DNA sequencing using multiple fluorescent labels being...

Details DNA sequencing using multiple fluorescent labels being... DNA sequencing using multiple fluorescent labels being...

2002-06-24

2004-04-06

Riley, Jezia (Department: 1637)

Chemical apparatus and process disinfecting, deodorizing, preser

Analyzer, structured indicator, or manipulative laboratory...

Means for analyzing liquid or solid sample

C435S006120, C055S423000, C055S423000, C055S423000

Reexamination Certificate

active

06716394

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns the analysis of mixtures of compounds. More particularly, the present invention involves tagging individual compounds with unique fluorescent markers having different fluorescence lifetimes. The analysis of the mixture is then accomplished by distinguishing individual compounds by their unique fluorescence lifetime.
BACKGROUND OF THE INVENTION
In numerous fields, including organic chemistry, forensics, medical diagnosis and molecular biology there is a growing need for safe, efficient and cost-effective methods for identifying compounds of interest within a mixture of compounds. Mixtures of compounds frequently arise as the product of an organic synthetic cycle, during the isolation of a product of biological origin and during the chemical or enzymatic sequencing of polymeric compounds such as polypeptides, proteins, polysaccharides and nucleic acids.
Accurately determining nucleic acid base sequence is a prerequisite to further understanding the structure and function of the proteins produced by the encoded information. One such method, DNA sequencing, involves determining the order in which the nucleic acid bases are arranged within a length of DNA. Two DNA sequencing techniques which are widely known and in current use, are the chemical degradation procedure according to Maxam and Gilbert (
Proc. Natl. Acad. Sci. USA
74:560 (1977)) and the enzymatic dideoxy chain termination method of Sanger et al (
Proc. Natl. Acad. Sci. USA
74:5463 (1977)). Additionally, reference is made to,
Current Protocols in Molecular Biology
, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (Supplement 37, current through 1997) (Ausubel), particularly, Chapter 7, which is incorporated herein by reference, for a description of DNA sequencing in general and various DNA sequencing techniques.
Traditional methods of DNA sequencing utilize a radiolabeled oligonucleotide primer to synthesize a nucleic acid having a sequence complementary to the sequence under analysis. Alternatively, a radiolabeled nucleotide is incorporated directly into the growing nucleic acid strand. Following synthesis, the radioactive nucleic acids are separated by a method such as gel electrophoresis and the positions of the nucleic acids are visualized by autoradiography. Although this technique provides sensitive detection, the use of radioisotopes and autoradiography requires extended exposure times and presents waste disposal problems.
Fluorescent-labeled oligonucleotide primers have been used in place of radiolabeled primers for sensitive detection of DNA fragments (U.S. Pat. No. 4,855,225 to Smith et al.). Additionally, DNA sequencing products can be labeled with fluorescent dideoxynucleotides (U.S. Pat. No. 5,047,519 to Prober et al.) or by the direct incorporation of a fluorescent labeled deoxynucleotide (Voss et al.
Nucl. Acids Res.
17:2517 (1989)). As currently practiced, fluorescent sequencing reactions circumvent many of the problems associated with the use of radionuclides.
In an attempt to increase laboratory throughput and to further decrease exposure of laboratory workers to harmful reagents, various strategies have been developed. For example, robotic introduction of fluids onto microtiter plates is commonly performed to speed mixing of reagents and to enhance experimental throughput. More recently, microscale devices for high throughput mixing and assaying of small fluid volumes have been developed. For example, U.S. Ser. No. 08/761,575 entitled High Throughput Screening Assay Systems in Microscale Fluidic Devices by Parce et al. provides pioneering technology related to microscale fluidic devices, especially including electrokinetic devices. The devices are generally suitable for assays utilizing fluorophores which relate to the interaction of biological and chemical species, including enzymes and substrates, ligands and ligand binders, receptors and ligands, antibodies and antibody ligands, as well as many other assays. Because the devices provide the ability to mix fluidic reagents and assay mixing results in a single continuous process, and because minute amounts of reagents can be assayed, these microscale devices represent a fundamental advance for laboratory science.
The application of fluorogenic and non-fluorogenic assays utilizing fluorescent labels in flowing microfluidic systems are provided in Kopf-Sill et al. U.S. Ser. No. 09/093,542 “Apparatus and Methods For Correcting for Variable Velocity in Microfluidic Systems,” filed Jun. 8, 1998. A fluorogenic assay is an assay in which a product of the assay emits a label distinct from those of the reactants of the assay. A non-fluorogenic assay is an assay in which the mobility of a product differs from those of labeled reactants (e.g., in a flowing electrokinetic system), but the emitted label is still the same as the label found on a reactant. Detection of non-fluorogenic assay products is possible in an electroosmotically driven microfluidic device using periodic injections of reaction mixture into a separation channel, in which reactants and products are separated by electrophoresis due to changes in the electrophoretic mobility resulting from the reaction (see also, A. R. Kopf-Sill, T. Nikiforov, L. Bousse, R. Nagel, & J. W. Parce, “Complexity and performance of on-chip biochemical assays,” in Proceedings of Micro- and Nanofabricated Electro-Optical Mechanical Systems for Biomedical and Environmental Applications, SPIE, Vol. 2978, San Jose, Calif., February 1997, p. 172-179).
Closed-loop biochemical microfluidic devices especially adapted to sequencing nucleic acids, as well as for high-throughput screening are described in U.S. Ser. No. 09/054,962 entitled “Closed-loop Biochemical Analyzers” by Knapp et al., filed Apr. 3, 1998. In brief, in the integrated systems described, it is possible to use the results of a first sequencing reaction or set of sequencing reactions to select appropriate reagents, reactants, products, or the like, for additional analysis. For example, the results of a first sequencing reaction can be used to select primers, templates or the like for additional sequencing, or to select related families of compounds for screening in high-throughput assay methods. These primers or templates are then accessed by the system and the process continues.
Although sequencing and other assay methods that utilize fluorescent markers often represent, in many ways, an improvement over methods that utilize radioactive isotopes, current fluorescent methodologies are hampered by certain deficiencies. For example, in order to identify the individual nucleotides, each nucleotide must bear a fluorescent marker that has by a unique absorbance and/or emission spectrum with a different absorbance or emission maximum. Thus, to clearly distinguish the individual nucleotides based upon the fluorescence spectrum of their tags, the absorbance or emission maxima of each tag must be clearly resolved from those of every other tag. Further, fluorescence must be monitored at a number of different wavelengths in order to detect each of the maxima and a filtering system must be employed. This is cumbersome and increases the expense of the instrumentation. This situation is additionally complicated by the dependence of the absorption or emission maxima for a compound upon the environment surrounding that compound.
Thus, a method of detecting individual fluorescently labeled compounds within a mixture of compounds which relied on a characteristic of the fluorescent moiety other than its absorption and/or emission spectrum (e.g., maxima) would represent a significant advance in the art. The present invention provides such a method.
SUMMARY OF THE INVENTION
It has now been discovered that individual members of a mixture can be distinguished and identified through the selective use of a set of fluorescent labels displaying a range of unique fluorescence lifetimes. This method is versatile and it can be practiced with a wide ran

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