Patent AT-E455869-T1 (AT)
Inventor: RIGLER RUDOLF
Filed: 2005-08-05
Granted: 2010-02-15
Abstract: Sequencing (M1) nucleic acids, by: (a) contacting a support having nucleic acid-degrading enzyme molecules, with free nucleic acid molecules carrying fluorescent labeling groups; (b) progressively cleaving off individual nucleotide building blocks from free nucleic acids; and (c) determining sequence of nucleic acids on basis of time-dependent change, in fluorescence of nucleic acid molecules. Sequencing (M1) nucleic acids, comprises: (a) providing a support (2) having a multiplicity of nucleic acid-degrading enzyme molecules (4) immobilized on it, contacting the support with free nucleic acid molecules carrying several fluorescent labeling groups, progressively cleaving off individual nucleotide building blocks from the free nucleic acid molecules by the immobilized enzyme molecules, and simultaneously determining the base sequence of several nucleic acid molecules on the basis of the time-dependent change, caused when nucleotide building blocks are cleaved off, in the fluorescence of the nucleic acid molecules and/or of the cleaved-off nucleotide building blocks; or (b) providing a support having a multiplicity of nucleic acid-polymerizing enzyme molecules immobilized on it, contacting the support with free nucleic acid molecules to which a primer molecule is bound and nucleotide building blocks carrying a fluorescent labeling group, incorporating the nucleotide building blocks into the primer by the immobilized enzyme molecules, and simultaneously determining the base sequence of several nucleic acid molecules on the basis of the time-dependent change, caused when nucleotide building blocks are incorporated, in the fluorescence of the extended primer molecule and/or of the nucleotide building blocks. Independent claims are also included for the following: (1) an apparatus (I) for performing (M1), comprising: (a) a support comprising a multiplicity of enzyme molecules immobilized on it, modes for delivering free nucleic acid molecules carrying several fluorescent labeling groups to the support, or generating free nucleic acid molecules carrying several fluorescent labeling groups on the support, a reaction space for progressively cleaving off individual nucleotide building blocks from the nucleic acid molecules, and mode for determining simultaneously the base sequence of several nucleic acid molecules on the basis of the time-dependent change, caused when nucleotide building blocks are cleaved off, in the fluorescence of the nucleic acid molecules and/or the cleaved-off nucleotide building blocks; or (b) a support comprising a multiplicity of nucleic acid-polymerizing enzyme molecules immobilized on it, modes for providing free nucleic acid molecules to which a primer molecule is bound, and nucleotide building blocks carrying a fluorescent labeling group on the support, a reaction space for incorporating the nucleotide building blocks into the primer, and mode for determining simultaneously the base sequence of several nucleic acid molecules on the basis of the time-dependent change, caused when nucleotide building blocks are incorporated, in the fluorescence of the extended primer molecule and/or of the nucleotide building blocks; (2) studying (M2) enzyme-substrate interactions, comprising providing a support having a multiplicity of enzyme molecules immobilized on it, contacting the support with free substrate molecules carrying fluorescent labeling groups, reacting the substrate molecules with the immobilized enzyme molecules, and studying simultaneously the enzyme-substrate interaction of several individual enzyme and/or substrate molecules on the basis of the time-dependent change, caused during the enzymic reaction of substrate molecules, in the fluorescence of the substrate molecules and/or cleaved-off substrate portions and/or reaction products formed; and (3) an apparatus (II) for performing (M2), comprising a support comprising a multiplicity of enzyme molecules immobilized on it, modes for delivering free substrate molecules carrying fluorescent labeling groups to the support, or generating free substrate molecules carrying fluorescent labeling groups on the support, a reaction space for reacting the substrate molecules with the immobilized enzyme molecules, and mode for studying simultaneously several enzyme-substrate interactions of several enzyme and/or substrate molecules on the basis of the time-dependent change, caused during the enzymic reaction, in the fluorescence of the substrate molecules and/or the cleaved-off substrate portions and/or reaction products formed.