C1-C~ alkyl, -F, -Cl, -E3r, -1, Cl-C~ alkoxy, and -CN; B is selected from the group consisting of l-thyminyl, l-cytosinyl, 1-uracilyl, 9-adeninyl, and 9-guaninyl; R9 is selected f{om the group 18 1338a97 consisting of -H and -OH; and n - 0 to about 100.
These oligonucleotides can be used in automated fluorescence-based DNA sequencing, according to the methods described in Prober, et al., Science 238, 336-41 (1987) and An~orge, et al., J. Biochem. Biophys. Methods, 13, 315-23 (1986). Additionally, they can be used in DNA
amplification by the polymerase chain reaction (PCR) method, according to the methods described in U.S. 4,683,195 and U.S. 4,683,202.

EXAMPLES
General Procedure The following Examples illustrate, but do not limit, the compounds and utilities of the p~esent invention. Examples 1-4 demonstrate the preparation of the claimed reagents; Examples 5 and 6 disclose the claimed class of 5~-fluorescence-tagged oligonucleotides and describe a process by which they can be produced;
and Examples 7 and 8 refer to the utility of these reagents and 5'-fluorescence-tagged oligonucleotides.
The following diagram is referred to in the Examples. Structures 7a-d, 8a-d, and 9a-d are protected forms of the preferred xanthene reporters. The activatable phosphorus group, Q, which is P(OCH2CH2CN)(N(i-Pr)2) in the diagram, is added to the reporter between 8 and 9. The last structure summarizes the fluorescent-tagged oligonucleotides of the present invention.

19 133~597 Rs R5 Rs R5 ACO`~O`h,OAc AcO~,O~OAc R6 ~ R6 , R6 ~ R6 O O ON~
o~NyO O H
\J 8a-d 7a-d R5 Rs 1 5 R6~XOAc J OEt ~N~l~ ,l~CN

2 0 O `N~i-Pr)2 Rs Rs 9a-d R6~R' O~N--~ O B
~O-P---0~~
O ` '~ B

SFxxx-4HP-p Group ll n H O R

a: Rs = R6 = H. b: Rs = H, R6 = CH3. c: Rs = CH3, R6 = H. d: Rs = R6 = CH3.
xxx = 505. xxx = 512. xxx = Sl9. xxx = 526.

All temperatures are in degrees Celsius (25C refers to ambient or room temperature).
All parts and percentages not otherwise indicated are by weight except for mixtures of liquids, which are by volume. The following abbreviations are employed: DMF for dimethylformamide; DMSO
for dimethylsulfoxide; SF for succinylfluorescein; NMR for nuclear magnetic resonance spectrum; IR for infrared spectrum; UV
for ultraviolet spectrum or detection; TLC for thin layer chromatography on silica gel; HPLC for high pressure liquid chromatogrphy; GC for gas chromatography; mp for melting point; mp d for melting point with decomposition; and bp for boiling point. In reporting NMR data, chemical shifts are given in ppm and coupling constants (J) are given in Hertz. All melting points are uncorrected. Ion exchange resins were washed with appropriate aqueous and organic solvents prior to use. The identity of all compounds described herein was established by appropriate spectroscopic and ananlytical techniques. Unless other noted, purification by chromatography on silica gel was performed as described by Still, et al., J. Org. Chem. 43, 2923-26 (1978).

Example 1 Preparation of Phosphoramidite 9a Step 1: Preparation of N-(3-(3,6-diacetoxy-9-ethoxy-9H-xanthen9-yl)propionyl)-4-hyroxypiperidine, 8a.
A solution of 4-hydroxypiperidine (506 mg, 5.00 mmol, 2.5 eq, Aldrich) and 9-(2-(N-succinimidyloxy-carbonyl))ethyl)-3,6-diacetoxy-9-ethoxy-9H-xanthene, 7a (1.023 g, 2.00 mmol, prepared according to Prober, et al., EP-A
252,683) in dry dichloromethane (20 ml) was stirred for 30 min. The reaction was added to 1 M aqueous potassium phosphate buffer (30 mL; pH
- 7) and extracted with dichloromethane ~3 x 30 mL). The organic extracts were dried over calcium sulfate, concentrated, and dried under vacuum overnight to afford crude amide 8a (1.039 g, 104%) as an off-white foam. This material was > 90% pure according to NM~ and TLC and was used without further purification in the next step.
lH-NMR (DMSO-d6): 7.58 (m, 2H, ArH), 7.04 (m, 4~, ArH), ~.66 (d, J-4, lH, OH), 3.72 (br m, lH, NCH2 ), 3.57 (app octet, lH, CHOH), 3.17 (br m, lH, NCH2b), 2.88 (q, J-7, 2H, OCH2CH3), 2.83 (m, 2H, NCH2 and NCH2 ), 2.29 (s, 6H, OAc), 2.22 (app dist t, J-8, 2H, CH2CO), 1.73 (app dist dd, 2H, CH2Ar), 1.58 (m, 2H, CH2-CHOH), 1.11 (m, 2H, CH2bCHOH), and 1.03 (t, J-7, 3H, OCH2C~3). TLC (9:1 dichloromethane-methanol;
UV): starting material 7, Rr ~ 0.87;
amide product 8a, 0.49.

Step 2: Preparation of 2-cyanoethyl (N-(3-(3,6-diacetoxy-9-ethoxy-9H-xanthen-9-yl)propionyl)-piperidin-4-yl)oxy N,N-diisopropyl phosphoramidite, 9a.
Crude amide 8a (1.00 g, ca. 1.98 mmol) was coevaporated with dry pyridine (1 x 10 mL) and dry toluene (2 x 10 mL) and then vacuum dried. Dry dichloromethane (15 mL), dry diisopropylamine (0.14 mL, 1.00 mmol, 0.5 eq), tet~zole (70.0 mg, 1.00 mmol, 0.5 eq, Aldrich ~old Label), and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.76 mL, 2.41 mmol, 1.2 eq, Aldrich) were added sequentially.

22 1338~97 -After stirring the resulting b,olution for 2 hours, the reaction mixture was added to 30 mL of 1 M aqueous potassium phosphate buffer (30 mL, pH
~ 7) and extracted with ether (3 x 30 mL). The ether extracts were dried over calcium sulfate and concentrated. The residue was chromatographed on silica ~el (100 g) with a 70:28:2 mixture of dichloromethane, ethyl acetate and pyridine. The first UV absorbing component to elute was concentrated, coevaporated with dry toluene (2 x 30 mL), and vacuum dried to afford phosphoramidite 9a, (893 mg, 65%). Except for the presence of residual toluene (16 mole%), this material was > 95% pure by lH- and 31 P-NMR and lS TLC. According to 31 P-NMR, this material was stable in dry DMSO-d6 in a normally sealed NMR
tube for at least a week at room temperature.
lH-Decoupled lP-NMR (DMSO-d6): 146.8 (s). P-Coupled lH-NMR (DMSO-d6): 7.58 (d, 2H, ArH), 7.23 (m, 0.32H, toluene), 7.18 (m, 0.48H, toluene), 7.04 (m, 4H, ArH), 3.94 (m, lH, CHOP), 3.68 (m, 2H, CH2OP), 3.57 (m, 2H, NCH), 3.49 (br m, lH, NCH2-), 3.18 (br m, 2H, NCH2b and NCH2'), 2.96 (br m, lH, NCH2 ), 2.88 (q, J~7, 2H, OCH2CH3), 2.74 (t, J-6, 2H, CH2CN), 2.31 (s, 0.48H, toluene), 2.30 (s, 6H, OAc), 2.23 (app dist t, J-8, 2H, CH2CO), 1.57 (app dist dd, 2H, ArCH2), 1.63 (br m, 2H, CH2-COP), 1.37 (br m, 2H, CH2 COP), 1.13 (app dist t, 12H, CH(CH3 )2 ) ~ and 1.03 (t, J~7, 3H, OCH2CH3).

Examples 2-4 Preparation of Phosphoramidites 9b-9d Phosphoramidites 9b-9d were prepared from N-hydroxysuccinimidyl esters 7b-7d as 23 1338~97 described for phosphoramidite 9a in Example 1.
The final phosphoramidites were purified on silica gel in the presence of pyridine. Pyridine (4%) in toluene was found to be the preferred eluent. Chromatography fractions were preferably analyzed by TLC on silica gel plates that had been deactivated by treatment with 5% pyridine in pentane. Fractions containing pure phosphor-amidite were combined, coevaporated with dry pyridine (4 x 10 mL) and dry toluene (2 x 10 mL), and vacuum dried.
H-Decoupled 1 P-NMR ( DMSO-d6 ) -:
9b 147.0 (s) and 146.9 ~s).
9c 146.9 (s) 9d 147.0 (s) and 146.9 (s).
In the cases where two phosphorus signals were observed by NMR, the two signals coalesced to a single signal upon warming to 70. (The NMR
spectra of all of the above compounds were best explained by variable restricted rotation about the amide bond.) Example 5 Preparation of SF505-4HP-pGTTTTCCCAGTCACGAC, An Oligonucleotide with the Fluorescent Reporter SF505-4HP-p Attached to the 5'-Oxygen Automated oligonucleotide synthesis was performed on a Du Pont Code ~ 300 according to the general methods described in the operator's manual. The sequence "5' XGTTTTCCCAGTCACGAC 3"' was entered and the following options were ~elected: ( 1 ) Use capping ~tep? YES .
(2) Remove 5' terminal DMT? NO. (3) Collect DMT? YES . ( q ) Synthesis scale 1 ~mole. The instrument was charged with reagents supplied 24 1338a97 commercially by Du Pont and an 0.1 M solution of phosphoramidite 9a in dry acetonitrile was placed on the ~X" port using a manual line purge (3 x 50 ~L) instead of the automated bottle change function. The starting material in the synthesis column was N-anisoyl-5'-dimethoxy-trityl-2'-deoxycytidine (1 ~mol) on long chain alkylamine controlled pore glass. Automated synthesis was run without any modification during or after the use of phosphoramidite 9a. Analysis of the dimethoxytrityl cation released indicated that the overall yield of untagged 17-mer on the ~olid support was 75% before phosphoramidite 9a was used. (The average coupling efficiency per cycle therefore was 98.3%.) After automated synthesis was complete, the solid support was removed from the synthesis column and stirred for 1 hour with concentrated ammonium hydroxide (4 mL). The solid support was removed by filtration through a plug of glass wool into a vial and the vial was topped off with additional concentrated ammonium hydroxide (approximately 1 mL). The vial was tightly sealed and heated at 55 for 4 hours. (Longer deprotection can cause production of a significant amount of nonfluorescent oligonucleotide side product.) After cooling, the ammonia solution was concentrated under vacuum.
The deep orange residue was dissolved in water (1000 ~L). An aliquot (10 ~L) of this solution and 0.05 M aqueous Tris buffer (990 ~L, pH 8.2) were placed in a 1 cm pathlength UV cell.
The absorbance was O . 813 and O .186 at the maxima at 260 and 493 nm respectively, indicating that the crude product amounted to 81.3 ODU (260 nm) or lB.6 ODU (493 nm). Assuming the absorption coefficient of the chromophore (72,600 at 493 nm and 23,000 at 260 nm; see Prober, et al., Science 238, 336-41 (1987)~ is unchanged by attachment to an oligonucleotide, the yield of crude fluorescent oliqonucleotide was 25%.
Analysis of the crude product by HPLC (2~ cm C8 reverse phase column eluted at 1 mL/min for 25 minutes with a gradient of 0-25% acetonitrile in 0.1 M aqueous triethylammonium acetate) with detection at 260 nm and 500 nm showed that the largest peak at 260 nm was the only sig~ificant product ~ > 90%) absorbing at 500 nm.
The remainder of the crude product was purified by preparative HPLC on a 300 A C8 Dynamax~ column (1 x 25 cm). The column was eluted with a 5-20% gradient of acetonitrile in 0.1 M aqueous triethylammonium acetate over 35 minutes with a flow rate of 5 mL/min. The major peak (22 minutes) was collected and dried under vacuum. The purified oligonucleotide was dissolved in water and assayed by UV as before.
The yield was 38.6 ODU (260 nm) and 15~2 ODU (493 nm). Assuming the absorption coefficient of the oligonucleotide product is the sum of the absorption coefficients of its subunits (192,000 at 260 nm), the yield of purified product was 20%. ~he ratio of the absorbances observed at 493 and 260 (2.54:1) for the purified product was within experimental error of theoretical ratio (2.64:1) calculated for a product containing one SF505 chromophore per oligonucleotide. The product was lyophilized, dissolved in sterile di6tilled water, and stored frozen at -25 until used.

26 1338~97 Example 6 Preparation of Other Fluorescently Tagqed Oligonucleotides Following the procedures of Example 5, SF512-4HP-p-, SF519-4HP-p-, and SF526-4HP-p-groups were attached to the 5'-end of the same oligonucleotide with phosphoramidites 9b-9d. The UV maxima of the products were 500, 511, and 518 nm respectively.
Subsequently, following the procedures of Example 5, succinylfluorescein dyes were attached to oligonucleotides with different sequences of bases.

Example 7 DNA Sequencing with an SF505-4HP-p-Tagged Primer Using a Modified Sanger Chain Elongation Protocol The following components were mixed in each of four 1.5 ~L Eppendorf tubes: 12 ~L M13 mpl8 single-stranded DNA template (N. E. BioLabs;
0.25 ~g/~L), 3 ~L sF5o5-4Hp-pGTTTTcccAGTcAcGAc primer solution (Example 5; 0.30 ODU (260 nm)/~L - 1.5 ~M), 3 ~L 10X Taq polymerase reaction buffer (166 mM (NH~ )2S~; 670 mM
Tris-HCl, pH 8.8; 67 mM MgCl2; 100 mM
b-mercaptoethanol; 67 ~M EDTA; 1.7 mg/mL bovine serum albumin), and 5 ~L H2O. Each tube was heated for two minutes in a boiling water bath, then allowed to cool to room temperature over a 15--minute period. Five ,uL of a nucleotide solution were added to the four tubes; one tube receiving the ddA mix, another the ddC mix, another the ddG mix, and another the ddT mix.

27 13 3 8 ~ 9 7 These mixes contain the following concentrations of dideoxy- and deoxynucleotide triphosphates:

ddA Mix ddC Mix ddG Mix ddT Mix ddATP 300 mM -- -- --ddC~P -- 100 m -- --ddG~P -- -- 150 mM --ddTTP -- -- -- 500 mM
dATP25 mM250 mM 250 mM250 mM
dCTP250 mM25 mM 250 mM250 mM
dGTP250 mM250 mM 25 mM250 mM
dTTP250 mM250 mM 250 mM25 mM

Reactions were initiated by adding 1 ~L
40 mM dithiothreitol and 1 ~L Taq DNA polymerase (Cetus; 5 units/~L) to each tube. The reactions wece incubated at 65 for 30 minutes. Each reaction was passed through a G-50 Select-D spin column (5 Prime~3 Prime, Inc.; Paoli, PA), which had been prewashed with H2O. Each column effluent was collected and vacuum dried. Each pellet was resuspended in 5 ~L 95% (v/v) formamide and incubated at 68 for ten minutes.
~5 ~hree ~L of each sample were loaded on an 8%
polyacrylamide-8 M urea sequencing gel and analyzed on the Genesi ~ 2000 DNA Analysis System (Du Pont) following the manufacturer's instructions.
A portion of the fluorescent emission detected during electrophoresis of the four samples of this example is shown in Figure 1.
The pattern of chain terminations allows the sequence of a portion of M13 mpl8 to be accurately deduced. This demonstrates (i) that the presence of the SF505-4HP moeity did not 28 1338a97 interfere with the ability of the primer to hybridize with the DNA template, (ii) that the SF505-4HP primer is a substrate that can be extended by a DNA polymerase, and (iii) that the primer products can be detected by their characteristic fluoresence.

Example 8 Polymerase Chain Reaction with an SF505-4HP-p-Tagged Primer An SF505-4HP-pGTTTTCCCAGTCACGAC primer (Example 5) was used as part of a polymerase chain reaction, which is a process for amplifying specific segments of DNA and i~ described in detail in U.S. 4,683,195 and U.S. 4,683,202. The sequence to be amplified was a 337 base pair segment of M13 mpl8. The second primer used in the polymerase chain reaction was an unlabeled 17-mer oligodeoxynucleotide with the sequence AAACCACCCTGGCGCCC (5'~3') prepared by standard automated procedures. The following c~omponents were mixed: 62 ~L H2O, 10 ~L 10X Taq polymerase reaction buffer (166 mM (NH~)2SO~; 670 mM
Tri~-HCl, pH 8.8; 67 mM MgCl2; 100 mM
~-mercaptoethanol; 67 ~M EDTA; 1.7 mg/mL
bovine serum albumin), 16 ~L deoxynucleotide triphosphate solution (1.25 mM dATP; 1.25 mM
dCTP; 1.25 mM dGTP; 1.25 mM dTTP ), 5 ~L
SF505-4HP-pGTTTTCCCAGTCACGAC primer solution (Example 5; 0.30 ODU (260 nmol)/~L - 1.5 ~M), S~L
AAACCACCCTGGCGCCC primer solution (0.30 ODU (260 nm)/mL ~ 1.8 ~M~ ), 1 ~L M13 mpl8 sinqle-stranded DNA (N. E. BioLabs, diluted to a concentration of 1 ng/~L), and 1 ~L Taq DNA polymerase (Cetus; 5 units/~L).

This reaction mixture was heated to 94 for one minute, cooled to 45 and incubated for two minutes, then heated to 72 and incubated for three minutes. This temperature cycle was repeated an additional 24 times. The DNA in the reaction was precipitated by adding 100 ~L 5 M
ammonium acetate plus 500 ~L ethanol. The precipitated DNA was collect by centrifugation, dried under vacuum, resuspended in 100 ~L 95%
(v/v) formamide, and incubated at 68 for 10 minutes. One ~L of this sample was loaded on an 8% polyacrylamide-8 M urea sequencing gel and analyzed on the Genesis~ 2000 DNA Analysis System (~u Pont) following the manufacturer's instructions.
Fluorescent emissions detected during electrophoresis of the polymerase chain reaction products are shown in Figure 2. Also shown are size markers that consist of labeled restriction fragments electrophoresed in a parallel lane.
The size markers are pBR322 ~I restriction fragments (N. E. BioLabs) end-labeled~with SF519-ddCTP (New England Nuclear) by standard procedures. Figure 2 shows that the major product of the polymerase chain reaction is, as expected, a DNA fragment approximately 337 base pairs in size. This demonstrates (i) that the presence of a 5'-sF505-4HP moeity does not interfere with the use of an oligodeoxynucleotide in the polymerase chain reaction and (ii) the products of the polymerase chain reaction can be detected by their characteristic fluorescence.

Claims (29)

1. A chemical compound of the formula reporter-A-Q
wherein the reporter is a protected fluorescent reporter or an unprotected fluorescent reporter selected from the group consisting of fluoresceins, benzamidizoles, ethidiums, propidiums, anthracyclines, mithramycins, actinomycins, merocyanines, coumarins, pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes, salicyclic acids, benz-2-oxadiazoles, and fluorescamine;
A is selected from the group consisting of -O-, -S-, -NR, and -CRR1, wherein R and R1 are independently selected from the group consisting of H, C3-C10 branched alkyl, C1-C10 unbranched alkyl, C6-C10 aryl, C7-C12 alkaryl, and C7-C12 aralkyl; and Q is selected from the group consisting of , , , , and and salts thereof, wherein R2 and R3 are independently selected from the group consisting of C3-C10 branched alkyl and C1-C10 unbranched alkyl; R4 is selected from the group consisting of -(CRR1)m(A)n(CRR1)m-, , and wherein R, R1, and A are as defined above, m = 1-6, n = 0-1, p = 1-10, and q = 0-10, provided that 2m+n 12 and further provided 2 n+p+q 13; X is selected from the group consisting of -F, -Cl, -Br, -I, imidazol-1-yl, 1,2,4-triazol-1-yl, tetrazol-1-yl, and 1-hydroxybenzotriazol-O-yl; and Y is any phosphate-protecting group.
2. A compound according to Claim 1 wherein A
is -O-.
3. A compound according to Claim 1 wherein Y
is selected from the group consisting of 4-Cl-C6H4-O-, 2-Cl-C6H4-O-, 4-NO2-C6H4-O-, 4-NO2-C6H4CH2CH2-O-, 2,4-NO2-C6H3CH2CH2-O-, 2,4-Cl2-C6H3-O-, 2,3-Cl2-C6H3-O-, NCCH2CH2O-, NCCH2C(CH3)2-O-, CH3O-, (Z)3CCH2-O-, (Z)3CC(CH3)2-O-, R'S-, R'SCH2CH2-O-, R'SO2CH2CH2-O-, and R'NH-, wherein Z is selected from the group consisting of Cl, Br, and I, and R' is selected from the group consisting of H, C3-CH10 branched alkyl, C1-C10 unbranched alkyl, C6-C10 aryl, C7-C12 alkaryl, and C7-C12 aralkyl.
4. A compound according to Claim 3 wherein Y
is selected from the group consisting of NCCH2CH2O-, CH3O-, CH3O-, and 2-Cl-C6H4-O-.
5. A compound according to Claim 1 wherein the reporter is selected from the group consisting of fluoresceins and coumarin.
6. A compound according to Claim 2 wherein the reporter is selected from the group consisting of fluoresceins and coumarin.
7. A compound according to Claim 3 wherein the reporter is selected from the group consisting of fluoresceins and coumarin.
8. A compound according to Claim 4 wherein the reporter is selected from the group consisting of fluoresceins and coumarin.
9. A compound according to Claim 5 wherein Q
is selected from the group consisting of
10. A compound according to Claim 6 wherein Q
is selected from the group consisting of
11. A compound according to Claim 7 wherein Q
is selected from the group consisting of
12. A compound according to Claim 8 wherein Q
is selected from the group consisting of
13. A compound according to Claim 9 wherein the reporter is a fluorescein.
14. A compound according to Claim 10 wherein the reporter is a fluorescein.
15. A compound according to Claim 11 wherein the reporter is a fluorescein.
16. A compound according to Claim 12 wherein the reporter is a fluorescein.
17. A compound according to Claim 13 wherein the fluorescein is of the structure wherein R5 and R6 are independently selected from the group consisting of -H, C1-C4 alkyl, -F, -Cl, -Br, -I, C1-C4 alkoxy, and -CH; R7 is C1-C4 alkyl; and R8 is selected from the group consisting of alkyl or aryl.
18. A compound according to Claim 14 wherein the fluorescein is of the structure wherein R5 and R6 are independently selected from the group consisting of -H, C1-C4 alkyl, -F, -Cl, -Br, -I, C1-C4 alkoxy, and -CH; R7 is C1 -C4 alkyl; and R8 is selected from the group consisting of alkyl or aryl.
19. A compound according to Claim 15 wherein the fluorescein is of the structure wherein R5 and R6 are independently selected from the group consisting of -H, C1-C4 alkyl, -F, -Cl, -Br, -I, C1-C4 alkoxy, and -CH; R7 is C1-C4 alkyl; and R8 is selected from the group consisting of alkyl or aryl.
20. A compound according to Claim 16 wherein the fluorescein is of the structure wherein R5 and R6 are independently selected from the group consisting of -H, C1-C4 alkyl, -F, -Cl, -Br, -I, C1-C4 alkoxy, and -CH; R7 is C1-C4 alkyl; and R8 is selected from the group consisting of alkyl or aryl.
21. A compound according to Claim 17 wherein R
and R3 are -CH(CH3)2.
22. A compound according to Claim 18 wherein R
and R3 are -CH(CH3)2.
23. A compound according to Claim 19 wherein R
and R3 are -CH(CH3)2.
24. A compound according to Claim 20 wherein R2 and R3 are -CH(CH3)2.
25. A compound according to Claim 9 wherein the reporter is a coumarin.
26. A compound according to Claim 10 wherein the reporter is a coumarin.
27. A compound according to Claim 11 wherein the reporter is a coumarin.
28. A compound according to Claim 12 wherein the reporter is a coumarin.
29. A chemical compound of the structure wherein R5 and R6 are independently selected from the group consisting of -H, C1-C4 alkyl, -F, -Cl, -Br, -I, C1-C4 alkoxy, and -CN: B is selected from the group consisting of 1-thyminyl, 1-cytosinyl, 1-uracilyl, 9-adeninyl, and 9-guaninyl; R9 is selected from the group consisting of -H and -OH; and n = 0 to about 100.
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Families Citing this family (115)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5262536A (en) * 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5654442A (en) 1989-11-14 1997-08-05 The Perkin-Elmer Corporation 4,7-dichlorofluorescein dyes as molecular probes
US5188934A (en) * 1989-11-14 1993-02-23 Applied Biosystems, Inc. 4,7-dichlorofluorescein dyes as molecular probes
US5391785A (en) * 1990-01-16 1995-02-21 La Jolla Pharmaceutial Company Intermediates for providing functional groups on the 5' end of oligonucleotides
US5210015A (en) * 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
CA2051217C (en) * 1990-09-14 2003-12-09 Wolfgang Pfleiderer Process for the chemical synthesis of oligonucleotides
US5371241A (en) * 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
GB9118785D0 (en) * 1991-09-03 1991-10-16 Amersham Int Plc Labelled primers for nucleic acid probes
US6048975A (en) * 1991-09-12 2000-04-11 Hoechst Aktiengesellschaft Process for the chemical synthesis of oligonucleotides
WO1993014224A1 (en) * 1992-01-14 1993-07-22 Applied Biosystems, Inc. Size-calibration dna fragment mixture and method
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US5795714A (en) * 1992-11-06 1998-08-18 Trustees Of Boston University Method for replicating an array of nucleic acid probes
US20060084092A1 (en) * 1993-01-05 2006-04-20 Gelfand David H Homogeneous assay system
US6194144B1 (en) 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry
DE69430909T2 (en) * 1993-03-19 2003-02-27 Sequenom Inc DNA SEQUENCE DETERMINATION BY MASS SPECTROMETRY ON THE WAY OF DEGRADATION WITH EXONUCLEASE
US5545535A (en) * 1993-04-13 1996-08-13 Molecular Probes, Inc. Fluorescent assay for bacterial gram reaction
US5445946A (en) * 1993-04-13 1995-08-29 Molecular Probes, Inc. Intravacuolar stains for yeast and other fungi
US5658751A (en) * 1993-04-13 1997-08-19 Molecular Probes, Inc. Substituted unsymmetrical cyanine dyes with selected permeability
US5436134A (en) * 1993-04-13 1995-07-25 Molecular Probes, Inc. Cyclic-substituted unsymmetrical cyanine dyes
DE4326466A1 (en) * 1993-08-06 1995-02-09 Boehringer Mannheim Gmbh Infrared dye-labeled nucleotides and their use in nucleic acid detection
JP3419850B2 (en) * 1993-10-26 2003-06-23 株式会社日立製作所 Nucleic acid sorting method
US8236493B2 (en) * 1994-10-21 2012-08-07 Affymetrix, Inc. Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA
US6974666B1 (en) * 1994-10-21 2005-12-13 Appymetric, Inc. Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA
US7803529B1 (en) 1995-04-11 2010-09-28 Sequenom, Inc. Solid phase sequencing of biopolymers
US20060063193A1 (en) * 1995-04-11 2006-03-23 Dong-Jing Fu Solid phase sequencing of double-stranded nucleic acids
US6864059B2 (en) * 1996-01-23 2005-03-08 Affymetrix, Inc. Biotin containing C-glycoside nucleic acid labeling compounds
US20010018514A1 (en) * 1998-07-31 2001-08-30 Mcgall Glenn H. Nucleic acid labeling compounds
US6613508B1 (en) 1996-01-23 2003-09-02 Qiagen Genomics, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
US7423143B2 (en) * 1996-01-23 2008-09-09 Affymetrix. Inc. Nucleic acid labeling compounds
US20040210045A1 (en) * 1996-01-23 2004-10-21 Mcgall Glenn Nucleic acid labeling compounds
US6965020B2 (en) 1996-01-23 2005-11-15 Affymetrix, Inc. Nucleic acid labeling compounds
US6312893B1 (en) 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US7282327B2 (en) * 1996-01-23 2007-10-16 Affymetrix, Inc. Nucleic acid labeling compounds
US7291463B2 (en) * 1996-01-23 2007-11-06 Affymetrix, Inc. Nucleic acid labeling compounds
EP0880598A4 (en) 1996-01-23 2005-02-23 Affymetrix Inc Nucleic acid analysis techniques
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
US6162931A (en) * 1996-04-12 2000-12-19 Molecular Probes, Inc. Fluorinated xanthene derivatives
DE19637042A1 (en) 1996-09-12 1998-03-19 Boehringer Mannheim Gmbh Heterocyclic compounds and their use in the detection of nucleic acids
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5830912A (en) * 1996-11-15 1998-11-03 Molecular Probes, Inc. Derivatives of 6,8-difluoro-7-hydroxycoumarin
US5696157A (en) * 1996-11-15 1997-12-09 Molecular Probes, Inc. Sulfonated derivatives of 7-aminocoumarin
ATE273381T1 (en) * 1997-02-12 2004-08-15 Eugene Y Chan METHOD FOR ANALYZING POLYMERS
US6130101A (en) * 1997-09-23 2000-10-10 Molecular Probes, Inc. Sulfonated xanthene derivatives
US6268131B1 (en) 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
US6153411A (en) * 1998-10-30 2000-11-28 American Water Works Company, Inc. Methods and kits for detection of Cryptosporidium parvum using immunomagnetic separation and amplification
US6664047B1 (en) 1999-04-30 2003-12-16 Molecular Probes, Inc. Aza-benzazolium containing cyanine dyes
ATE415409T1 (en) 1999-05-24 2008-12-15 Invitrogen Corp A METHOD FOR DEPROTECTING LABELED OLIGONUCLEOTIDES
US7097973B1 (en) 1999-06-14 2006-08-29 Alpha Mos Method for monitoring molecular species within a medium
US6531591B1 (en) 1999-07-07 2003-03-11 Exiqon A/S Synthesis of stable quinone and photoreactive ketone phosphoramidite reagents for solid phase synthesis of photoreactive-oligomer conjugates
DE60006680T2 (en) * 1999-07-07 2004-09-16 Exiqon A/S SYNTHESIS OF STABLE CHINONE- AND PHOTOREACTIVE KETONE-PHOSPHORAMIDITE REAGENT FOR SOLID-PHASE SYNTHESIS OF PHOTOREACTIVE OLIGOMER CONJUGATES
EP1136569A3 (en) 2000-03-24 2004-01-28 Bayer Corporation Nucleic acid probes having highly hydrophilic non-nucleosidic tags with multiple labels, and uses thereof
US7019129B1 (en) 2000-05-09 2006-03-28 Biosearch Technologies, Inc. Dark quenchers for donor-acceptor energy transfer
US20070172866A1 (en) * 2000-07-07 2007-07-26 Susan Hardin Methods for sequence determination using depolymerizing agent
AU8288101A (en) * 2000-07-07 2002-01-21 Visigen Biotechnologies Inc Real-time sequence determination
US7169922B2 (en) 2000-08-04 2007-01-30 Invitrogen Corporation Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings
EP2045252B1 (en) 2000-08-04 2013-05-01 Life Technologies Corporation Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings
AU2001294859A1 (en) 2000-09-29 2002-04-08 Molecular Probes, Inc. Modified carbocyanine dyes and their conjugates
EP1354064A2 (en) 2000-12-01 2003-10-22 Visigen Biotechnologies, Inc. Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity
AU2002258502A1 (en) * 2001-03-12 2002-09-24 Affymetrix, Inc. Nucleic acid labeling compounds
CZ20033143A3 (en) * 2001-05-21 2004-04-14 Aclara Biosciences, Inc. Methods and compositions for analyzing proteins
US7668697B2 (en) * 2006-02-06 2010-02-23 Andrei Volkov Method for analyzing dynamic detectable events at the single molecule level
US20030148391A1 (en) * 2002-01-24 2003-08-07 Salafsky Joshua S. Method using a nonlinear optical technique for detection of interactions involving a conformational change
US20040229380A1 (en) * 2002-05-21 2004-11-18 Po-Ying Chan-Hui ErbB heterodimers as biomarkers
US7005518B2 (en) * 2002-10-25 2006-02-28 Li-Cor, Inc. Phthalocyanine dyes
US20040091850A1 (en) * 2002-11-08 2004-05-13 Travis Boone Single cell analysis of membrane molecules
US20090186343A1 (en) * 2003-01-28 2009-07-23 Visigen Biotechnologies, Inc. Methods for preparing modified biomolecules, modified biomolecules and methods for using same
US7402398B2 (en) * 2003-07-17 2008-07-22 Monogram Biosciences, Inc. Measuring receptor homodimerization
BRPI0413471A (en) 2003-08-11 2006-10-17 Monogram Biosciences Inc methods for detecting one or more protein complexes and for detecting a complex in a biological sample
EP2298312B1 (en) 2003-10-31 2018-09-26 Molecular Probes Inc. Fluorinated resorufin compounds and their application in detecting hydrogen peroxide
US20050214807A1 (en) * 2003-11-19 2005-09-29 Iain Johnson Environmental sensitive fluorogenic compounds and their application for singlet oxygen and protein detection
EP1720944B1 (en) * 2003-12-05 2013-07-17 Life Technologies Corporation Cyanine dye compounds
US7776529B2 (en) 2003-12-05 2010-08-17 Life Technologies Corporation Methine-substituted cyanine dye compounds
US8039642B2 (en) 2003-12-09 2011-10-18 Life Technologies Corporation Pyrenyloxysulfonic acid fluorescent agents
WO2005064336A1 (en) * 2003-12-09 2005-07-14 Molecular Probes, Inc. Pyrenyloxysulfonic acid fluorescent agents
US7939267B2 (en) * 2004-11-04 2011-05-10 Laboratory Corporation Of America Holdings Detection of activation of endothelial cells as surrogate marker for angiogenesis
EP1885718B1 (en) 2005-05-11 2017-03-15 Life Technologies Corporation Fluorescent chemical compounds having high selectivity for double stranded dna, and methods for their use
US20070134685A1 (en) * 2005-09-06 2007-06-14 Invitrogen Corporation Control of chemical modification
WO2007120265A2 (en) 2005-11-14 2007-10-25 Applera Corporation Coded molecules for detecting target analytes
ES2332139T3 (en) 2005-11-23 2010-01-27 F. Hoffmann-La Roche Ag POLINUCLEOTIDOS WITH PHOSPHATE MIMETIC.
US20080241951A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Method and apparatus for moving stage detection of single molecular events
US20080241938A1 (en) * 2006-07-20 2008-10-02 Visigen Biotechnologies, Inc. Automated synthesis or sequencing apparatus and method for making and using same
US20080091005A1 (en) * 2006-07-20 2008-04-17 Visigen Biotechnologies, Inc. Modified nucleotides, methods for making and using same
US7888506B2 (en) * 2007-01-26 2011-02-15 Duquesne University Of The Holy Spirit Composition, synthesis, and use of a new class of fluorophores
US8586743B2 (en) 2007-01-30 2013-11-19 Life Technologies Corporation Labeling reagents and methods of their use
WO2009070772A1 (en) * 2007-11-27 2009-06-04 Monogram Biosciences, Inc. Enhanced method for detecting and/or quantifying an analyte in a sample
CA2711843C (en) * 2007-12-20 2018-11-13 Laboratory Corporation Of America Holdings Her-2 diagnostic methods
MX2010010881A (en) 2008-04-01 2011-03-04 Biosearch Tech Inc Stabilized nucleic acid dark quencher-fluorophore probes.
US9182406B2 (en) * 2008-08-04 2015-11-10 Biodesy, Inc. Nonlinear optical detection of molecules comprising an unnatural amino acid possessing a hyperpolarizability
WO2011026085A2 (en) 2009-08-31 2011-03-03 Promega Corporation Reactive cyanine compounds
WO2011087707A1 (en) 2009-12-22 2011-07-21 Elitech Holding B.V. Hypertheromostable endonuclease iv substrate probe
US8623324B2 (en) 2010-07-21 2014-01-07 Aat Bioquest Inc. Luminescent dyes with a water-soluble intramolecular bridge and their biological conjugates
AU2011305445B2 (en) 2010-09-24 2017-03-16 The Board Of Trustees Of The Leland Stanford Junior University Direct capture, amplification and sequencing of target DNA using immobilized primers
WO2012129347A1 (en) 2011-03-21 2012-09-27 Biodesy, Llc Classification of kinase inhibitors using nonlinear optical techniques
US8809238B2 (en) 2011-05-09 2014-08-19 Fluidigm Corporation Probe based nucleic acid detection
EP2710379B1 (en) 2011-05-19 2016-12-14 Laboratory Corporation of America Holdings Methods for determining the likelihood of survival and for predicting likelihood of metastasis in cancer patients
WO2013048583A2 (en) 2011-05-24 2013-04-04 Elitech Holding B.V. Detection of methicillin-resistant staphylococcus aureus
US9056885B2 (en) 2011-11-21 2015-06-16 Promega Corporation Carboxy X rhodamine analogs
EP2814857A1 (en) 2012-02-17 2014-12-24 NVS Technologies Inc. Polymer scaffolds for assay applications
CA2888132A1 (en) 2012-10-12 2014-04-17 NVS Technologies, Inc. Polymers having orthogonal reactive groups and uses thereof
US9914979B2 (en) 2013-03-04 2018-03-13 Fry Laboratories, LLC Method and kit for characterizing microorganisms
US20140287945A1 (en) 2013-03-14 2014-09-25 NVS Technologies, Inc. Surface oxidation for sequestering biomolecules and related methods
WO2014152298A1 (en) 2013-03-15 2014-09-25 Promega Corporation New silicon and germanium dyes for use in genetic identity
US20160041171A1 (en) 2013-04-05 2016-02-11 Laboratory Corporation Of America Holdings Systems and methods for facilitating diagnosis, prognosis and treatment of cancer based on detection of her3 activation
KR102360517B1 (en) 2014-05-09 2022-02-09 바이오서치 테크날로지즈, 인크. Cosmic quenchers
EP3143022B1 (en) 2014-05-16 2021-12-22 Life Technologies Corporation Carbopyronone compounds useful as diagnostic adjuvants
WO2016094162A1 (en) 2014-12-12 2016-06-16 Elitechgroup B.V. Methods and compositions for detecting antibiotic resistant bacteria
EP3230468B1 (en) 2014-12-12 2020-09-16 ELITechGroup, Inc. Methods and kits for detecting antibiotic resistant bacteria
WO2016106286A1 (en) 2014-12-23 2016-06-30 Biodesy, Inc. Attachment of proteins to interfaces for use in nonlinear optical detection
ES2924731T3 (en) 2015-04-15 2022-10-10 Biosearch Tech Inc Probes with double extinguisher
CN108780089B (en) 2016-03-15 2020-09-08 美国控股实验室公司 Method for evaluating protein interactions between cells
JP2019512573A (en) 2016-03-28 2019-05-16 エーエーティー バイオクエスト インコーポレイテッド Polyfluoreno [4,5-cde] oxepin conjugates and their use in analyte detection methods
EP3478702A4 (en) 2016-06-27 2020-03-18 Dana-Farber Cancer Institute, Inc. Methods for measuring rna translation rates
US20230131000A1 (en) 2020-01-30 2023-04-27 Aat Bioquest, Inc. UV Excitable Polyfluorene Based Conjugates and Their Use in Methods of Analyte Detection
CA3223867A1 (en) 2021-06-30 2023-01-05 Gerassimos Makrigiorgos Compositions and methods for enrichment of nucleic acids using light-mediated cross-linking
CA3229091A1 (en) 2021-10-26 2023-05-04 Caribou Biosciences, Inc. Exonuclease-coupled real-time endonuclease activity assay

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4275149A (en) * 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4374925A (en) * 1978-11-24 1983-02-22 Syva Company Macromolecular environment control in specific receptor assays
US4547569A (en) * 1982-11-24 1985-10-15 The United States Of America As Represented By The Department Of Health And Human Services Intercalating agents specifying nucleotides
JPS60197698A (en) * 1983-12-20 1985-10-07 カリフオルニア・インステイテユ−ト・オブ・テクノロジ− Synthesis of amino derived oligonucleotide
GB8509880D0 (en) * 1985-04-17 1985-05-22 Ici Plc Testing device
IT1202321B (en) * 1985-06-06 1989-02-02 Uni Degli Studi Di Parmo BIOLOGICALLY ACTIVE FLUORESCENT CYCLIC NUCLEOTIDES
US4739044A (en) * 1985-06-13 1988-04-19 Amgen Method for derivitization of polynucleotides
CA1340806C (en) * 1986-07-02 1999-11-02 James Merrill Prober Method, system and reagents for dna sequencing
US4873355A (en) * 1987-05-29 1989-10-10 E. I. Du Pont De Nemours And Company Process for regioselectively preparing phosphorylated inositols and other cyclitols
GB8720394D0 (en) * 1987-08-28 1987-10-07 Ici Plc Nucleotide probes
US4965349A (en) * 1987-12-24 1990-10-23 Applied Biosystems, Inc. Method of synthesizing oligonucleotides labeled with ammonia-labile groups on solid phase supports
AU622899B2 (en) * 1988-05-04 1992-04-30 F. Hoffmann-La Roche Ag Rutheniumcomplex-compounds

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