Methods of analyzing polynucleotides employing energy transfer dyes

A method of analyzing a polynucleotide, comprising the steps of:

• (a) forming a plurality of differently sized fluorescently labeled extended oligonucleotide primers that are complementary to the polynucleotide by enzymatically extending an oligonucleotide primer that is complementary to a region of the polynucleotide and that is labeled with an energy transfer dye in the presence of the polynucleotide, nucleotides suitable for effecting template-dependent primer extension and at least one terminating nucleotide, such that the labeled primer is extended until the terminating nucleotide is incorporated into the labeled extended primer;
• (b) separating the fluorescently labeled extended oligonucleotide primers based upon their sizes;
• and
• (c) detecting the fluorescently labeled extended primers based upon their fluorescence, wherein the energy transfer dye comprises:
• (i) a xanthene donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto;
• (ii) a 4,7-dichlororhodamine acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto;
• and
• (iii) a non-nucleosidic linker linking the xanthene donor dye to the 4,7-dichlororhodamine acceptor dye.