Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-11
2004-08-17
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C435S287200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06777184
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of detection of fluorescence polarization, e.g., in microfluidic devices.
BACKGROUND OF THE INVENTION
Detection of single nucleotide polymorphisms (SNPs) and other genetic phenomena is an increasingly important technique in molecular biology and medicine. For example, in medical contexts, polymorphism detection is useful for diagnosing inherited diseases and susceptibility to diseases. The detection of SNPs and other polymorphisms can also serve as a basis for tailoring or targeting treatment, i.e., where certain allelic forms of a polymorphism are associated with a response to a particular treatment. In molecular biology, polymorphism detection is fundamental in a variety of contexts, including molecular marker assisted breeding (e.g., of important crop varieties such as Zea and other Graminea, soybeans, etc.), the study of gene diversity, gene regulation and other genetic, epigenetic or para-genetic phenomena.
Many techniques exist for measuring nucleic acid hybridization for polymorphism detection, as well as for other purposes. In addition to standard Southern and northern blotting, complex arrays of nucleic acid probes are available from a variety of commercial sources, as are solution based detection methods such as those utilizing fluorescence resonance energy transfer (FRET), molecular beacons, or other real-time solution-based hybridization detection methods. These hybridization methodologies typically involve the use of one or more probe, e.g., which includes a fluorophore or other label. Specific hybridization is detected by localization of probe label signals in solid phase hybridization methods such as Southern blotting, or array-based versions thereof, or by real time optical and/or spectroscopic methods which monitor changes in fluorescence in solution, e.g., as detected by FRET.
One additional technique has recently been used for detecting hybridization formation between nucleic acids, e.g., in the presence of polylysine. As described by the inventors in Nikiforov and Jeong “Detection of Hybrid Formation between Peptide Nucleic Acids and DNA by Fluorescence Polarization in the Presence of Polylysine” (1999)
Analytical Biochemistry
275:248-253, Fluorescence Polarization (FP) provides a useful method to detect hybridization formation between nucleic acids. This method is applicable to hybridization detection, e.g., to monitor SNPs.
Generally, FP operates by monitoring the speed of rotation of fluorescent labels, such as fluorescent dyes, e.g., before, during and/or after binding events between probes which comprise the labels and target molecules. In short, binding of the probe to a target molecule ordinarily results in a decrease in the speed of rotation of the bound probe, resulting in a change in FP.
For example, when a fluorescent molecule is excited by a polarized light source, the molecule will emit fluorescent light in a fixed plane; that is, the emitted light is also polarized, provided that the molecule is fixed in space. However, because the molecule is typically rotating and tumbling in space, the plane in which the fluoresced light is emitted varies with the rotation of the molecule (also termed the rotational diffusion of the molecule). Restated, the emitted fluorescence is generally depolarized. The faster the molecule rotates in solution, the more depolarized it is. Conversely, the slower the molecule rotates in solution, the less depolarized, or the more polarized it is. The polarization value (P) for a given molecule is proportional to the molecule's “rotational correlation time,” or the amount of time it takes the molecule to rotate through an angle of 57.3° (1 radian). The smaller the rotational correlation time, the faster the molecule rotates, and the less polarization will be observed. The larger the rotational correlation time, the slower the molecule rotates, and the more polarization will be observed. Rotational relaxation time is related to viscosity (&eegr;), absolute temperature (T), molar volume (V), and the gas constant (R). The rotational correlation time is generally calculated according to the following formula:
Rotational Correlation Time=3&eegr;
V/RT
(1)
As can be seen from the above equation, if temperature and viscosity are maintained constant, then the rotational relaxation time, and, therefore, the polarization value, is directly related to the molecular volume. Accordingly, the larger the molecule, the higher its fluorescent polarization value, and conversely, the smaller the molecule, the smaller its fluorescent polarization value.
In the performance of fluorescent binding assays, a typically small, fluorescently labeled molecule, e.g., a ligand, antigen, etc., having a relatively fast rotational correlation time, is used to bind to a much larger molecule, e.g., a receptor protein, antibody etc., which has a much slower rotational correlation time. The binding of the small labeled molecule to the larger molecule significantly increases the rotational correlation time (decreases the amount of rotation) of the labeled species, namely the labeled complex over that of the free unbound labeled molecule. This has a corresponding effect on the level of polarization that is detectable. Specifically, the labeled complex presents much higher fluorescence polarization than the unbound, labeled molecule.
Generally, the fluorescence polarization level is calculated using the following formula:
P=[I
(∥)−
I
(⊥)]/[
I
(∥)+
I
(⊥)] (2)
Where I(∥) is the fluorescence detected in the plane parallel to the excitation light, and I(⊥) is the fluorescence detected in the plane perpendicular to the excitation light.
In addition to Nikiforov and Jeong (1999), above, other references which discuss fluorescence polarization and/or its use in molecular biology include Perrin (1926). “Polarization de la lumiere de fluorescence. Vie moyenne de molecules dans l'etat excite.”
J Phys Radium
7, 390; Weber (1953) “Rotational Brownian motion and polarization of the fluorescence of solutions”
Adv Protein Chem
8, 415; Weber (1956).
J Opt Soc Am
46, 962; Dandliker and Feigen (1961), “Quantification of the antigen-antibody reaction by the polarization of fluorescence”
Biochem Biophys Res Commun
5, 299; Dandliker and de Saussure (1970) (Review Article) “Fluorescence polarization in immunochemistry”
Immunochemistry
7, 799; Dandliker W B, et al. (1973). “Fluorescence polarization immunoassay. Theory and experimental method.”
Immunochemistry
10, 219; Levison S A, et al. (1976), “Fluorescence polarization measurement of the hormone-binding site interaction”
Endocrinology
99, 1129; Jiskoot et al. (1991), “Preparation and application of a fluorescein-labeled peptide for determining the affinity constant of a monoclonal antibody-hapten complex by fluorescence polarization”
Anal Biochem
196, 421; Wei and Herron (1993), “Use of synthetic peptides as tracer antigens in fluorescence polarization immunoassays of high molecular weight analytes”
Anal Chem
65, 3372; Devlin et al. (1993), “Homogeneous detection of nucleic acids by transient-state polarized fluorescence”
Clin Chem
39, 1939; Murakami et al. (1991), Fluorescent-labeled oligonucleotide probes detection of hybrid formation in solution by fluorescence polarization spectroscopy.”
Nuc. Acids Res
19, 4097. Checovich et al. (1995), “Fluorescence polarization-a new tool for cell and molecular biology” (product review),
Nature
375, 354-256; Kumke et al. (1995), “Hybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancement”
Anal Chem
67:21, 3945-3951; and Walker et al. (1996), “Strand displacement amplification (SDA) and transient-state fluorescence polarization detection of mycobacterium tuberculosis DNA”
Clinical Chemistry
42:1, 9-13.
One difficulty in the use of FP to monitor hybridization of nucleic acids is that the change in FP which occurs simply upon binding of a labeled p
Jeong Sang
Nikiforov Theo T.
Caliper Life Sciences, Inc.
McKenna Donald R.
Siew Jeffrey
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