4,7-dichlorofluorescein dyes as molecular probes

1 . In a method of detecting a plurality of electrophoretically separated classes of DNA fragments, an improvement comprising labelling DNA fragments of at least one class with a 4,7-dichlorofluorescein dye.

2 . The method of claim 1 wherein said 4,7-dichlorofluorocein is difined by the formula:

wherein:

A′ is hydrogen, fluoro, chloro, a linking functionality, or a group that may be converted to a linking functionality;

B′ is fluoro, chloro, or an acidic anionic group;

X′ is hydrogen, fluoro, or chloro;

Z 1 is hydrogen or, when taken with Z 2 , benzo;

Z 2 , when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, or a group that may be converted to a linking functionality, or when taken with Z 1 , benzo;

Z 3 and Z 4 are separately hydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, or a group that may be converted to a linking functionality;

Z 5 , when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, or a group that may be converted to a linking functionality, or when taken with Z 6 , benzo;

Z 6 is hydrogen or, when taken with Z 5 , benzo; and wherein at least one of A, Z 2 , Z 3 , Z 4 , and Z 5 is a linking functionality or a group that may be converted to a linking functionality.

3 . The method of claim 2 wherein:

A′ is carboxyl, sulfonyl, isothlocyanato, succinimidyl carboxytate, phosphoramidite, or amino;

B′ is carboxyl or sulfonyl;

X′ is hydrogen or chloro;

Z 2 , when taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro;

Z 3 , and Z 4 are separately hydrogen, methyl, ethyl, methoxy, ethoxy, chloro, carboxyl, sulfonyl, isothiocyanate, succinimidyl carboxylate, phospboamidite, or methylamino:

Z 5 , when taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy or chloro; and

wherein only one of A′, Z 3 , and Z 4 is carboxyl, sulfonyl, metylamino, isothiocyanate, succinimidyl carboxylate, phosphoramidite, or amino.

4 . The method of claim 3 wherein Z 3 , and Z 4 are separately hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro.

5 . The method of claim 4 wherein Z 2 , when taken alone, is hydrogen, methoxy, ethoxy, or chloro;

Z 3 , and Z 4 are separately hydrogen, methoxy, ethoxy, chloro; and

Z 5 when taken alone, is hydrogen, methoxy, ethoxy, or chloro.

6 . The method of claim 5 wherein B′ is carboxy and A′ is carboxy, succinimidyl, carboxylate, or phosphoramidite.

7 . A compound having the formula:

wherein

A′ is hydrogen, fluoro, chloro, a linking functionality or a group that may be converted to a linking functionality;

B′ is fluoro, chloro, or an acidic anionic group;

X′ is hydrogen, fluoro, or chloro;

Z 3 and Z 4 are separately hydrogen, halo, a linking functionality, or a group that may be converted to a linking functionality; and

wherein at least one of A′, Z 3 , and Z 4 is a linking functionality or a group that may be converted to a linking functionality.

8 . The compound of claim 7 wherein A′ is carboxyl, sulfonyl, isothiocyanate, succinimidyl carboxylate, phosphoramidite, or amino: B′ is carboxyl or sulfonyl: X′ is hydrogen; Z 3 and Z 4 are separately hydrogen, halo, carboxyl, sulfonyl, or methylamino.

9 . The compound of claim 8 wherein only one of A′, Z 3 , and Z 4 is carboxyl, sulfonyl, methylamino, or amino.

10 . The compound of claim 9 wherein A′ and B′ are carboxyl, Z 3 is hydrogen or chloro, and Z 4 is hydrogen or chloro.

11 . A kit for detecting a plurality of electrophoretically separated classes of DNA fragments comprising an oligonucleotide labelled with a 4,7-dichlorofluorescein dye.

12 . The kit of claim 11 further compounding:

an enzyme selected from the group consisting of acid polymerase and nucleic acid ligase; and

a reaction buffer.

13 . The kit of claim 12 wherein said enzyme is a nucleic acid polymerase and wherein said kit further includes a nucleoside triphosphate mix.

14 . The kit of claim 12 wherein said enzyme is a nucleic acid ligase.

15 . A kit for sequencing DNA comprising:

an oligonucleotide with a 4,7-dichlorofluorescein dye;

a nucleic acid polymerase;

a reaction buffer; and

a nucleoside triphosphate mix.

16 . The kit of claim 15 wherein said 4,7-dichlorofluorescein dye is defined by the formula:

wherein:

A′ is hydrogen, fluoro, chloro, or a group that may be converted to a linking functionality;

B′ is fluoro, chloro, or an acidic anionic group;

X′ hydrogen, fluoro, or chloro;

Z 1 is hydrogen or, when taken with Z 2 , benzo;

Z 2 when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, or a group that may be converted to a linking functionality, or when taken with Z 1 , benzo;

Z 3 and Z 4 are separately hydrogen, halo, lower alkyl, lower alkyloxy, or a group that may be converted to a linking functionality:

Z 5 , when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, or a group that may be converted to a linking functionality, or when taken with Z 6 , benzo;

Z 6 hydrogen or, when taken with Z 5 , benzo; and wherein at least one of A′, Z 2 , Z 3 , Z 4 , and Z 5 is a group that may be converted to a linking functionality.

17 . The kit of claim 16 wherein:

A′ and B′ are carboxy;

X′ hydrogen or chloro;

Z 2 , when taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro

Z 3 , and Z 4 are separately hydrogen, methyl, ethyl, methoxy, ethoxy, chloro; and

Z 5 , when taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro.

18 . A kit for sequencing DNA comprising:

a dye-terminator mix wherein at least one dye-terminator is labelled with a 4,7-dichlorofluorescein dye;

a nucleic acid polymerase;

a nucleoside triphosphate mix; and

a reaction buffer.

19 . The kit of claim 18 wherein said dye-terminator mix comprise dideoxynudeotide triphosphates selected from the group consisting of dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate wherein each of said dideoxynudeotide triphosphates is separately labelled with a dye 3elected from the group consisting of 5- and carboxyfluorescein. 5- and carboxyl-4,7-dichlorofluorescein, 2′,7′-dimethoxy-5- and carboxy4,7-dichlorofluorocein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and 6-carboxyfluorescein. 2′,7′-dimethoxy-4′,5′-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-4′,5′-dichloro-5- and 6-carboxy-4,7dichlorofluorescein, 2′,7′-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2′,4′,5′,7′-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein.

20 . The kit of claim 19 wherein said dideoxythymidine triphosphate is labelled with 6-carboxyfluorescein, said dideoxycytidine triphosphate is labelled with 2′,4′,5′,7′-tetrachloro-5-carboxyfluorescein, said dideoxyadenosine triphosphate is labelled with 2′,4′,5′,7′-tetrachloro-4,7-dichloro-5-carboxyfluorescein, and said dideoxyguanosine triphosphate is labelled with 1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein.

21 . The kit of claim 20 wherein said nucleic acid polymerase is Sequenase™.