Mobility-modifying cyanine dyes

Abstract

The present invention provides a novel class of fluorescent cyanine dye compounds that are modified at one of the hetercyclic ring nitrogen atoms with a mobility-modifying moiety that permits the electrophoretic mobilities of polynucleotides labeled with the mobility-modifying cyanine dyes to be adjusted or tuned in a predictable fashion while retaining enzymatic activity. The ability to predictably tune the relative electrophoretic mobilities of the dyes permits the creation of sets of mobility-matched fluorescent dyes of a variety of structures for a variety of applications, including fluorescence-based 4-color nucleic acid sequencing reactions.

Description

1. CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a divisional of U.S. application Ser. No. 09 / 477,270, filed Jan. 4, 2000 which is incorporated herein by reference.2. FIELD OF THE INVENTION [0002] The present invention relates generally to fluorescent dye compounds that are useful as molecular probes. In particular, the present invention relates to fluorescent cyanine dye compounds that are mobility modified for use in nucleic acid sequencing reactions. 3. BACKGROUND OF THE INVENTION [0003] The advent of automated four-color Sanger-type DNA sequencing has revolutionized the speed with which stretches of DNA can be reliably sequenced. In four-color Sanger-type DNA sequencing, a single-stranded target DNA of interest is hybridized with a complementary primer and the primer enzymatically extended with a DNA polymerase in the presence of a mixture of 2′-deoxyribonucleotides capable of supporting continuous primer extension (e.g., dATP, dGTP, dCTP and dTTP or dUTP) an...

AI Technical Summary

Benefits of technology

The cyanine dye compounds allow for predictable adjustment of electrophoretic mobilities, reducing ambiguities in DNA sequencing by ensuring that labeled polynucleotides have matched mobilities, thus improving the accuracy of sequence determination without compromising spectral properties or enzymatic activity.

Problems solved by technology

While sets of terminators that impart primer extension products with similar mobility shifts are available, rationally designing such sets of “mobility matched” terminators is currently virtually impossible.

To date, no methods exists whereby one can predictably alter the electrophoretic mobilities imparted by terminators labeled with desirable dyes without altering the spectral properties of the dyes and / or jeopardizing the abilities of the labeled terminators to act as substrates for polymerizing enzymes.

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