WO1987007611A1

WO1987007611A1 - Process for dna labelling

Info

Publication number: WO1987007611A1
Application number: PCT/EP1987/000287
Authority: WO
Inventors: Brian Sproat
Original assignee: Europäisches Laboratorium Für Molekularbiologie (E
Priority date: 1986-06-03
Filing date: 1987-06-03
Publication date: 1987-12-17
Also published as: DE3642939A1

Abstract

A process for labelling DNA fragments (oligonucleotides) using fluorescent dyes. The labelling is carried out by converting the DNA fragment into its 5'-[HS-(Y)z]-derivative, and the latter is reacted with a derivative of a fluorescent dye, which together with the 5'-[HS-(Y)z]-derivative forms a DNA fragment of the formula (I) fluorophore-X-S-(Y)z-CH$(2,6)$ -oligonucleotide, wherein X and Y independently are a group comprising one or more carbon atoms and/or hetero atoms, z = 0 or 1, and fluorophore is the residue of a fluorescent dye. In a preferred embodiment the DNA fragment is converted into its 5'-(S-triphenylmethyl-3-mercaptopropyl phospho)-derivative and the 5'-(3-mercaptopropyl phospho)-derivative obtained after cleavage of the triphenylmethyl group is reacted with a (5- and/or 6)-iodoacetamino-rhodamine or -fluorescein.

Description

Process for DNA labelling

The invention relates to a process for labelling DNA frag¬ ments using fluorescent dyes and to its application in the sequential analysis of DNA.

Various methods for the sequential analysis of DNA are known. The majority of processes for determining the DNA sequence are based either on chemical decomposition by base-specific chemical reactions (cf . A.M. Maxa and W. Gilbert, Proc . Natl. Acad. Sci. USA 7 (1977) 560-564) , or on enzymatic me¬ thods of sequential determination on the basis of the elon¬ gation of DNA chains catalysed by DNA polymerase (cf . F. San- ger and A. R. Coulson, J. Mol . Biol. 9± (1975) 441-448; F. Sanger et al, .Nature 265 (1977) 687-695) . According to the so-called "dideoxy-method" as specific terminators of the DNA chain elongation there are used 2 ' ,3 '-dideoxynucleoside- triphosphates, e.g. ddA TP, (F. Sanger et al, Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467); cf. in summary also R. Knippers, "Molekulare Genetik", 4th edition, Thieme-Verlag Stuttgart, New York 1985, pages 402-407) . The enzymatic me¬ thods are particularly suitable for determining long DNA se¬ quences, because only low labour costs are incurred for each individual sequencing reaction.

For the determination of DNA fragments it is necessary in all sequencing methods for the DNA fragments to be labelled specifically. This can be carried out by labelling with ra-

3 dioactive isotopes (radioactive labelling) . In particular H,

C, S and P are used as radioactive isotopes. Very in- tensive labelling is necessary for some determination methods, e.g. the "Southern Blot" method. Therefore, because of the low radiation intensity of 3H and 14C only the phosphorus lsoto- pe 32P has been considered. However, the use of this sub¬ stance entails many disadvantages which have hitherto preven- ted in particular the application of the "Southern Blotting" method to routine medical diagnosis: 32P is very expensive, it has a half-life of only 14.3 days (it thus has to be con¬ sumed rapidly and stock-keeping is not possible) and after use has to be disposed of at great cost. Moreover, extensive safety precautions are imperative both for personnel and la¬ boratory equipment. As a rule the time taken for results to be obtained is very long, since gel electrophoretically sepa- rated DNA fragments labelled with P 32 mostly have to lie se- veral days to weeks on an X-ray film before signals become visible.

This .time aspect is even more significant with respect to the

3 isotope H which is predominantly used for "in situ hybri¬ dization", namely the direct identification of DNA sequences in histological preparations. Here the exposure times fre¬ quently last several months.

Therefore, to overcome these problems it has been proposed to replace radioactive labelling by labelling with nucleotides which are coupled to a biotin molecule via the 5 position of the pyrimidine ring and an allyla ine branch; nucleotides of this type behave like their unsubstituted analogues in the "nick-translation" used for the "Southern Blot" method, wherein the biotin residues can be readily identified by way of an antibody reaction (cf. e.g. "Nachrichten Chem. Tech.

Lab. 34 (1986) page 430). By optimizing the experimental con¬ ditions this method could be optimized in such a way that its sensitivity is comparable to the radioactive labelling pro¬ cess.

According to another type of DNA labelling, applying the "dideoxy-method", the M13 primer is labelled with a fluores¬ cent dye in each of the four batches, namely with fluorescein isothiocyanate (FITC) , 4-chloro-7-nitrobenzo-2-oxa-1-diazole (NBD chloride) , tetramethylr odamine-isothiocyanate (TMRITC) and with Texas red (cf. Bio Technology 3_ (1985) 395-396; "Nachrichten Chem. Tech. Lab. 3_4 (1986) pages 430-431). The DNA fragments thus labelled can then be separated with a po- lyacrylamide gel and determined photometrically with a laser. A major problem in this method is the optical sensitivity of the photometric system, because each DNA fragment contains only one molecule of the fluorescent labeller, from which there results a concentration of the fluorescent dye in the gel of only about 10 -10 M. Only synthetic oligonucleotides have been analyzed hitherto in accordance with this method.

L.I_Ϊ. Smith et al (Nucleic Acids Research _13_ (1985) 2399-2412, GB-A-2155176) describe the synthesis of oligonucleotides with an aliphatic amino group at their 5' position. This amino group can be reacted with a plurality of electrophilic reagents. In this way fluorescent dyes can be bonded covalently to synthesized DNA-primer oligonucleotides.

The present invention relates to a process for labelling DNA fragments (oligonucleotides) using fluorescent dyes, charac¬ terized in that the DNA fragment is converted into its 5'-[HS-(Y) - derivative, and the latter is reacted with a derivative of a fluorescent dye, which together with the 5'-[HS-(Y) ^"] -derivative forms a DNA fragment of the formula (I) fluorophore-X-S-(Y) -CH -oligonucleotide, wherein X and Y independently are a group comprising one or more carbon atoms and/or hetero atoms, z = 0 or 1 , and fluorophore is the residue of a fluorescent dye. Preferably as the fluorescent dyes rhodamines are used, e.g. rhoda ine, tetraethylrhodamine (rhodamine B) , rhodamine 6G, and especially tetramethylrhodamin, or fluoresceins , e.g. tetrabromfluorescein (eosin) , and especially fluorescein. i

In formula (I) X and Y can be the same or, preferably, different.

Preferably X is the group and especially

Preferably Y is the group -(CH₂) wherein n is an

integer 3, and particularly 3.

A preferred embodiment of the present invention relates to a process for labelling DNA fragments (oligonucleotides) using fluorescent dyes, which is characterized in that the DNA fragments (oligonucleotides) are converted into their 5' - (S-triphenylmethyl-3-mercapto-propyl phospho)-derivatives and the 5 '- (3-mercapto-propyl phospho ) -derivative obtained after cleavage of the triphenylmethyl group (trityl group) is reacted with a (5- and/or 6)-iodoacetamino-rhodamine or 5 -fluorescei .

Any rhodamines can be used as the rhodamine skeleton in the iodoacetamino derivatives, for example rhodamine, tetraethyl- 0 rhodamine ⁽rhodamine B) , rhodamine 6G, and any fluoresceins can be used as the fluorescein skeleton, for example fluores¬ cein or tetrabromofluorescein (eosin) .

In accordance with the invention, (5- and/or 6) -iodoacetamino 5 tetramethylrhodamine is preferably used because of its fluorescence properties. It has very high extinction coeffi- cients, a high quantum yield and an emission in the long wave range (Λ = 560 nm) with a band-width at half maximum of 52 nm. A preferred (5- and/or 6)-iodoacetamino-fluorescein derivative is 5-iodoacetamino-fluorescein.

The subject matter of the present invention also concerns the compound of formula (I), wherein X, Y, z and fluorophore have the meanings stated above.

Formulae la and lb show the structure of a DNA fragment labelled with tetramethylrhodamine (la) and with fluorescein (lb) in accordance with a preferred embodiment of the process of the invention.

Θoiide-

oligonucleotide.

Because of their properties the DNA fragments labelled in ac¬ cordance with the process of the invention are extremely sui¬ table for the sequential analysis of DNA. Therefore, the sub¬ ject-matter of the invention is also the use of the process according to the invention for the labelling of DNA fragments for the sequential analysis of DNA.

Preferably, the process according to the invention is uti¬ lised for sequential analysis in accordance with the dideoxy- method, wherein there is used as primer in particular an M13 primer with fluorescent labelling, which can be obtained by reacting its 5'- (S-triphenylmethyl-3-mercapto-propyl phospho)- derivative after cleavage of the triphenylmethyl group with a (5- and/or 6)-iodoacetamino rhodamine or fluorescein. However, labelling of other oligonucleotides is also possible for instance by con¬ verting them into their corresponding 5 '-(S-triphenylmethyl- 3-mercapto-propyl phospho)-derivative and reacting the latter, after cleavage of the triphenylmethyl group (trityl group) , with a (5- and/or 6)-iodoacetamino rhodamine or fluorescein.

The process according to the invention represents a reliable, simple and economical method of labelling DNA fragments, with which the disadvantages of the state of the art can be sub¬ stantially obviated. In view of the sensitivity to the la- belled DNA fragments, the process is also very well suited for laser-induced photometric determination of the labelled DNA fragments in automatic apparatus, for example in the apparatus described in the German patent application P 36 18 605.8 by the same Applicant, as well as for other bioresearch and biotechnology processes using labelled DNA fragments.

The subject-matter of the invention also concerns the use of (5- and/or 6)-iodoacetamino rhodamines or fluoresceins in the fluorescent labelling of organic compounds, in particular DNA fragments. The preparation of the 5'-[HS-(Y) 7 -oligonucleotides and the reaction with the derivative of a fluorescent dye can be effected in a per se known manner, preferably the prepara¬ tion of the 5'-[HS- (Y)_Z] - oligonucleotides is carried out via the corresponding 5'- s-triphenylmethyl-(Y) 1 - deriva¬ tive, and cleavage of the triphenylmethyl group.

For instance, preparation of 5'- (S-triphenylmethyl-3-mercapto- propyl-phospho)-oligonucleotides

can be effected by condensation of triethyl ammonium|_S-triphenylmethyl-3-mercaptopropyl 2- (1-methylimidazole-2-yl)phenyl phosphateJ with the 5 '-terminal hydroxy group of an oligonucleotide linked to a support (e.g. long-chain alkylamine/glass) - cf. B.S. Sproat et al, Nucleic Acids Research 1_4 (1986) 1811-1824) . Preferably the prepara¬ tion is effected following the phosphoramidite method (cf. for example B.A. Connolly and P. Rider, I.e.) by using (S- Trityl-3-mercaptopropyloxy)-(2-cyanoethoxy) - (N,N-diisopropyl =amino) -phosphine; this method can be carried out directly in a commercial automated DNA-synthesiser (e.g. Applied Bio- systems) .

The cleavage of the triphenylmethyl group (detritylation) can be effected, for example, with silver nitrate analogously to the method described by B.A. Connolly and P. Rider (Nucleic Acids Res. V3_ (1985) 4485-4502). The resultant compound with free SH group is then reacted at a pH value of about 8.5 with (5- and/or 6) -iodoacetamido-rhodamine or -fluorescein.

The following examples are intended to illustrate the inven¬ tion .in greater detail but without implying any restriction thereto. Unless otherwise indicated, the percentage and weight values refer to percentage by weight and parts by weight, while the temperature refers to the Celsius scale.

Examples

The solvents and reagents for solid phase oligodeoxyribo- nucleotide synthesis in accordance with the phosphotriester method were prepared in the manner described by B.S. Sproat et al, loc. cit. The S-triphenylmethyl-3-mercaptopropanol was prepared in accordance with B.A. Connolly and P. Rider, loc. cit. The (5- and/or 6) -iodoacetamino tetramethylrhodamine was supplied by Molecular Probes, Inc., Junction City, Oregon, USA. The NMR spectra were taken on a Bruker AM250 spectrometer.

Examole 1

Preparation of triethylammonium- s-triphenylmethyl-3-mercapto- propyl, 2- (1-methylimidazole-2-yl)phenyl_, phosphate]

S-triphenylmethyl-3-mercaptopropanol (3.34 g, 10 mm ole) was converted into the title compound using the method described by B.S. Sproat et al, loc. cit. The crude product was applied on to a silica gel 60H-column (170 g, 8 cm x 7 cm; dichloro- methane/triethylamine = 99/1 , v/v) and the column was then eluted with dichloromethane/triethylamine (0.5 1, 99/1; v/v), ethanol/dichloromethane/triethylamine (0.5 1, 4/95/1; v/v) and, finally, with ethanol/dichloromethane/triethylamine (-1.5 1, 10/89/1; v/v) using a nitrogen pressure. The fractions containing the pure product (determined by silica gel thin- layer chromatography; R_f = 0.11, using ehanol/chloroform/ triethylamine = 10/89/1; v/v as eluant; spraying the plate with 70 % perchloric acid/ethanol = 3/2; v/v gave a yellow/ orange stain) were collected and evaporated under vacuum, in which case a slightly hygroscopic white foam (1.6 g, 24 % yield) remained. The resultant product exhibited the corres- ponding 31P and 13C NMR spectra,

Example 2

5 ' - ( S-triphenylmethyl-3-mercaptopropyl phospho ) -d [GTAAAACGACGGCCAGT]

Fully protected [GTAAAACGACGGCCAGT] was prepared using a highly effective phosphotriester method (cf . B . S . Sproat et al , loc . cit . ) on a 1 μmole scale on a support (long-chain alkylamine/ porous glass) . Subsequently , a further reaction cycle was carried out , in which triethylammonium[s-triphenyl-methyl-3- mercaptopropyl , 2- ( 1 -methylimidazole-2yl) phenyl-phosphate] was condensed with the 5 ' -terminal hydroxy group of the oligodeoxyribonucleotide linked to the support. The modified oligodeoxyribonucleotide was freed of the protective groups and separated from the support, as described by B.S. Sproat et al, loc. cit., using oxi ate, followed by 25 % ammonia. After the ammonia stage, the crude product was desalinated by dialysis and the desired 5' - (S-triphenyl-methyl-3-mercapto- prophyl phospho )-oligodeoxyribonucleatide is purified by re- versed-phase high pressure liquid chromatography (HPLC) on μ-Bondapa,-C.g using 0.1M triethyl ammonium acetate (pH = 7)/ acetonitrile as eluant. The S-triphenylmethyl compound was elu- ted at a buffer composition of about 29% acetonitrile. The solution containing the product was evaporated under vacuum to dryness and left behind a white glass (about 160 nmole, determined by UV spectroscopy) .

Example 3

Preparation of d [GTAAAACGACGGCCAGTj (Formula I) labelled with tetramethylrhodamine

5 '- (S-triphenylmethyl-3-mercaptopropyl phospho) d [GTAAAACGAC GGCCAGTj (about 20 nmole) was detritylated with silver nitra¬ te in accordance with the method described by B.A. Connolly and P. Rider, loc. cit., and after removal of the silver ions with dithiothreitol a small amount of the solution was ana¬ lyzed by reversed-phase high pressure liquid chromatography (HPLC) on μ,-Bondapak-C..₈. The 5'- (3-mercaptopropyl phospho) oligodeoxyribonucleotide was eluted with an acetonitrile com¬ position of about 14 %. The pH value of the solution contai- ning the mercaptooligodeoxyribo-nucleotide was adjusted to 8.5 with sodium bicarbonate solution and a solution of (5- and/or 6)-iodoacetaminotetramethylrhodarαine (200 nmole) in N,N-dimethylformamide (50 μl) was added. The solution was mixed carefully and left for one hour in darkness at room temperature. The rose-coloured solution was then dialyzed against water and the oligodeoxyribonucleotide labelled with tetramethylrhodamine was purified by ion-exchange high pres- sure liquid chromatography (HPLC) on Partisil 10SAX using a concentration gradient of potassium dihydrogenphosphate, pH = 6.3, in formamide/water (6/e, v/v) as eluant. The purified oligonucleotide labelled with tetramethylrhodamine was then desalinated by dialysis and stored in darkness at -20°C.

The resultant fluorescence-labelled M13 primer of Formula I was used to carry out improved standard dideoxy methods, as described by S.A. Williams et al, Biotechniques 4_ (1986) 138- 147, wherein the dideoxy/deoxy ratio was optimized in such a way as to ensure satisfactory labelling of the first 300 to 400 bases. Very good results were obtained.

Example 4- •

Preparation of d /GTAAAACGACGGCCAGT/ (Formula II) labelled with fluorescein

As described in example 3, 5 '- (3-mercaptopropyl phospho ) - oligodeoxyribonucleotide was reacted with 5-iodoacetamino fluorescein instead of iodoacetamino tetramethylrhodamine. The oligodeoxyribonucleotide labelled with fluorescein was then firstly purified by ion-exchange high pressure liquid chromatography (HPLC). After desalination by dialysis, the fluorescein-labelled oligodeoxyribonucleotide was then fur¬ ther purified by reverse-phase HPLC on an Aquapore RP-300 „ column. The product (fluorescence-labelled M13 primer) elutes with an acetonitrile content of about 25 % and is then desalinated by dialysis.

Example 5

Preparation of S-Trityl-3-mercaptopropanol

Sodium hydroxide ( 4 .4 g , 1 10 nmol ) was dissolved in H_0 ( 25 ml ) , and added to a solution of triphenylmethylmercaptan (27 .6 g , 100 nmol ) in ethanol (250 ml) . Added freshly dis- tilled 3-bromopropanol (9.9 ml 110 mmol) and left 1 h at RT stirring (T.L.C. in CHC give product R_f ca 0.3) . Solution filtered and filtrate evaporated in vacuo. Residue dissolved in CHC1₃ (250 ml) and washed with H O (3 x 250 ml) . Organic layer dried (Na_S04) , filtered and evaporated in vacuo. Crude S-trityl-3-mercaptopropanol was recryst. 2 x from diethyl ether to give 23.4 g (70%) of pure product (R_f 0.8 on silica gel run in diethyl ether) .

Example 6

Preparation of (S-Trityl-3-mercaptopropyloxy) ,2-cyanoethoxy N,N-diisopropylaminophosphine

S-Trityl-3-mercaptopropanol (668 mg, 2 mmol) was dissolved in dry dichloromethane (10 ml) under dry argon. Added diiso- propylammonium tetrazolide (171 mg, 2 mmol) , followed by 2-cyanoethosy bis (N,N-diisopropylamino) phosphine (488 μl, 2.2 mmol) . After 1 h at room temperature T.L.C. shows com- plete reaction (petrol / CH-Cl as eluant cont. 3% trietyl- amine) . Diluted mixture with CH₂C1₂ (100 ml) and washed with saturated NaHCO . Organic layer dried (Na_S04) filtered and evaporated in vacuo. Product purified on silica gel (20 g) using dichloromethane / petrol (1:1) containing 1% triethyl- amine as eluant. Pure fractions pooled and evaporated in vacuo to give an oil (0.7 g, 65%). Product pure by T.L.C. 13C and 31P N.M.R.

Example 7

DNA synthesis

The compound of example 6 can be used directly in an automa¬ ted DNA synthesiser using the -cyanoethyl phosphoramidite chemistry. It is added like an extra base at the 5 '-end of a support bound oligodeoxyribonucleotide. The 5 '- (S-trityl-3- mercaptopropylphospho) oligonucleotide is cleaved from the support with NH_ solution. Overnight at 60°C with NH solution cleaves the base protecting groups. The solution is evapora¬ ted in vacuo and the S-trityl compound purified by reversed phase HPLC on μ-Bondapak C18 using a gradient of CH-CN in 0.1 M aqueous triethylammonium acetate pH 7. The S-trityl peak elutes at an acetonitrile composition of about 25%. Product peak collected, evaporated in vacuo and lyophilised twice with water.

Example 8

Detritylation and labelling with fluorescein

The 5'-(S-Triphenylmethyl-3-mercaptop-:Opyl phospho; oligo- nucleotide of example 7 (TrS-oligo) (100 mmol) is dissolved in 0.5 ml of 50 mM triethylammonium acetate solution pH 7 in an Eppendorf. Added 50 /ul of 10 mM AgN03 solution, mixed and left 1 h at room temperature. Added 50 /ul of fresh 14 mM dithiothreitol solution, mixed and left 30 min at RT. Centri- fuged to remove the insoluble silver salt (reversed phase

HPLC run shows an early eluting peak, at ca 10% CH-CN) . Trans¬ ferred supernatant containing free sulfhydryloligo to a fresh Eppendorf and added 100 /ul of 1 M NaHCO- buffer pH 9. Imme¬ diately added a solution of 5-iodoacetamidofluorescein (10 / ug) in H₂0/DMF (400 /ul, 1:1 v/v). Left solution overnight in dark at room temperature. Dialysed solution in the dark to remove most of the excess dye. A substantial amount of dye still remains associated with the•covalently dye-labelled oligonucleotide. Evaporate the solution and purify the dye labelled oligo on ion-exchange HPLC (Partisil SA X, eluting with KH_P04 gradient in forma ide / H₂0 (6:4 v/v); M13 primer elutes at about 0.2 M salt (monitor at 260 nm for oligo and 495 nm for fl orescein) . A very large dye peak elutes at about 80 mM salt. Remove phosphate and formamide by dialysis and repurify the dye-labelled oligonucleotide by reversed phase HPLC on μ-Bondapak C18. Pure fluorescein labelled oligonucleotide elutes at an acetonitrile composition of about 20%. Remove CH CN and salt by lyophilisation or dia¬ lysis in 3.500 MW cut off dialysis tubing. Store product frozen in the dark.

Claims

C l a i m s

1. A process for labelling a DNA fragment using fluorescent dyes, c h a r a c t e r i z e d in that the DNA fragment is converted into its 5'-£HS-(Y) - derivative, and the latter is reacted with a derivative of a fluorescent dye, which to¬ gether with the 5'- HS- (Y) 1 -derivative forms a DNA fragment of the formula (I) fluorophore-X-S-(Y) -CH 5₂ '-oligonucleotide, wherein X and Y independently are a group comprisingone or more carbon atoms and/or hetero atoms, z = 0 or 1 , and fluorophore is the residue of a fluorescent dye.

2. A process according to claim 1, c h a r a c t e r i z e d in that the fluorescent dye is a rhodamine or a fluorescein.

3. A process according to claim 2, c h a r a c t e r i z e d in that tetramethylrhodamine is used as the rhodamine.

4. A process according to claim 2, c h a r a c t e r i z e d in that fluorescein is used as the fluorescein dye.

5. A process according to any of the preceding claims, c h a r a c t e r i z e d in that X is the group -NH-CO-CH₂-

6. A process according to any of the preceding claims, c h a r a c t e r i z e d in that Y is the group

-(CH.) -0-P(0)-0-, 2 n wherein n is an integer 3 , and preferably 3.

7. A process according to any of the preceding claims, c h a r a c t e r i z e d in that the DNA fraσment is conver¬ ted into its 5'-[s-triphenylmethyl- (Y) J -derivative and the latter is converted into the corresponding 5'-[_,HS-(Y) J -deri- vative by cleavage of the triphenylmethyl group.

8. A process according to any of the preceding claims, c h a r a c t e r i z e d in that the DNA fragment is conver¬ ted into its 5'- (S-triphenylraethyl-3-mercaptopropyl phospho)- derivative and the 5'- (3-mercaptopropyl phospho )-derivative obtained after cleavage of the triphenylmethyl group is reac¬ ted with a (5- and/or 6)-iodoacetamino-rhodamine or-fluores- cein.

9. A process according to claim 8, c h a r a c t e r i z e d in that (5- and/or 6) -iodoaceta¬ mino tetramethylrhodaiαine is used as (5- and/or 6) -iodoacet¬ amino rhodamine.

10. A process according to claim 8, c h a r a c t e r i z e d in that 5-iodoacetamino fluores¬ cein is used as (5- and/or 6)-iodoacetamino fluorescein.

11. A process according to any of claims 8 to 10, c h a r a c t e r i z e d in that the DNA fragment is conver¬ ted into its 5'- (S-triphenylmethyl-3-mercaptopropyl phospho)- derivative by reaction with triethylammonium-]TS-triphenylme- thyl-3-mercaptopropyl, 2-(1- methylimidazole-2-yl)phenyl- phosphatej.

12. A process according to any of claims 8 to 10, c h a r a c t e r i z e d in that the DNA fragment is con¬ verted into its 5'-(S-triphenylmethyl-3-mercaptopropyl phos¬ pho)-derivative by reaction with (S-Trityl-3-mercaptopro- pyloxy) ,2-cyanoethoxy N,N-diisopropylaminophosphine.

13. Use of the process according to any of claims 1 to 12 in ^& the sequential analysis of DNA.

14. Use according to claim 13, c h a r a c t e r i z e d in that the sequential analysis is carried out in accordance with the dideoxy method. 0

15. Use according to claim 14, c h a r a c t e r i z e d in that as primer an M13 primer with fluorescence labelling is used, which is obtained by reacting its 5 '- (S-triphenyl-methyl-3-mercaptcpropyl phospho) 5 derivative with a (5- and/or 6)-iodoacetamino rhodamine or fluorescein.

16. The process described in the Examples for labelling DNA fragments. 0

17. Use of (5- and/or 6)-iodoacetamino rhodamine or fluores¬ cein in the fluorescence labelling of organic compounds, in particular DNA fragments.

5

18. A compound of formula (I)

5 i fluorophore-X-S- (Y)_z~CH₂-oligonucleotide (I) , wherein X and

Y independently are a group comprising one or more carbon atoms and/or hetero atoms, z = 0 or 1 , and fluorophore is the residue of a fluorescent dye.

19. A compound of formula (I) according to claim 18, c h a r a c t e r i z e d in that the fluorescent dye is a rhodamine or a fluorescein.

20. A compound of formula (I) according to claim 19, c h a r a c t e r i z e d in that the rhodamine is tetra¬ methylrhodamine.

21. A compound of formula (I) according to claim 19, c h a r a c t e r i z e d in that the fluorescein dye is fluorescein.

22. A compound according to any of claims 18 to 21, c h a r a c t e r i z e d in that X is the group

23. A compound according to any of claims 18 to 22, c h a r a c t e r i z e d in that Y is the group

0~ I - (CH₂ ) -0-P(0)-0-, wherein n is an integer * 3, and prefe¬ rably 3

24. A compound of formula (I) according to any of claims 18 to 23, c h a r a c t e r i z e d in that it has the structure (la) :

25. A compound of formula (I) according to any of claims 18 to 23, c h a r a c t e r i z e d in that it has the structure (lb) :

leotide

26. The compound of formula (I) according to any of claims 18 to 25, wherein "oligonucleotide" has the meaning d [GTAAAACGACGGCCAGTJ.