Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-21
2001-10-16
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007200, C435S091500, C536S025300, C530S300000, C530S350000, C530S387100
Reexamination Certificate
active
06303309
ABSTRACT:
BACKGROUND OF THE INVENTION
Today many powerful techniques, derived from research in molecular biology, are ready to be used in routine diagnostic or forensic applications. Prominent examples are the polymerase chain reaction, PCR (Saiki, R. K. et al.,
Science
, 230, 1350-1355), based on a cyclic, template-directed primer extension reaction; the analysis of restriction fragment length polymorphisms (Kan, Y. W. and A. M. Dozy,
Lancet
, 2, 910-912); and the ligase chain reaction (Barany, F. (1991) Proc. Natl. Acad. Sci. USA, 88, 189-193) to detect known point mutations at the ligation site of adjacent oligonucleotides.
It appears likely that in the near future, application of these techniques for analysis of DNA will augment or supplant conventional diagnostic procedures based, for example, on the detection of disease-associated metabolites.
Currently, the most common tool for the analysis of DNA is fragment separation by gel electrophoresis. However, in many cases, electrophoretic analysis and subsequent detection of labeled fragments is more time-consuming than performing the enzymatic reaction, and therefore is a time-limiting step.
Detection techniques are under development which will enhance signal acquisition and provide automated and parallel sample processing, and will likely lead to cost-efficient and time-saving sample processing in diagnostic and forensic applications. Also, large DNA sequencing projects, such as the Human Genome Initiative, that seek to sequence genes or entire genomes, for research or diagnostic purposes, require automated techniques with a high throughput to ensure timely completion of the project.
A promising tool which meets at least some of these criteria is the analysis of DNA fragments by matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (Karas, M. and Hillenkamp, F. (1988)
Anal. Chem
., 60, 2299-2301).
The biotin-streptavidin system is a common and useful tool for the purification of biotinylated materials (X. Tong and L. M. Smith (1992)
Anal. Chem
., 64, 2672-2677), e.g., products from PCR or sequencing reactions. Streptavidin (and also avidin) are bacterial proteins which form tight complexes with biotin, including biotin conjugated to other molecules such as nucleic acids. The stability of the biotin-streptavidin complex during intensive washing permits removal of non-specifically bound and non-biotinylated material, which is of great importance for the success of reaction product analysis. The properties of the biotin-streptavidin complex can be used in systems employing biotin bound to streptavidin on a solid support and to yield immobilized biotinylated molecules. The solid phase, including the complexed biotinylated molecules, can be physically collected for further manipulations, including i) removal of excess reaction components like buffer salts, enzymes or deoxynucleotide triphosphates (dNTPs) or ii) performance of enzymatic reactions like nucleolytic digests and solid phase sequencing.
A broad spectrum of applications for the biotin-streptavidin system is known and even techniques not yet developed will be adaptable to this system (see, e.g., Stahl et al.
Nucleic Acids Research
(1988) 16, 3025-3038; Hultman et al.
Nucleic Acids Res
. (1989) 17, 4937-4946; Hornes et al.
Genet. Anal
. (1990) 7, 145-150).
Although the biotin-streptavidin complex is the result of non-covalent bonding, the affinity of streptavidin for biotin is about one million times more powerful than that of most antibody-antigen interactions. However, for the analysis of reaction products it is important to provide conditions for an effective dissociation of the complex while recovering the analyte molecules without modification.
Currently the recovery of biotinylated substances is based on biotin-streptavidin complex dissociation using substances like phenol, urea, or, most preferably 95% formamide at temperatures between 25 and 100° C. (Cocuzza et al., U.S. Pat. No. 5,484,701, 1996).
However, the use of formamide has been shown to be harmful for sample crystallization, a necessary process for MALDI-TOF analysis, and for various enzymatic reactions (e.g. reactions employing alkaline phosphatase). Therefore methods based on the use of formamide are only useful for subsequent gel electrophoretic analysis, but are harmful if enzymatic reactions should be performed involving the isolated material or other analytical tools are to be applied. The endo- or exonucleolytic digest, for example, of PCR products would benefit from a method allowing isolation of single-stranded PCR products which can be digested after purification.
Gel electrophoresis, the time and sample throughput-limiting factor in DNA diagnostics, will be replaced by more efficient techniques in the near future. These applications suggest that efficient methods linking the biotin-streptavidin technology to, for example, MALDI-TOF MS, are strongly required.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for dissociating a complex comprising a biotin compound and a biotin-binding compound. The method includes contacting the complex with an effective amount of an amine, under conditions such that the complex is dissociated, thereby forming a biotin compound and a biotin-binding compound.
In preferred embodiments, the complex is contacted with an amine at a temperature of between about 25° C. and about 100° C. In preferred embodiments, the biotin compound is a biotinylated macromolecule. In preferred embodiments, the biotinylated macromolecule is selected from the group consisting of a biotinylated nucleic acid sequence, a biotinylated protein, a biotinylated carbohydrate, and a biotinylated lipid. In preferred embodiments, the biotin-binding compound is selected from the group consisting of avidin, streptavidin, and derivatives thereof.
In preferred embodiments, the biotin-binding compound is immobilized on a solid support. In preferred embodiments, the solid support is a magnetic bead.
In certain preferred embodiments, after the contacting step, the biotin compound is separated from the biotin-binding compound. In preferred embodiments, after the separation step, the biotin compound is purified. In preferred embodiments, the biotin compound is purified by a method selected from the group consisting of lyophilization, precipitation, filtration, and dialysis.
In preferred embodiments, prior to the contacting step, the complex is purified.
In other preferred embodiments, the biotin compound is immobilized on a solid support.
In preferred embodiments, after dissociation of the complex, at least one of the biotin compound and the biotin-binding compound is analyzed by mass spectrometry.
In certain preferred embodiments, after dissociation of the complex, the biotin-binding compound retains biotin-binding activity. In preferred embodiments, after dissociation of the complex, the biotin moiety of the biotin compound remains substantially intact.
In particularly preferred embodiments, the amine is ammonia. In other preferred embodiments, the amine is a primary amine.
In another aspect, the invention provides a method for analyzing a biotinylated nucleic acid. The method includes the steps of contacting the biotinylated nucleic acid with a biotin-binding compound, thereby forming a biotinylated nucleic acid:biotin-binding compound complex and contacting the complex with an effective amount of an amine, under conditions such that the complex is dissociated, thereby releasing a biotinylated nucleic acid and a biotin-binding compound; and analyzing the biotinylated nucleic acid.
In preferred embodiments, the nucleic acid is DNA. In preferred embodiments, the biotin-binding compound is immobilized. In preferred embodiments, the biotinylated nucleic acid is analyzed by mass spectrometry. In preferred embodiments, the amine is ammonia.
Thus, in one embodiment, the subject method provides a process for isolating biotinylated single-stranded or double-stranded DNA from enzymatic reactions for the purpose of purification and sample conditioning, which ca
Jurinke Christian
Koster Hubert
Van Den Boom Dirk
Heller Ehrman White & McAuliffe LLP
Low Christopher S. F.
Seidman Stephanie L.
Sequenom Inc.
Tu Stephen
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