4,7-dichlorofluorescein dyes as molecular probes

A method of distinguishing polynucleotides having different terminal dideoxynucleotides in the chain termination method of DNA sequencing, the method comprising the steps of:

• forming a mixture of a first, a second, a third, and a fourth class of polynucleotides,
• each polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled with a first dye selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 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, and 1',2',7',8'-dibenzo-4',5'-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein,
• each polynucleotide in the second class having a 3'-terminal dideoxythymidine and being labeled with a second dye selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 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, and 1',2',7',8'-dibenzo-4',5'-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein,
• each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled with a third dye selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 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, and 1',2',7',8'-dibenzo-4',5'-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein,
• each polynucleotide in the fourth class having a b 3'-terminal dideoxycytosine and being labeled with a fourth dye selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 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, and 1',2',7',8'-dibenzo-4',5'-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein,
• wherein the first, second, third, and fourth dyes are spectrally resolvable from one another;
• electrophoretically separating on a gel the polynucleotides in the mixture so that bands of similarly sized polynucleotides are formed;
• illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the dyes to fluoresce;
• and
• identifying the class of the polynucleotides in the bands by the fluorescence or absorption spectrum of the dyes.

In a chain termination method of DNA sequencing, the method of the type wherein four classes of DNA fragments are formed such that DNA fragments of the same class have the same terminating base and are labelled with the same fluorescent dye, an improvement comprising:

• labelling DNA fragments of at least one class with a 4,7-dichlorofluorescein dye selected from the group defined by the formula:
• ##STR12## wherein:
• A' is hydrogen, fluoro, chloro, or a group that may be converted to a linking functionality;
• D' is fluoro, chloro, or an acidic anionic group;
• X' is hydrogen, fluoro, or chloro;
• Z' is hydrogen or, when taken with Z.sub.2, benzo;
• Z.sub.2, when taken along, is hydrogen, halo, lower alkyl, lower alkyloxy, or a group that may be converted to a linking functionality, or when taken with Z.sub.1, Z.sub.2 is benzo;
• Z.sub.3 and Z.sub.4 are separately hydrogen, halo, lower alkyl, lower alkyloxy, or a group that may be converted to a linking functionality;
• Z.sub.6 is hydrogen or, when taken with Z.sub.5, benzo;
• Z.sub.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.sub.6, benzo;
• and wherein only one of A', A.sub.2, Z.sub.3, Z.sub.4, and Z.sub.5 is a group that may be converted to a linking functionality.