4,7-dichlorofluorescein dyes as molecular probes

a technology of dimerization and fluorescence, which is applied in the field of fluorescent labelling techniques, can solve the problems of difficult to find three or more dyes with no significant overlap of emission bands, and the requirement is particularly difficult to satisfy, and achieves high fluorescence efficiency

Inactive Publication Date: 2005-04-21
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0018] The invention is based in part on the discovery that the fluorescent properties of 4,7-chloro-5-(and 6-)carboxyfluorescein and related dyes are highly favorable for use as molecular probes. Their emission band widths are generally 20-30 percent narrower than analogs lacking the 4,7-dichloro derivatives, their emission and absorption maxima are at wavelengths generally about 10-30 nm high r than analogs lacking the 4,7-dichloro derivatives, and their fluorescent efficiencies are high, in some cases being nearly triple those of analogs lacking the 4,7-dichloro derivatives.

Problems solved by technology

This requirement is particularly difficult to satisfy in DNA sequence analysis where 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 width for organic fluorescent dyes is about 4080 nanometers (nm) and the width of the visible strum is only about 350400 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.

Method used

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  • 4,7-dichlorofluorescein dyes as molecular probes
  • 4,7-dichlorofluorescein dyes as molecular probes
  • 4,7-dichlorofluorescein dyes as molecular probes

Examples

Experimental program
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Effect test

example 1

4,7-dichloro-5-(and 6-)carboxyfluorescein (“ALF”)

[0044] 0.58 g of 3,6-dichlonttrimellitic acid, 0.72 g of resorcinol, 0.5 ml concentrated suifuti acid, and 3 ml of propionic acid were refluxed 12 hours under argon. The reaction mixture w8 poured into 150 ml water; the precipitate was dried, taken into 3 ml pyridine and acetylated with 2 ml acetic anhydride for 1 hour. The acetylation mbiture was taken into 100 ml ethyl acetate, washed with 1 N hydrochloric acid, water, and evaporated to dryness. The residue was placed on 15 grams of silica gel and eluted with 50 ml ethyl acetate, then 4:1 ethyl acetate:methanol. Fractions containing UV active material with Rf of about 0.2 (4:1 ethyl acetate:methano / silica gel) were evaporated to dryness. This resldue was dissolved in 10 ml methanol and then 1 ml of 4 N sodium hydroxide was added. After 10 minutes, the reaction mixture was diluted to 200 ml with water and then 0.5 ml of concentrated hydrochloric acid was added. The total mixture was...

example 2

4,7-dichloro-5-(and 6-) carboxvfluomscein N-hvdroxvsuccinimide (NHS) ester

[0045] 13.7 ng of fluorescein from Example 1, 3.3 mg of 30 NHS, 6,4 mg DCC and 1 ml ethyl acate were stired 0.5 hours. The solid was filtered, and the supematnt was washed three times with 1:1 bine:water, dried with sodium sulfate, and evaporated to dryness yielding 15 mg of NHS ester.

example 3

Conjugation of 4,7-dichloro-5-(and 6-)carboxyfluorescein with aminoalkyloligonucleotides

[0046] 5 mg of NHS ester from Example II were dissolved 5 in 20 ul of DMSO: 3 ul of this solution were added to a solution consisting of 20 ul of 1.0 mM 5′-aminophosphate oligonucleotide (an 18-mer) in water and 10 ul of 1 M sodium bkarbond / sodium carbonate buffer. pH 9.0. After one hour in the dark, the solution was passed through a 10 ml Sophedox G 25 (medium) column with 0.1 M triethylammonium acetate buffer, pH 7.0. The band of colored material eluting in the exclusion volume was collected. Reverse phase HPLC showed two major fluent peaks, corresponding to the 5- and 6-isomers of the dye conjugated onto the DNA. The peaks were collected, and the fluorescence speera in 50% urea at pH 8.0 showed full width at half m of 34 nm with the emission maxima at 528 nm.

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Abstract

Long wavelength, narrow emission bandwidth fluorescein dyes are provided for detecting specially overlapping target substances. The dyes comprise 4,7-dichlorofluorescein, and particularly 2′, 4′,5′,7′-tetrachloro-4,7-dichloro-5-(and 5-) 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. 09 / 949,444 filed Sep. 7, 2001, which is a continuation of application Ser. No. 09 / 580,754 filed May 30, 2000, now Pat. No. 6,403,812 which is a continuation of application Ser. No. 09 / 273,655 filed Mar. 23, 1999, now Pat. No. 6,096,723 which is a continuation of application Ser. No. 08 / 905,855 filed Aug. 4, 1997, now Pat. No. 5,885,778, which is a continuation of application Ser. No. 08 / 400,780 filed Mar. 8, 1995, now 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 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 the use of 4,7-dichlorofluosceins for detecting multiple target substances i...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07D311/82C07H21/00C09B11/08C12Q1/68
CPCA61K48/00Y10T436/145555C07H21/00C09B11/08C12Q1/6816C12Q1/6869C07D311/82Y10T436/143333Y10T436/147777Y10S436/80Y10S435/968C12Q2563/107
Inventor MENCHEN, STEVENLEE, LINDACONNELL, CHARLESHERSHEY, N.CHAKERIAN, VERGINEWOO, SAMFUNG, STEVEN
Owner APPLERA
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