CA2117583A1 - Modified nucleotides - Google Patents
Modified nucleotidesInfo
- Publication number
- CA2117583A1 CA2117583A1 CA 2117583 CA2117583A CA2117583A1 CA 2117583 A1 CA2117583 A1 CA 2117583A1 CA 2117583 CA2117583 CA 2117583 CA 2117583 A CA2117583 A CA 2117583A CA 2117583 A1 CA2117583 A1 CA 2117583A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleic acid
- rhodamine
- fluorescein
- enzyme
- jeffamine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Saccharide Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides novel modified nucleotide compounds having the general formula: X-n(J)-(d or r)NTP where N is adenosine, guanosine or cytidine; X is H, a fluorophore, a chromophore, a luminescent compound, a ligand or a hapten; n is an integer of 7 or more; and J is a Jeffamine (TM Texaco, Inc.) substituent.
These compounds are substantially superior substrates for incorporating label into nucleic acids during enzyme-catalyzed synthesis. Use of a Jeffamine-linked labelled nucleotide derivative as a partial substitute for unlabelled substrate therefore leads to significantly greater incorporation, hence increased label density per unit length of nucleic acid, than the same label joined by a prior art linker. Jeffamine-modified ribo- or deoxyribo-nucleotides are also provided for synthesis of Jeffamine-modified nucleic acids. The invention further provides an improved method of synthesizing labelled nucleic acids. This method provides greater frequency and higher efficiency of label incorporation, thus requiring lower amounts of nucleotide derivative in the reaction mixture.
These compounds are substantially superior substrates for incorporating label into nucleic acids during enzyme-catalyzed synthesis. Use of a Jeffamine-linked labelled nucleotide derivative as a partial substitute for unlabelled substrate therefore leads to significantly greater incorporation, hence increased label density per unit length of nucleic acid, than the same label joined by a prior art linker. Jeffamine-modified ribo- or deoxyribo-nucleotides are also provided for synthesis of Jeffamine-modified nucleic acids. The invention further provides an improved method of synthesizing labelled nucleic acids. This method provides greater frequency and higher efficiency of label incorporation, thus requiring lower amounts of nucleotide derivative in the reaction mixture.
Description
WO 9:~t907~ PCI~/US93/023~0 ~ 2 i i /S83 MOI:IIF:CED NTJCLEOTIDES
BACKGROUND AND PRIOR ART
:"
The invention concerns novel compounds for nucleic acid labelling and methcd for making nucleic acids incorporatin~ the ~el compounds~
Techniques for labelling nucleic acids with a reporter molecule generally fall into one of two categories: incorporating the label int~ the nucleic acid during syn~hesis, or post~
synthetically modifying the nucleic acidO The form r is commonly accomplished by providing a labelled derivati~e of one of the preCurcor nucleotide triphosphates as a partial o~ complete subs itute for the nsrmal precursor during the enzyme-catalyzed synthesis of the nucleic acid. The labelled nucleotide deriv~tive must meet certain criteria. The derivative must continue to be recognized by the ~nzyme as a substrate, it must not inter~ere with or inhibit the enzyme and it must participate in ~he normal hydrogen-bonding in~eractions of base pairing, adenine with thymine (or uracil), guanine with cytosine.
ZO Post-synthetic modification labelling is commonly accomplished by modifying the end group of a nucleiG acid by means of a chemical reaction or by ~n~enzyme such as terminal trans~erase.
A wide v~riety of reporter molecules have ~een incorporated in~o nucleic acids. Besides radioactive labels, which are usually incorporated during synthesis, fluorescent labels, chromatic labels, luminPscent labels, ligands and haptens have C A ~
been employed. Rhodamine and fluorescein have been used for fluorescent labelling. Nitroblue tetrazolium and BCIP ~5-bromo-4-chloro-3-indolylphosphate) (Gibco BRL~ have been us~d as chromophores. Firefly luciferin and PPD (~-methoxy-4-(3-phosphatenephenyl)spiro~l,2 dioxetane-3,2'-ad3mantane~ (Gibco 8RL) have been used as luminescent labels. Biotin has been used as a ligand to bind labelled streptavidin. Dinitrophenol and digoxigenin have been used as hapten }abels to bind antibody and take ad~antage of immunoassay methods. The foregoing examples are illustrative only and not limiting.
Radioactive labels have the advantage of pr~viding high ~ensitivit~, however they have the disadYantagss of being expensive~ of having short shelf life in sQme cases, and of ~ssenting safety and disposal problems. While n~n-radioactive labels lack the disadvantages of radioactivity, they pose other difficulties for the potential user. ~any of the reporter compounds are large, bulky molecules rela~ive to the nucleotides themselves, and their size can sterically interfere with incorporation. Low levels of incorporat~on c~use loss of detection sensiti~ity by limiting the level of detectab~e signal per nucleic acid molecule. The reaction conditions used for post~synthetic nucleic acid labelling can be incompatible with nucleic acid integrity and can in~olve specialized ch2mistry that requires equipment and reagents not normally found in the la~ora~ories of many potential end users o~ the labelled nucleic acid.
Steric interference by large reporter compounds has been alleviated by the use of linkers, linear chains of, typically four to twelve atoms, usually a saturated or partially unsat~rated aliphatic chain, occasionally containing an amide group. The function of linkers has been considered to be to act as a spacer be~ween *he nucleotide base and the label. Any linkers capable of providing adequate spacing and flexibility have been considered functionally equi~al~nt.
W093/1907X CA2 i i -15~3 PCT/~S93/02390 The use of linkers has also made possible a hybrid labelling technique whereby precursor nucleotides modif ied to possess a linker moiety are incorporated into the nucleic acid during synthesis, to yield linker-modified nucleic acid. The linker groups suitable for such modification must have a reactive group at the free end of the linker hain. Th2 linker-modified nucleic acid is post-synthetically coupled with a reporter compound at the reactive ends of the incorpor~ted linkers. (See, e.g., Jett et al., U.S. Application Serial Number 07/765,277).
SUMMARY ~F_THE INVENTION
.
The pres~nt invention is based on the discovery that Jeffamine (TM Texaco, Inc.~-linked nucleoside triphosphates can substantially superior subskrates for incorpora~ing label into nucleic acids during enzyme-catalyzed synthesis. Use ~f a Jeffamine-linked labelled nucleoside triphv~phate derivative as a partial substitute for unlabelled substrate leads to significantly greater incorporation, hence increased label density per unit length of nucleic acid, than the same label joined by a p~ior art linker. Jeffamine linkers have been found to have the property of permitting higher levels of incorporation of any label.
Accordingly, novel Jeffamine-linked deoxy- or ribo-nucleotide derivatives are provided for any sort of nucleic acid label desired: fluorescent, chromatic, bio- or chemi-~5 luminescent, ligand or hapten. Simi~arly, Jeffamine~modifiedribo- or deoxyribo-nucleotides are provided for synthesis of Jef~amine-modified nucleic acids. The lat~er can be post-synthetically modified by attachmènt of any desired label or combination of labels.
.
The invention further provides an impro~ed method of synthesizing labelled nucleic acids, whereby greater frequency of label incorporation than heretofore is achieved. Because the Jeffamine-linked deoxy- and ribo-nucleotides of the invention are W~ g3/lgO78 PC~USg3tO2390 `` C~2~ , 75~3 incorporated with such high efficiency, the method also provides conventional levels of label using lower amounts of nucleotide derivative in the reacti~n mixture, thereby pr~viding substantial savings in conventional uses of labelled nucleic acids.
S DETAILED DESCRIPTION OF THE INVENTION
Jeffamines arP polyglycol diamines having a general formula H2N tCH2)n~tO~(C~2)n]~~N~2r where n is 2 or more, preferably 2 to 4, and m is 1 to 10, preferably 1-5, m~st preferably 2 or 3.
Where m is greater than 1I there can be a different value of n for each ~O (CH23~- group. However, typically and most conveniently, the value of n will be ~he same f or each ( C~2) n group in the compound. Useful Jeffamines of the invention are -, triD or tetra- and higher ethylene, n-propylene, or n-butylene glycol diamines. Although Jeffamines having branched (CH2)n groups are known, those preferred herein have linear (CH2)n groups. The structure ~f triethylene glycol diamine can be diagrammed as H2N-~H2c~2-o-cH2c~2-o-c~2c~2 NH2 The choice of any particular Jeffamine is one which can be made readily by those skilled in the art, within limits disclosed herein.
One of the Jeffamine amino groups is used to rP ct with the base moiety of a nucleotide, the other amino group can reacted with a reporter moiety either before or after nucleic acid synthesis. R~action of a Jeffamine with adenine can be carried out at the 8 position or preferably, at the 6 position of adenine. A Je~f amine derivative of guanine at the 8 position of guanine is the only suitable derivative known. Cytosine can be reacted at the 4 position of cytosine. Jeffamine derivatives of uracil and thymine are only feasible by indirect linkage. Such indirect linkage can be achieved, for example~ by f irst forming allylamine derivatives of the pyrimidine ring at the 5 or 6 WO93/19078 C A 2 i I / 5 ~ 3 PCT/USg3/02390 position, followed by subse~uent reaction with a Jeffamine.
Derivatives of cytosine can also be formed in like manner.
The labelled nucleotides of the invention therPfore have three parts, the first being a deoxy- or ribo- nucleotide, abbreviated dN or rN (or d or rNTP if in the triphosphate form) which can be either d or r- adenosine (dA or rA), d- or r-guanosine (dG or rG) or d- or r-cytosine (dC or rC). The s~cond par~ is a Jeffamine-based linker, which will have a chain length of 7 Qr more atoms, depending on the Jeffamine used, and abbreviated herein as n(J) where n is an integer of 7 or more.
The third part is the reporter, designated X~ X can be any molecule use~ul for labeling nucleic acid and having the ability to f~rm stabl compounds with a primary amine group. X can be ~fluorophore, for example, rhodamine or fluorescein. X can be a chromoph~re, for example, Nitro blue tetraæolium, or BCIP. X
can be a luciferin or other luminescent reporter, such as PPD.
X can be a ligand, for example, biotin, having the property of bindi~g another, readily detectable, molecule. Similarly, a ligand such as an enzyme cofactor can be detected by the activity of the enzyme which binds it, the enzyme-catalyzed reaction providing an amplifica~ion factor to enhance sensitivi.ty. X can also be a hapten, for example dinitrophenol or digoxigenin, detectable by immunochemical means.
A labelled nucleotide of the invention is therefore 2~ abbreviated herein as X-n(J)-(d or r)NTP.
The term "modified nucleotide" is de~ined herein as a nucleotide having a Jeffamine substituent but no reporter moiety (X is H). A modified nucleotide is useful for post-synthetic labelling of DNA in which thP modified nu~leotide has been incorporated. Such a modified nucleotide is abbreviated n(J)-~d or r~NTP, where n is 7 or more, and N is adenosine, guanosine or cytidine.
WO93/19078 C A 2 i i ~5 8 3 PCT/US93/02390 Enzyme-catalyzed addition of labelled nucleotides of the invention to the end of a nucleotide chain can also be accomplished. Using terminal deoxynucleotide transferase, Rho-lO(J)-dCTP was successfully added to DNA, using reaction conditions disclosed in U.S. Pat~nt 4,878,979 for end-labelling DNA with Biotin-14-dATP.
The invention is exemplified by comparing two compounds of the invention, Rhodamine-lO(J)-dCTP and Fluorescein-~O(J)-dCTP, with pri~r art compounds, demonstrating surprisingly higher incorporation efficiency than heretofore possible with prior art compounds. The structures of Rhodamine-10(~)-dCTP and the corresponding FluorescPin-lO(J)-dCTP are shown in Formula 1.
Formulas 2-5 show the structures of Fluorescein- and Rhodamine-8~dATP, Fluor~scein- and Rhodamine-4-dUTP, Rhodamine-8-~CTP, Fluorescein-(15)-dCTP, and Rhodamine-~15)-dCTP, respPctively.
WO93/1907~ CA 2 i I /583 P~US93/0:~3~0 FORMUI,A 1 - N H /~/O o~N
O
Rho--lO (J) -dCTP: R = TETlRAMETHYL--RHODZ~MINE
Fl--lO (J~--dCTP: R = FLUORESCEIN
X = DEOXYRIBOSE-S '--TR:l PHOSPHAT~E
OR RIBOSE--5 '--TRIPHOSPH~TE
~YO93/19078 PC~/US93/02390 C~2i ! /-5~3 FOR~LA 2 N H ( C H ~ ) 6 N H ~ C O - R
. Nl~N
, I
Rh~-8-dATP: R = TETRAMETHYL-RH~DAMINE
Fl-8-dATP: R = FLUO~ESCEIN
X = DEOXYR BOSE-5'-TRIPHOSPHATE OR
RIBOSE
WO ~3~1~0~8 ` C A 2 i i 7 5 8 3 PCr/lJS93/02390 FORMUI~ 3 O ~) H N~ N H J~` R
'~ o N
Rho-4-dUTP: R = TETRl~IEmYL-RHODAMINE
Fl-4-dU~P: R = ~?1UORESCEIN
X = DEOXYRIBOSE-5 ' -TRIPHOSPHATE
WO 93~19078 C A 2 i i l 5 ~ 3 PCr~US93/02390 FORMUL~ 4 NH ~, ~ NH~R
1 ~
O
X
Rho-8-dCTP: R = TETRAMETHYL--RHODAMINE
1~
WO 93/19~78 ~ A 2 i I / 5 8 3 P~/US~3/0239~ ^
NH /\, NH~1~R
Il o~3 X
-Rho-15--dCrP: R = - ( CH2) 5NH-CO--TETRAMETHYL RHI:)D~IINE
Fl 15--dCTP- R = - ~CH2) 5NH--CO--FLU5:~RESCEIN
X = DEOXYRIBOSE-5 ' -TRIPHOSPHATE OR
RIBOSE
WO 93~19û7B ` P~/US93/02390 EXAMPLES C h 2 i I ~ 3 Example 1. PreParation of N4-triethYlene ql~rcol amine~
deoxv~rtidine-5'-triphosPhate flOr3~-dCTP2 To triethylene glycol diamine (6.55 mmol, 1 ml) at 0C was added hydrochloric a id (9.5 mmol, 0.8 ml, 36%) drop-wise. To this solution was added sodium meta-bisulfite (2.2 mol, 430 mg) and water to a final volume of 3 ml. Deoxycytosine-5'-triphosphate (O.1 mmol, 50 ~g~ was di~solved in the above solution of dimine-bisulfite freshly prepared and filtered. ~o thls solution was added hydroquinone (~ mg in 10 ~l of ethanol) and the reaction mixture stirred for 2 days a~ ~5C under argon.
HPLC trace, after adjustment of ~he aliqu~t to pH = ~.5, (DuPont Zorb~x oligo c~lumn, 25% acetonitrile, Q.25 ~ NH4H2P04) 2 ml/min.
isocratic mode~ shows no starting material plus a new ma~or peak (68% of total). The crude mixture was ad~usted to pH = B.5 with sodium hydroxide, diluted to 500 ml with water and loaded on a DEAE anion exchange column equilibrated with triethyl ammonium biearbonate tTEAB). The column was eluted with a linear gradient of T~AB (0,01 - 1.0 M; 600/600 ml). After desalting of the major peak fractions, 6006 mg of pure desired product were isolated ~x = ~71-~72nm.
xample 2. Labellin~ of amino-nucleoside triPhosphates with fluorescent dyes.
The Je~famine-nucleoside triphosphates (10-20 ~mol) e.g., Z5 lO~ dCTP, were dissolved in sodium bicarbonate tO.4 M, 500 ~l) or sodium borate solution (0.1 M) and treated with a 3 to 5 fold molar excess of the N-hydroxysu~cinimide ester of the dye ~e.g., C ~ 3 fluorescein or rhodamine) in anhydrous dimethyl formamide (500 ~l). The mixture was reacted for 3 - 18 hr. at room temperature.
The reaction was monitored by thin layer chromatography (silica gel; butanol: acetone: acetic acid: 5~ ammonium hydroxide:
water/70:50:30:30:20) and/or by HPLC. The crude mixtures were diluted in water (200 - 300 ml), loaded on a lO - 15 cm long, by l cm diameter column of mild anion exchange resins and eluted, sequen~ially, with O.Ol, 0.2 and 0.5 ~ triethylammonium bicarbonate until the fraction containing the fluorescent dNTP
was collected. After desalting of thP appropriate column ~raction, TLC, HPLC and capillary electrophoresis analysis was used to assess the purity and characteristic elution patterns of the desired product. The compounds were characterized by thPir U.V. spectra as the o~erlapp~ng sp~ctra of the starting amino ~dified base and the dyes. Yields of fluorescent nucleotides were 50 - 60~.
Example 3. Use of Rhodamine-lO(J~-dCTP in Nucleic Acid Labelin~
Five fluorescent nucleotides were initially screened ~or enzymic incorporation into DNA using random primer extension with Klenow fragment of DNA polymerase I. The five fluore cent nucleotides were fluorescein-8-dCTP, rhodamine-~ d~TP, rhodamine-8-dCTP, rhodamine-lO(J)-dCTP, and rhodamine 4 dUTP. All but rhodamine-lO(J) dCTP (which has two ether linkages) have alkylamine linkers.
Template DNA (lO0 ng) was denatured in a dilute buffer such as TE (lO mM Tris-HCl, pH 7-5i 1 mM EDTA) by heating at 100C fsr 5 min. Reaction components were added to final concentxations as follows: 5~ mM Tris-HCl (pH 6.8), 5 mM MgCl2, lO mM 2-mercaptoethanol, 400 ~g/ml BSA, 300 ~g/ml random octamers, 200~M
dCTP, dGTP, and dTTP,lO0 ~M dCTP and lO0 ~M rhodamine-lO(J)-dCTP, 10 ~Ci ~- [32p~ - dATP ~3900Ci/mmol) and 40 units Klanow fragment in a final volume of 50 ~l. After incubation at 37~C for 1-2 hours, 5 ~l 0.~ M EDTA (pH 7.5) was added to terminate the reaction. Incorporation was determined by trichloroacetic acid W093/1~07~ PCT~US~3/02390 CA2ii~583 (TCA) precipitation. Diluted aliquots from the reaction were spotted on glass f iber f ilters in duplicate and dried . One of the duplicate filters was washed four times in cold 5~ TCA, 20 mM sodium pyrophosphate, then rinsed in 70% ethanol and dried and counted in a liquid scintillation counter ~incorporated counts).
The second filter was counted directly in the scintil~ ation counter (total counts). Incorporation of radioactive label was 40-50% indicating syn~hesis of several micrograms of fluorescent DNA probe. When other modified nucleotides were tested a mixture of 100 ~M unmodified dNTP and 100 ~M flusrescent dNTP was used in place of the dCTP/rhodamine-lO(J)-dCTP above. When the modified nucleotide was a dATP derivative, ~-[32P~-dCTP was used as a trace label.
Fluorescent dNIP% Incorvorati~onnq DNA S~nthesi2ed Fluorescein-8-dATP 0.6 79 Rhodamine-8-dATP 0.5 66 Rhodamine-8-dCTP 0.1 13 Rhodamine-lOtJ)-dCTP42.6 5623 Rhodamine-4-dUTP 0.5 6~
The percentage of rhodamine-lO~J)-dCTP was varied in subsequent experiments from 75%- to 100%. Incorporation decreased with increasing *luorescent nucleotide concentration.
Incorporation wa5 increased at higher percent ges by increasing the absolute concentration of unmodified dNTP to at least 50 ~M.
At a percentage of 95%, incorporation wa~ approximately 15% and at a percentags of 97~5%, incorporation was reduced to about 6%.
~NA probes prepared at 50% and 9%5 both functioned in in situ chromosome hybridizations.
W0 93~19~78 C ~ / 5 ~ 3 PCr/US93/~23~0 ExamPle 4 . Use of Rhodamine-10 fJ) -dCTP in Nucleic_ Acid La~el inq Template DNA (500 ng) was denatured in a dilute buffer such as TE (10 mM Tris-HCl, pH 7.5; 1 mM EDTA) by h~ating a~ 100~C for 10 min. Reaction components were added to final concentrations as f~llows: 5~ mM Tri~-HCl (pH 6.8), 5 mM MgC12, 10 mM 2-mercaptoethanol, 400 ~g/ml BSA, 300 ~m/ml random octamers~ 1~0 ~M each of dA~P, dGTP, dTTP, and ~CTP, 10 ~Ci ~-[32P]-dATP (3000 Ci/mmol), ~nd 40 units Klenow fragment in a final volume of 50 ~1. When testing modified nucleotides the c~rresponding unmodified dNTP was rsplaced with 100 ~M of the modified nucleoside triphosphates. In some experiments ~e.g., Table 2), diff~r~nt mixtures of modified and unmodified nucleotides were used. The perc~ntage of modified nucleotides to the total modified plus corresponding unmodified nucleotide was varied ~rom 25% to 100%. IN all cases J the total concentration of each nucleotide was kept at 100 ~M which resulted in a total nucleotide concentra~ion of 400 ~M. After incubation at 37C for 2 hours, 5 ~1 0.2 M EDTA (pH 7.5~ was added to terminate the r~action. Incorp~ration was determined by trichloroaceti~ acid (~CA~ preripitation~ Diluted aliquots from the reacti~n wer~
spotted on glass fiber filters in duplicate and dried. One of the duplicate filters was washed four times in cold 5% TCA, 20 mM sodium pyrophosphate, then rinsed in 70% ethanol and dried and count~d in a liquid scintillation counter (incorporated rounts).
The second fi~ter was counted directly in the -~cintillation counter (total count~). Incorporation of radioactive label was used to dete~mine sy~thesis of fluorescent DN~ probe. When the modified nucleotide was a dATP derivative, ~-[32P~-dCTP was used as a trace label.
WO 93/1~078 P~/US93/02390 C~ 583 Fluor. ng DNA
Fluorascent dNTP dNTP ~ % Incorpsvnthesized Rhodamine-10 (J) - lOO~M --- 5 . 7~ 37 dCTP 75~ 25~M 24 . 51617 50,uM 50~M 33 . 92237 25,uM 75~M 51. 83149 ~luorescein-l tJ) - lOOl M --- 5, gb38,9 dCTP 75~5 25,u~l 13 . 8 911 50~M 50~M 36. 22389 2S~M 75~M 54 . ~3623 ~ - b a = average of 7 experiments. = average of 3 experiments 15Below, Table 3, is a li~t of additional modified nucleotides - that have been screened for use in enzymatic incorpora~ion into DNA using random primer extension with ~lenow fragm~nt of DNA
polym~race .
O
~"9~9e~ % Incor~orationq DNA Synthesized Fluorescein-8-dATP 1.2 79 Rhodamine-8-dATP 1.8 1~9 Rhodamine-8-dCTP 0.4~ 32 Rhodamine-lO(J)-dCTP 5.7 (avg o~ 7~ 376 Fluorescein-lO(J)-dCTP 5.9 (avg 9~ 3) 389 Fluor~scein-(15)-dCTP 1.5 148 Fluoresc~in-4-dUTP 1.7 112 Rhodamine- (12) -du~rp 12 825 Similar experiments were also performed using T5 DNA
polymerase. The reaction conditions were identical to those of Example 4 tTa~le 2) except that T5 DNA psl~merase was used and WO93/19078 C~ 8~ PCT/US93/0~390 the buffer composition for the reaction was 50 mM HEPES (pH 7.3), 10 mM MgCl2, 50 mM ammonium sulfate, 5 mM DTT. It was found that using Rhodamine-lO(J)-dCTP and Fluorescein-~O(J)-dCTP as the modified nucleotides, 7.8~ and 7.4% of nucleotides were incorporated into TCA precipitable material, respectively. This corresponds to 5l5 and 488 ng of total DNA synthesized, for Rhodamine-lO(J)-dCTP and Fluorescein-lO(J)-dCTP respe~tively.
BACKGROUND AND PRIOR ART
:"
The invention concerns novel compounds for nucleic acid labelling and methcd for making nucleic acids incorporatin~ the ~el compounds~
Techniques for labelling nucleic acids with a reporter molecule generally fall into one of two categories: incorporating the label int~ the nucleic acid during syn~hesis, or post~
synthetically modifying the nucleic acidO The form r is commonly accomplished by providing a labelled derivati~e of one of the preCurcor nucleotide triphosphates as a partial o~ complete subs itute for the nsrmal precursor during the enzyme-catalyzed synthesis of the nucleic acid. The labelled nucleotide deriv~tive must meet certain criteria. The derivative must continue to be recognized by the ~nzyme as a substrate, it must not inter~ere with or inhibit the enzyme and it must participate in ~he normal hydrogen-bonding in~eractions of base pairing, adenine with thymine (or uracil), guanine with cytosine.
ZO Post-synthetic modification labelling is commonly accomplished by modifying the end group of a nucleiG acid by means of a chemical reaction or by ~n~enzyme such as terminal trans~erase.
A wide v~riety of reporter molecules have ~een incorporated in~o nucleic acids. Besides radioactive labels, which are usually incorporated during synthesis, fluorescent labels, chromatic labels, luminPscent labels, ligands and haptens have C A ~
been employed. Rhodamine and fluorescein have been used for fluorescent labelling. Nitroblue tetrazolium and BCIP ~5-bromo-4-chloro-3-indolylphosphate) (Gibco BRL~ have been us~d as chromophores. Firefly luciferin and PPD (~-methoxy-4-(3-phosphatenephenyl)spiro~l,2 dioxetane-3,2'-ad3mantane~ (Gibco 8RL) have been used as luminescent labels. Biotin has been used as a ligand to bind labelled streptavidin. Dinitrophenol and digoxigenin have been used as hapten }abels to bind antibody and take ad~antage of immunoassay methods. The foregoing examples are illustrative only and not limiting.
Radioactive labels have the advantage of pr~viding high ~ensitivit~, however they have the disadYantagss of being expensive~ of having short shelf life in sQme cases, and of ~ssenting safety and disposal problems. While n~n-radioactive labels lack the disadvantages of radioactivity, they pose other difficulties for the potential user. ~any of the reporter compounds are large, bulky molecules rela~ive to the nucleotides themselves, and their size can sterically interfere with incorporation. Low levels of incorporat~on c~use loss of detection sensiti~ity by limiting the level of detectab~e signal per nucleic acid molecule. The reaction conditions used for post~synthetic nucleic acid labelling can be incompatible with nucleic acid integrity and can in~olve specialized ch2mistry that requires equipment and reagents not normally found in the la~ora~ories of many potential end users o~ the labelled nucleic acid.
Steric interference by large reporter compounds has been alleviated by the use of linkers, linear chains of, typically four to twelve atoms, usually a saturated or partially unsat~rated aliphatic chain, occasionally containing an amide group. The function of linkers has been considered to be to act as a spacer be~ween *he nucleotide base and the label. Any linkers capable of providing adequate spacing and flexibility have been considered functionally equi~al~nt.
W093/1907X CA2 i i -15~3 PCT/~S93/02390 The use of linkers has also made possible a hybrid labelling technique whereby precursor nucleotides modif ied to possess a linker moiety are incorporated into the nucleic acid during synthesis, to yield linker-modified nucleic acid. The linker groups suitable for such modification must have a reactive group at the free end of the linker hain. Th2 linker-modified nucleic acid is post-synthetically coupled with a reporter compound at the reactive ends of the incorpor~ted linkers. (See, e.g., Jett et al., U.S. Application Serial Number 07/765,277).
SUMMARY ~F_THE INVENTION
.
The pres~nt invention is based on the discovery that Jeffamine (TM Texaco, Inc.~-linked nucleoside triphosphates can substantially superior subskrates for incorpora~ing label into nucleic acids during enzyme-catalyzed synthesis. Use ~f a Jeffamine-linked labelled nucleoside triphv~phate derivative as a partial substitute for unlabelled substrate leads to significantly greater incorporation, hence increased label density per unit length of nucleic acid, than the same label joined by a p~ior art linker. Jeffamine linkers have been found to have the property of permitting higher levels of incorporation of any label.
Accordingly, novel Jeffamine-linked deoxy- or ribo-nucleotide derivatives are provided for any sort of nucleic acid label desired: fluorescent, chromatic, bio- or chemi-~5 luminescent, ligand or hapten. Simi~arly, Jeffamine~modifiedribo- or deoxyribo-nucleotides are provided for synthesis of Jef~amine-modified nucleic acids. The lat~er can be post-synthetically modified by attachmènt of any desired label or combination of labels.
.
The invention further provides an impro~ed method of synthesizing labelled nucleic acids, whereby greater frequency of label incorporation than heretofore is achieved. Because the Jeffamine-linked deoxy- and ribo-nucleotides of the invention are W~ g3/lgO78 PC~USg3tO2390 `` C~2~ , 75~3 incorporated with such high efficiency, the method also provides conventional levels of label using lower amounts of nucleotide derivative in the reacti~n mixture, thereby pr~viding substantial savings in conventional uses of labelled nucleic acids.
S DETAILED DESCRIPTION OF THE INVENTION
Jeffamines arP polyglycol diamines having a general formula H2N tCH2)n~tO~(C~2)n]~~N~2r where n is 2 or more, preferably 2 to 4, and m is 1 to 10, preferably 1-5, m~st preferably 2 or 3.
Where m is greater than 1I there can be a different value of n for each ~O (CH23~- group. However, typically and most conveniently, the value of n will be ~he same f or each ( C~2) n group in the compound. Useful Jeffamines of the invention are -, triD or tetra- and higher ethylene, n-propylene, or n-butylene glycol diamines. Although Jeffamines having branched (CH2)n groups are known, those preferred herein have linear (CH2)n groups. The structure ~f triethylene glycol diamine can be diagrammed as H2N-~H2c~2-o-cH2c~2-o-c~2c~2 NH2 The choice of any particular Jeffamine is one which can be made readily by those skilled in the art, within limits disclosed herein.
One of the Jeffamine amino groups is used to rP ct with the base moiety of a nucleotide, the other amino group can reacted with a reporter moiety either before or after nucleic acid synthesis. R~action of a Jeffamine with adenine can be carried out at the 8 position or preferably, at the 6 position of adenine. A Je~f amine derivative of guanine at the 8 position of guanine is the only suitable derivative known. Cytosine can be reacted at the 4 position of cytosine. Jeffamine derivatives of uracil and thymine are only feasible by indirect linkage. Such indirect linkage can be achieved, for example~ by f irst forming allylamine derivatives of the pyrimidine ring at the 5 or 6 WO93/19078 C A 2 i I / 5 ~ 3 PCT/USg3/02390 position, followed by subse~uent reaction with a Jeffamine.
Derivatives of cytosine can also be formed in like manner.
The labelled nucleotides of the invention therPfore have three parts, the first being a deoxy- or ribo- nucleotide, abbreviated dN or rN (or d or rNTP if in the triphosphate form) which can be either d or r- adenosine (dA or rA), d- or r-guanosine (dG or rG) or d- or r-cytosine (dC or rC). The s~cond par~ is a Jeffamine-based linker, which will have a chain length of 7 Qr more atoms, depending on the Jeffamine used, and abbreviated herein as n(J) where n is an integer of 7 or more.
The third part is the reporter, designated X~ X can be any molecule use~ul for labeling nucleic acid and having the ability to f~rm stabl compounds with a primary amine group. X can be ~fluorophore, for example, rhodamine or fluorescein. X can be a chromoph~re, for example, Nitro blue tetraæolium, or BCIP. X
can be a luciferin or other luminescent reporter, such as PPD.
X can be a ligand, for example, biotin, having the property of bindi~g another, readily detectable, molecule. Similarly, a ligand such as an enzyme cofactor can be detected by the activity of the enzyme which binds it, the enzyme-catalyzed reaction providing an amplifica~ion factor to enhance sensitivi.ty. X can also be a hapten, for example dinitrophenol or digoxigenin, detectable by immunochemical means.
A labelled nucleotide of the invention is therefore 2~ abbreviated herein as X-n(J)-(d or r)NTP.
The term "modified nucleotide" is de~ined herein as a nucleotide having a Jeffamine substituent but no reporter moiety (X is H). A modified nucleotide is useful for post-synthetic labelling of DNA in which thP modified nu~leotide has been incorporated. Such a modified nucleotide is abbreviated n(J)-~d or r~NTP, where n is 7 or more, and N is adenosine, guanosine or cytidine.
WO93/19078 C A 2 i i ~5 8 3 PCT/US93/02390 Enzyme-catalyzed addition of labelled nucleotides of the invention to the end of a nucleotide chain can also be accomplished. Using terminal deoxynucleotide transferase, Rho-lO(J)-dCTP was successfully added to DNA, using reaction conditions disclosed in U.S. Pat~nt 4,878,979 for end-labelling DNA with Biotin-14-dATP.
The invention is exemplified by comparing two compounds of the invention, Rhodamine-lO(J)-dCTP and Fluorescein-~O(J)-dCTP, with pri~r art compounds, demonstrating surprisingly higher incorporation efficiency than heretofore possible with prior art compounds. The structures of Rhodamine-10(~)-dCTP and the corresponding FluorescPin-lO(J)-dCTP are shown in Formula 1.
Formulas 2-5 show the structures of Fluorescein- and Rhodamine-8~dATP, Fluor~scein- and Rhodamine-4-dUTP, Rhodamine-8-~CTP, Fluorescein-(15)-dCTP, and Rhodamine-~15)-dCTP, respPctively.
WO93/1907~ CA 2 i I /583 P~US93/0:~3~0 FORMUI,A 1 - N H /~/O o~N
O
Rho--lO (J) -dCTP: R = TETlRAMETHYL--RHODZ~MINE
Fl--lO (J~--dCTP: R = FLUORESCEIN
X = DEOXYRIBOSE-S '--TR:l PHOSPHAT~E
OR RIBOSE--5 '--TRIPHOSPH~TE
~YO93/19078 PC~/US93/02390 C~2i ! /-5~3 FOR~LA 2 N H ( C H ~ ) 6 N H ~ C O - R
. Nl~N
, I
Rh~-8-dATP: R = TETRAMETHYL-RH~DAMINE
Fl-8-dATP: R = FLUO~ESCEIN
X = DEOXYR BOSE-5'-TRIPHOSPHATE OR
RIBOSE
WO ~3~1~0~8 ` C A 2 i i 7 5 8 3 PCr/lJS93/02390 FORMUI~ 3 O ~) H N~ N H J~` R
'~ o N
Rho-4-dUTP: R = TETRl~IEmYL-RHODAMINE
Fl-4-dU~P: R = ~?1UORESCEIN
X = DEOXYRIBOSE-5 ' -TRIPHOSPHATE
WO 93~19078 C A 2 i i l 5 ~ 3 PCr~US93/02390 FORMUL~ 4 NH ~, ~ NH~R
1 ~
O
X
Rho-8-dCTP: R = TETRAMETHYL--RHODAMINE
1~
WO 93/19~78 ~ A 2 i I / 5 8 3 P~/US~3/0239~ ^
NH /\, NH~1~R
Il o~3 X
-Rho-15--dCrP: R = - ( CH2) 5NH-CO--TETRAMETHYL RHI:)D~IINE
Fl 15--dCTP- R = - ~CH2) 5NH--CO--FLU5:~RESCEIN
X = DEOXYRIBOSE-5 ' -TRIPHOSPHATE OR
RIBOSE
WO 93~19û7B ` P~/US93/02390 EXAMPLES C h 2 i I ~ 3 Example 1. PreParation of N4-triethYlene ql~rcol amine~
deoxv~rtidine-5'-triphosPhate flOr3~-dCTP2 To triethylene glycol diamine (6.55 mmol, 1 ml) at 0C was added hydrochloric a id (9.5 mmol, 0.8 ml, 36%) drop-wise. To this solution was added sodium meta-bisulfite (2.2 mol, 430 mg) and water to a final volume of 3 ml. Deoxycytosine-5'-triphosphate (O.1 mmol, 50 ~g~ was di~solved in the above solution of dimine-bisulfite freshly prepared and filtered. ~o thls solution was added hydroquinone (~ mg in 10 ~l of ethanol) and the reaction mixture stirred for 2 days a~ ~5C under argon.
HPLC trace, after adjustment of ~he aliqu~t to pH = ~.5, (DuPont Zorb~x oligo c~lumn, 25% acetonitrile, Q.25 ~ NH4H2P04) 2 ml/min.
isocratic mode~ shows no starting material plus a new ma~or peak (68% of total). The crude mixture was ad~usted to pH = B.5 with sodium hydroxide, diluted to 500 ml with water and loaded on a DEAE anion exchange column equilibrated with triethyl ammonium biearbonate tTEAB). The column was eluted with a linear gradient of T~AB (0,01 - 1.0 M; 600/600 ml). After desalting of the major peak fractions, 6006 mg of pure desired product were isolated ~x = ~71-~72nm.
xample 2. Labellin~ of amino-nucleoside triPhosphates with fluorescent dyes.
The Je~famine-nucleoside triphosphates (10-20 ~mol) e.g., Z5 lO~ dCTP, were dissolved in sodium bicarbonate tO.4 M, 500 ~l) or sodium borate solution (0.1 M) and treated with a 3 to 5 fold molar excess of the N-hydroxysu~cinimide ester of the dye ~e.g., C ~ 3 fluorescein or rhodamine) in anhydrous dimethyl formamide (500 ~l). The mixture was reacted for 3 - 18 hr. at room temperature.
The reaction was monitored by thin layer chromatography (silica gel; butanol: acetone: acetic acid: 5~ ammonium hydroxide:
water/70:50:30:30:20) and/or by HPLC. The crude mixtures were diluted in water (200 - 300 ml), loaded on a lO - 15 cm long, by l cm diameter column of mild anion exchange resins and eluted, sequen~ially, with O.Ol, 0.2 and 0.5 ~ triethylammonium bicarbonate until the fraction containing the fluorescent dNTP
was collected. After desalting of thP appropriate column ~raction, TLC, HPLC and capillary electrophoresis analysis was used to assess the purity and characteristic elution patterns of the desired product. The compounds were characterized by thPir U.V. spectra as the o~erlapp~ng sp~ctra of the starting amino ~dified base and the dyes. Yields of fluorescent nucleotides were 50 - 60~.
Example 3. Use of Rhodamine-lO(J~-dCTP in Nucleic Acid Labelin~
Five fluorescent nucleotides were initially screened ~or enzymic incorporation into DNA using random primer extension with Klenow fragment of DNA polymerase I. The five fluore cent nucleotides were fluorescein-8-dCTP, rhodamine-~ d~TP, rhodamine-8-dCTP, rhodamine-lO(J)-dCTP, and rhodamine 4 dUTP. All but rhodamine-lO(J) dCTP (which has two ether linkages) have alkylamine linkers.
Template DNA (lO0 ng) was denatured in a dilute buffer such as TE (lO mM Tris-HCl, pH 7-5i 1 mM EDTA) by heating at 100C fsr 5 min. Reaction components were added to final concentxations as follows: 5~ mM Tris-HCl (pH 6.8), 5 mM MgCl2, lO mM 2-mercaptoethanol, 400 ~g/ml BSA, 300 ~g/ml random octamers, 200~M
dCTP, dGTP, and dTTP,lO0 ~M dCTP and lO0 ~M rhodamine-lO(J)-dCTP, 10 ~Ci ~- [32p~ - dATP ~3900Ci/mmol) and 40 units Klanow fragment in a final volume of 50 ~l. After incubation at 37~C for 1-2 hours, 5 ~l 0.~ M EDTA (pH 7.5) was added to terminate the reaction. Incorporation was determined by trichloroacetic acid W093/1~07~ PCT~US~3/02390 CA2ii~583 (TCA) precipitation. Diluted aliquots from the reaction were spotted on glass f iber f ilters in duplicate and dried . One of the duplicate filters was washed four times in cold 5~ TCA, 20 mM sodium pyrophosphate, then rinsed in 70% ethanol and dried and counted in a liquid scintillation counter ~incorporated counts).
The second filter was counted directly in the scintil~ ation counter (total counts). Incorporation of radioactive label was 40-50% indicating syn~hesis of several micrograms of fluorescent DNA probe. When other modified nucleotides were tested a mixture of 100 ~M unmodified dNTP and 100 ~M flusrescent dNTP was used in place of the dCTP/rhodamine-lO(J)-dCTP above. When the modified nucleotide was a dATP derivative, ~-[32P~-dCTP was used as a trace label.
Fluorescent dNIP% Incorvorati~onnq DNA S~nthesi2ed Fluorescein-8-dATP 0.6 79 Rhodamine-8-dATP 0.5 66 Rhodamine-8-dCTP 0.1 13 Rhodamine-lOtJ)-dCTP42.6 5623 Rhodamine-4-dUTP 0.5 6~
The percentage of rhodamine-lO~J)-dCTP was varied in subsequent experiments from 75%- to 100%. Incorporation decreased with increasing *luorescent nucleotide concentration.
Incorporation wa5 increased at higher percent ges by increasing the absolute concentration of unmodified dNTP to at least 50 ~M.
At a percentage of 95%, incorporation wa~ approximately 15% and at a percentags of 97~5%, incorporation was reduced to about 6%.
~NA probes prepared at 50% and 9%5 both functioned in in situ chromosome hybridizations.
W0 93~19~78 C ~ / 5 ~ 3 PCr/US93/~23~0 ExamPle 4 . Use of Rhodamine-10 fJ) -dCTP in Nucleic_ Acid La~el inq Template DNA (500 ng) was denatured in a dilute buffer such as TE (10 mM Tris-HCl, pH 7.5; 1 mM EDTA) by h~ating a~ 100~C for 10 min. Reaction components were added to final concentrations as f~llows: 5~ mM Tri~-HCl (pH 6.8), 5 mM MgC12, 10 mM 2-mercaptoethanol, 400 ~g/ml BSA, 300 ~m/ml random octamers~ 1~0 ~M each of dA~P, dGTP, dTTP, and ~CTP, 10 ~Ci ~-[32P]-dATP (3000 Ci/mmol), ~nd 40 units Klenow fragment in a final volume of 50 ~1. When testing modified nucleotides the c~rresponding unmodified dNTP was rsplaced with 100 ~M of the modified nucleoside triphosphates. In some experiments ~e.g., Table 2), diff~r~nt mixtures of modified and unmodified nucleotides were used. The perc~ntage of modified nucleotides to the total modified plus corresponding unmodified nucleotide was varied ~rom 25% to 100%. IN all cases J the total concentration of each nucleotide was kept at 100 ~M which resulted in a total nucleotide concentra~ion of 400 ~M. After incubation at 37C for 2 hours, 5 ~1 0.2 M EDTA (pH 7.5~ was added to terminate the r~action. Incorp~ration was determined by trichloroaceti~ acid (~CA~ preripitation~ Diluted aliquots from the reacti~n wer~
spotted on glass fiber filters in duplicate and dried. One of the duplicate filters was washed four times in cold 5% TCA, 20 mM sodium pyrophosphate, then rinsed in 70% ethanol and dried and count~d in a liquid scintillation counter (incorporated rounts).
The second fi~ter was counted directly in the -~cintillation counter (total count~). Incorporation of radioactive label was used to dete~mine sy~thesis of fluorescent DN~ probe. When the modified nucleotide was a dATP derivative, ~-[32P~-dCTP was used as a trace label.
WO 93/1~078 P~/US93/02390 C~ 583 Fluor. ng DNA
Fluorascent dNTP dNTP ~ % Incorpsvnthesized Rhodamine-10 (J) - lOO~M --- 5 . 7~ 37 dCTP 75~ 25~M 24 . 51617 50,uM 50~M 33 . 92237 25,uM 75~M 51. 83149 ~luorescein-l tJ) - lOOl M --- 5, gb38,9 dCTP 75~5 25,u~l 13 . 8 911 50~M 50~M 36. 22389 2S~M 75~M 54 . ~3623 ~ - b a = average of 7 experiments. = average of 3 experiments 15Below, Table 3, is a li~t of additional modified nucleotides - that have been screened for use in enzymatic incorpora~ion into DNA using random primer extension with ~lenow fragm~nt of DNA
polym~race .
O
~"9~9e~ % Incor~orationq DNA Synthesized Fluorescein-8-dATP 1.2 79 Rhodamine-8-dATP 1.8 1~9 Rhodamine-8-dCTP 0.4~ 32 Rhodamine-lO(J)-dCTP 5.7 (avg o~ 7~ 376 Fluorescein-lO(J)-dCTP 5.9 (avg 9~ 3) 389 Fluor~scein-(15)-dCTP 1.5 148 Fluoresc~in-4-dUTP 1.7 112 Rhodamine- (12) -du~rp 12 825 Similar experiments were also performed using T5 DNA
polymerase. The reaction conditions were identical to those of Example 4 tTa~le 2) except that T5 DNA psl~merase was used and WO93/19078 C~ 8~ PCT/US93/0~390 the buffer composition for the reaction was 50 mM HEPES (pH 7.3), 10 mM MgCl2, 50 mM ammonium sulfate, 5 mM DTT. It was found that using Rhodamine-lO(J)-dCTP and Fluorescein-~O(J)-dCTP as the modified nucleotides, 7.8~ and 7.4% of nucleotides were incorporated into TCA precipitable material, respectively. This corresponds to 5l5 and 488 ng of total DNA synthesized, for Rhodamine-lO(J)-dCTP and Fluorescein-lO(J)-dCTP respe~tively.
Claims (16)
1. A modified nucleotide compound having the formula X-n(J)-(d or r)NTP
where N is adenosine, guanosine or cytidine; X is H, a fluorophore, a chromophore, a luminescent compound, a ligand or a hapten; and n is an integer of 7 or more.
where N is adenosine, guanosine or cytidine; X is H, a fluorophore, a chromophore, a luminescent compound, a ligand or a hapten; and n is an integer of 7 or more.
2. A compound according to claim 1 wherein X is H, fluorescein, rhodamine, nitroblue tetrazolium, BCIP, firefly luciferin, PPD, biotin, dinitrophenol or digoxigenin.
3. A compound according to claim 1 wherein X is rhodamine, N
is cytidine and n is 10.
is cytidine and n is 10.
4. A compound according to claim 1 wherein X is Fluorescein, N is cytidine and n is 10.
5. A compound according to claim 1 wherein X is biotin, and n is 10.
6. Nucleic acid comprising a modified nucleotide of the formula X-n(J)-(d or r)N
where N is adenosine, guanosine or cytidine; X is H, a fluorophore, a chromophore, a luminescent compound, a ligand or a hapten; and N is an integer of 7 or more.
where N is adenosine, guanosine or cytidine; X is H, a fluorophore, a chromophore, a luminescent compound, a ligand or a hapten; and N is an integer of 7 or more.
7. The nucleic acid of claim 6 wherein the nucleic acid-is DNA
and N is dA, dG or dC.
and N is dA, dG or dC.
8. The nucleic acid of claim 6 wherein the nucleic acid is DNA, N is dA, dG or dC and X is H, Fluorescein, rhodamine, nitroblue tetrazolium, BC1P, firefly luciferin, PPD, biotin, dinitrophenol or digoxigenin.
9. The nucleic acid of claim 6 wherein the nucleic acid is DNA, N is dC, X is fluorescein or rhodamine and n is 10.
10. In a method of nucleic acid synthesis by a reaction catalyzed by a nucleic acid polymerase enzyme wherein the polymerase is present in a reaction mixture comprising a template nucleic acid and one or more deoxy- or ribo-nucleotide triphosphates, the improvement comprising substituting for a portion of one of said nucleotide triphosphates X-n(J)-(d or r)NTP where N is adenosine, guanosine or cytidine; X is H, a fluorophore, a chromophore, a luminescent compound, a ligand or a hapten;
and n is an integer of 7 or more.
and n is an integer of 7 or more.
11. The method of claim 10 wherein the enzyme is a DNA
polymerase, the reaction mixture comprises deoxynucleotide triphosphates and X is H, fluorescein, rhodamine, nitroblue tetrazolium, BC1P, firefly luciferin, PPD, biotin, dinitrophenol or digoxigenin.
polymerase, the reaction mixture comprises deoxynucleotide triphosphates and X is H, fluorescein, rhodamine, nitroblue tetrazolium, BC1P, firefly luciferin, PPD, biotin, dinitrophenol or digoxigenin.
12. The method of claim 10 wherein the enzyme is a DNA
polymerase, the reaction mixture comprises deoxynucleotide triphosphates, X is fluorescein or rhodamine, N is C and n is 10.
polymerase, the reaction mixture comprises deoxynucleotide triphosphates, X is fluorescein or rhodamine, N is C and n is 10.
13. The method of claim 10 wherein the enzyme is the Klenow fragment of E. coli DNA polymerase, the reaction mixture comprises deoxynucleotide triphosphates, X is fluorescein or rhodamine N is C and n is 10.
14. The method of claim 10 wherein the enzyme is T5 polymerase, the reaction mixture comprises deoxynucleotide triphosphates, X is fluorescein or rhodamine, N is C and n is 10.
15. The method of claim 10 wherein the enzyme is terminal deoxynucleotide transferase, the reaction mixture comprises deoxynucleotide triphosphates, X is fluorescein or rhodamine, N is C and n is 10.
16. A kit for making a fluorescent nucleic acid having four ribo- or deoxynucleotides in its composition and having at least one of the ribo or deoxynucleotides partially substituted by a labelled ribo- or deoxynucleotide, comprising:
a) a nucleic acid synthesizing enzyme;
b) a modified nucleotide compound having the formula X-n(J)-(d or r)NTP
where N is adenosine, guanosine or cytidine; X is H, a fluorophore, a chromophore, a luminescent compound, a ligand or a hapten; and n is an integer of 7 or more.
a) a nucleic acid synthesizing enzyme;
b) a modified nucleotide compound having the formula X-n(J)-(d or r)NTP
where N is adenosine, guanosine or cytidine; X is H, a fluorophore, a chromophore, a luminescent compound, a ligand or a hapten; and n is an integer of 7 or more.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US85268892A | 1992-03-17 | 1992-03-17 | |
| US07/852,688 | 1992-03-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2117583A1 true CA2117583A1 (en) | 1993-09-30 |
Family
ID=25313973
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2117583 Abandoned CA2117583A1 (en) | 1992-03-17 | 1993-03-17 | Modified nucleotides |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0638087A1 (en) |
| CA (1) | CA2117583A1 (en) |
| WO (1) | WO1993019078A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE60141871D1 (en) | 2000-05-05 | 2010-06-02 | Wallac Oy | Oligonucleotide labels and their use |
| AU2002213125B2 (en) | 2000-10-11 | 2007-11-22 | Applied Biosystems, Llc. | Fluorescent nucleobase conjugates having anionic linkers |
| FR2893617A1 (en) * | 2005-11-24 | 2007-05-25 | Inst Nat Sante Rech Med | New photoactivable NADH, NADPH, NAD or NADP analogs, useful to study the time-resolved spectroscopy and crystallography mechanisms of the enzymes of a cell or a tissue |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4828979A (en) * | 1984-11-08 | 1989-05-09 | Life Technologies, Inc. | Nucleotide analogs for nucleic acid labeling and detection |
| US4962029A (en) * | 1987-10-02 | 1990-10-09 | Cetus Corporation | Covalent oligonucleotide-horseradish peroxidase conjugate |
| US4914210A (en) * | 1987-10-02 | 1990-04-03 | Cetus Corporation | Oligonucleotide functionalizing reagents |
| EP0407816A3 (en) * | 1989-07-14 | 1993-01-27 | Abbott Laboratories | Base modified nucleosides |
-
1993
- 1993-03-17 WO PCT/US1993/002390 patent/WO1993019078A1/en not_active Application Discontinuation
- 1993-03-17 CA CA 2117583 patent/CA2117583A1/en not_active Abandoned
- 1993-03-17 EP EP93907533A patent/EP0638087A1/en not_active Withdrawn
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| EP0638087A4 (en) | 1994-12-27 |
| WO1993019078A1 (en) | 1993-09-30 |
| EP0638087A1 (en) | 1995-02-15 |
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