Detection methods using probes labeled with energy transfer...

Details Detection methods using probes labeled with energy transfer... Detection methods using probes labeled with energy transfer...

1998-11-30

2001-01-23

Marschel, Ardin H. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S005000, C435S007100, C435S007200, C536S025400

Reexamination Certificate

active

06177247

ABSTRACT:

TECHNICAL FIELD
The field of this invention is fluorescent tags and their use as exemplified with DNA fragment analysis.
BACKGROUND
There is an increasing demand to be able to identify and quantify components of mixtures. The greater the complexity of the mixture, the greater the interest in being able to simultaneously detect a plurality of the components present. As illustrative of this situation is DNA sequencing and DNA fragment analysis, where it is desirable to efficiently excite from one to four or more fluorescently tagged components with a laser source at a single wavelength, while providing for fluorescent signal emission at a plurality of distinctive wavelengths. In this situation, the different labels should not adversely affect the electrophoretic mobility of DNA fragments to which they are attached.
Currently, there are four methods used for automated DNA sequencing: (1) the DNA fragments are labeled with one fluorophore and then the fragments run in adjacent sequencing lanes (Ansorge et al.,
Nucleic Acids Res
. 15, 4593-4602 (1987); (2) the DNA fragments are labeled with four different fluorophores and all the fragments are electrophoretically separated and detected in a single lane (Smith et al.,
Nature
321, 674-679 (1986); (3) each of the dideoxynucleosides in the termination reaction is labeled with a different fluorophore and the four sets of fragments are run in the same lane (Prober et al.,
Science
238, 336-341 (1987); or (4) the sets of DNA fragments are labeled with two different fluorophores and the DNA sequences coded with the dye ratios (Huang et al.,
Anal. Chem
. 64, 2149-2154 (1992). For fluorescence based PCR DNA fragments analysis, primers labeled with different fluorescent dyes were employed thereby permitting multiple target analysis (Andy et al. (1995) Biotechniques, 18, 116-121).
All of these techniques have significant deficiencies. Method 1 has the potential problems of lane-to-lane variations in mobility, as well as a low throughput. Methods 2, 3 and 4 as well as the multiple color PCR method require that the four dyes be well excited by one laser source and that they have distinctly different emission spectra. In practice, it is very difficult to find two or more dyes that can be efficiently excited with a single laser and that emit well separated fluorescent signals.
As one selects dyes with distinctive red-shifted emission spectra, their absorption maxima will also move to the red and all the dyes can no longer be efficiently excited by the same laser source. Also, as more different dyes are selected, it becomes more difficult to select all the dyes such that they cause the same mobility shift of the labeled molecules.
It is therefore of substantial interest that improved methods be provided which allow for multiplexing of samples, so that a plurality of components can be determined in the same system and in a single run. It is also desirable for each label to have strong absorption at a common wavelength, to have a high quantum yield for fluorescence, to have a large Stokes shift of the emission, that the various emissions be distinctive, and that the labels introduce the same mobility shift. It is difficult to accomplish these conflicting goals by simply labeling the molecules with a single dye.
SUMMARY OF THE INVENTION
The subject invention provides compositions and methods for analyzing a mixture using a plurality of fluorescent labels. To generate the labels, pairs or families of fluorophores are bound to a backbone, particularly a nucleic acid backbone, where one of the members of the families is excited at about the same wavelength. By exploiting the phenomenon of energy transfer, the other members of each of the families emit at detectably different wavelengths. The range of distances between donor and acceptor chromophores is chosen to ensure efficient energy transfer. Furthermore, labels used conjointly are selected to have approximately the same mobility shift in a separation system, where one of the labels may have two molecules of the same fluorescer, so as to provide the fluorescence emission of the single fluorescer, but the same mobility shift as the different donor-acceptor chromophore labels. This is achieved by changing the mobility shift of the labeled entity, by varying the distance between the two or more members of the family of fluorophores and by choosing labels with the same mobility. The subject invention finds particular application in DNA sequencing and DNA fragment sizing, where the fluorophores may be attached to universal or other primers and different fluorophore combinations used for the different dideoxynucleosides. Kits of combinations of labels are also provided.


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