4,7-dichlorofluorescein dyes as molecular probes

Abstract

Long wavelength, narrow emission bandwidth fluorecein dyes are provided for detecting spacially overlapping target substances. The dyes comprise 4,7-dichlorofluoresceins, and particularly 2′,4′,5′,7′-tetrachloro-4,7-dichloro-5- (and 6-)carboxyfluoresceins. Methods and kits for using the dyes in DNA analysis are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of application Ser. No. 10 / 714,407 filed Nov. 14, 2003, which is a continuation of application Ser. No. 09 / 949,444 filed Sep. 7, 2001, now U.S. Pat. No. 6,649,598, which is a continuation of application Ser. No. 09 / 580,754 filed May 30, 2000, now U.S. Pat. No. 6,403,812, which is a continuation of application Ser. No. 09 / 273,655 filed Mar. 23, 1999, now U.S. Pat. No. 6,096,723 which is a continuation of application Ser. No. which is a continuation of application Ser. No. 08 / 400,780 filed Mar. 8, 1995, now U.S. Pat. No. 5,654,442, which is a continuation of application Ser. No. 07 / 939,813 filed Sep. 3, 1992, Abandoned, which is a continuation-in-part of application Ser. No. 07 / 436,455 filed Nov. 14, 1989, now U.S. Pat. No. 5,188,934, which are all incorporated herein by reference.FIELD OF THE INVENTION [0002] The invention relates generally to fluorescent labelling techniques, and more particularly, to ...

AI Technical Summary

Benefits of technology

The invention is about a method and dyes for detecting multiple substances simultaneously using a technique called fluorescence resonance energy transfer (FRET). Specifically, the invention involves using 4,7-dichlorofluorescein dyes to detect spacially overlapping target substances, such as DNA fragments, using a method called DNA sequencing. The dyes have certain favorable properties that make them ideal for use as molecular probes, including narrow emission band widths, high fluorescent efficiencies, and favorable absorption and emission maxima. The invention also includes kits for carrying out the DNA sequencing method using the dyes. Overall, the invention provides a more efficient and accurate way to detect multiple substances simultaneously using FRET.

Problems solved by technology

This requirement is particularly difficult to satisfy in DNA sequence analysis at least four spectrally resolvable dyes are needed in most automated sequencing approaches.

Obtaining a set of dyes to label the different fragments is a major difficulty in such DNA sequencing systems.

First, it is difficult to find three or more dyes that do not have significantly overlapping emission bands, since the typical emission band halfwidth for organic fluorescent dyes is about 40-80 nanometers (nm) and the width of the visible spectrum is only about 350-400 nm.

Second, even when dyes with non-overlapping emission bands are found, the set may still be unsuitable for DNA sequencing if the respective fluorescent efficiencies are too low.

Third, when several fluorescent dyes are used concurrently, excitation becomes difficult because the absorption bands of the dyes are often widely separated.

When several dyes are used one is often forced to make a trade off between the sensitivity of the detection system and the increased cost of providing separate excitation sources for each dye.

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