Spectrally resolvable rhodamine dyes for nucleic acid sequence determination

A method of distinguishing polynucleotides having different 3'-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 going consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110;
• each polynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled with a second dye selected from the group consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110;
• each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled with a third dye selected from the group consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110;
• and each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled with a fourth dye selected from the group consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110;
• wherein each class of polynucleotide in the mixture is labeled with a different dye;
• 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 classes of the polynucleotides in the bands by the fluorescence or absorption spectrum of the dyes.