Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-14
2002-01-22
Whisenant, Ethan (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025320, C252S301160, C422S082060, C422S082070
Reexamination Certificate
active
06340750
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates in general to the field of DNA sequencing and, more particularly, to through bond energy transfer in fluorescent dyes for labelling biological molecules.
BACKGROUND OF THE INVENTION
Methods routinely applied for high throughput DNA sequencing have oscillated between two embodiments of the Sanger scheme. [A1, A2] Fluorescence detection dominates throughout, [A3] but the factor that distinguishes the approaches is that the labels can be situated in the primer (dye-primers) or in the terminating fragments (dye-terminators). Both methods have been, and continue to be, used. [A4-A6]
Early dye-primer technology featured one fluorescent flag per primer. Four reactions were performed with each of the ddNTP's using the “workhorse tags”, i.e., JOE, TAMRA, ROX, and FAM. These four reactions were mixed after production of a nested set of chain terminated DNA fragments, and analyses were performed via gel electrophoresis in one lane with a static detector.
Dye terminator strategies [A7] have the advantage that only one reaction is required to produce a nested set of chain terminated DNA fragments labeled with fluorescent groups appropriate to the four ddNTP's. The (unlabeled) primers used are also cheaper to produce than the corresponding fluorescently labeled ones. Moreover, in contrast to dye-primer strategies, pausing bands are invisible to fluorescence detection when the label is present only in the terminator. The disadvantage of dye-terminators is that not all of the relatively precious labeled component is incorporated into the complement whereas all the fluorescence is retained in the complement if the dye primer method is used.
A significant advance in dye-primer methodology occurred when it was realized that the fluorescence signal could be enhanced by approximately ten-fold when two labels were used in the following way. [A9] One was selected to absorb relatively high energy photons; energy transfer though space to the second fluorescent group would then lead to emission at a lower wavelength. Specifically, FAM was (and is) used to harvest the irradiation, then convey energy through space to either JOE, TAMRA, or ROX. The ten-fold enhancement obtained is significant because it facilitates use of less reagents (dye-primer, enzyme, dNTPs, ddNTPs, etc.), and/or lessens the need to concentrate the reactions before gel electrophoresis and detection.[A9-A13] Energy transfer enhancement of fluorescence is more efficient than other systems wherein two identical fluorescent labels per primer have been used to enhance sensitivity.[A14]
The utility of the dye-terminator approach has also been enhanced, but in this case the development was one in molecular biology. Tabor and Richardson showed that some mutated DNA polymerases favor incorporation of labeled ddNTPs. [A15] These enzymes are more expensive than the wild type, but they can be obtained in significant quantities via over-expression. Use of these DNA replicating enzymes leads to more efficient use of ddNTPs in Sanger sequencing, and this is particularly important when the ddNTP bears a label.
The state of the art in high throughput sequencing is such that both dye-primers and dye terminators are used. Typically, cloned genomic fragments are randomly sheared and subcloned into specialized sequencing vectors, i.e., the “shot-gun” approach. Doubly labeled dye-primers that complement the specialized vector arms are then used to begin the sequencing operation; a compelling advantage of this is that only a limited repertoire of these expensive primers is required. Primer walking is then used to extend the sequence information obtained. However, the primer walking steps, and sequencing of regions riot-covered by the shot-gun/primer walking process, require primers that are tailor made to those particular sequences (rather than to a restriction site sequences). Syntheses of many different doubly labeled dye-based primers cannot be justified, so a different approach is used. In fact, it is cost effective to use labeled ddNTPs/mutant DNA replicating enzymes at this stage, therefore obviating the need for extensive dye-primer syntheses.
SUMMARY OF THE INVENTION
Fluorescent energy transfer cassettes are reported. Unique features of these are that they allow through bond energy transfer and have a succinimidyl ester functionality suitable for attaching them to biomolecules. The relevance of this design concept to high throughout DNA sequencing is discussed.
This disclosure outlines a general design principle for new fluorescent dyes to be applied in high throughput DNA sequencing protocols (e.g., The Genome Project) and other applications in biotechnology.
Fluorescent dyes for DNA sequencing and other biotechnological applications can be produced in the following way. A UV-absorbing chromophore is selected that will absorb relatively strongly at the wavelength emitted by the source chosen for the application under consideration. Organic synthesis is then performed to incorporate this chromophore into a molecule wherein the chromophore is conjugated with a molecular entity having desirable fluorescence emission properties. In DNA sequencing, the latter group would be one with a strong, narrow bandwidth, emission at a distinctly different wavelength to the other dyes used in the sequencing method. The UV chromophore must absorb at a lower wavelength than the fluorescence emitter, and it is highly desirable that the chromophore and fluorescence emitter be placed at opposite ends of the conjugated system (not in the middle). In the anticipated mode of action of these dyes, the UV absorbing group would harvest radiation from the excitation source and transmit it through the conjugated system to the fluorescence emitter which would then fluoresce.
The new fluorescent dyes should also preferably have the following properties:
(i) manageable solubility characteristics;
(ii) functionality that allows them to be conveniently attached to nucleotides (or other biomolecules);
(iii) similar structures when used as sets for DNA sequencing, thus giving near tagged DNA fragments with similar gel mobilities;
(iv) chemical stability;
(v) chemical accessibility (i.e., can be obtained via convenient syntheses); and,
(vi) functional groups which facilitate convenient and economical incorporation of the labels.
According to one embodiment, the design principle disclosed here provides dyes that can be designed to:
harvest radiation (from lasers and similar devices) in regions of the electromagnetic spectrum that cannot be efficiently absorbed by the dyes currently used for DNA sequencing, thus allowing a wider variety of light source wavelengths to be used;
fluoresce in a greater wavelength range than the four dye detection system most often used at present (i.e., JOE, TAMRA, ROX, FAM) allowing greater resolution of the fluorescence emission from the dyes giving a more accurate read in DNA sequencing experiments;
give more intense fluorescent emission on irradiation with a usable source than is currently possible using JOE, TAMRA, ROX, and FAM, thus giving increased sensitivity and enabling smaller amounts of samples to be detected;
give fluorescence emission from a usable source that is comparable or superior to the through space energy transfer dyes introduced by Mathies, and by Gibbs, and their coworkers;
be introduced more conveniently and economically than the through space energy transfer dyes introduced by Mathies, and by Gibbs, and their co-workers; and,
be useful in both the “dye-primer” and the “dye-terminator” approaches to DNA sequencing.
Sets of fluorescent dyes would be prepared such that one UV absorbing group was paired with four different fluorescent emitter moieties, each with clearly different emission wavelengths. This would allow strong fluorescence at four clearly distinguishable wavelengths.
There is also potential for two different sets of sequencing reactions to be mixed and analyzed in a sin
Burgess Kevin
Gibbs Richard
Baker & Botts L.L.P.
Lu Frank
The Texas A&M University System
Whisenant Ethan
LandOfFree
Through bond energy transfer in fluorescent dyes for... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Through bond energy transfer in fluorescent dyes for..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Through bond energy transfer in fluorescent dyes for... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2833909