Detection of nucleic acid polymorphisms
OwnerRudolf Rigler, Lars Edman +1inventors
InventorsRudolf Rigler, Lars Edman, Zeno Foeldes-Papp
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Abstract
Methods are described methods functioning at the single-molecule level for characterizing nucleotide polymorphisms.
Claims33
A method for characterizing nucleic acid polymorphisms, including the steps:
- (a) provision of a nucleic acid template to be investigated,
- (b) annealing of at least one starting primer onto the nucleic acid template, with the 3′ end of the starting primer being located upstream of a nucleic acid polymorphism to be investigated,
- (c) extension of the starting primer with at least one fluorescence-labeled nucleotide and
- (d) detection of nucleotides incorporated into the starting primer by single-molecule determination.
The method as claimed in any of the preceding claims, characterized in that to carry out the single-molecule determination the starting primer is coupled to a carrier particle.
The method as claimed in any of the preceding claims, characterized in that an enzymatic elimination of nucleotides attached to the starting primer is effected by an exonuclease, in particular by T7 DNA polymerase as exonuclease, E. coli exonuclease I or E. coli exonuclease III.
The method as claimed in any of the preceding claims, characterized in that a single starting primer is used.
A method for characterizing nucleic acid polymorphisms in a microwell, including the steps:
- (a) provision of a carrier particle with a nucleic acid molecule which is immobilized thereon and consists of a single-stranded nucleic acid template and of a starting primer,
- (b) extension of the starting primer by a fluorescence-labeled chain-termination molecule,
- (c) where appropriate washing of the well to remove unincorporated labels and
- (d) detection of the fluorescent label incorporated into the starting primer.
The method as claimed in any of the preceding claims, characterized in that a carrier particle made of plastic, glass, quartz, metals, metalloids, metal oxides or of a composite material is used.
The method as claimed in any of the preceding claims, characterized in that the carrier particle has a diameter of from 1 nm to 10 μm.
The method as claimed in any of the preceding claims, characterized in that the the nucleic acid matrix is immobilized on the carrier particle via a 5′ terminus or the starting primer is immobilized via its 3′ terminus by means of bioaffinity interactions.
The method as claimed in any of the preceding claims, characterized in that the different fluorescent labels are distinguished on the basis of the wavelength, the lifetime of the excited state or a combination thereof.
The method as claimed in any of the preceding claims, characterized in that the determination takes place by confocal single-molecule detection or/and by time-resolved decay measurement.
The method as claimed in any of the preceding claims, characterized in that the determination includes the measurement of a cross-correlated signal which originates from a nucleic acid molecule, or nucleic acid molecule complex, comprising at least two different labels, especially fluorescent labels.
A method for increasing the detection efficiency in the detection of the fluorescence from single molecules, characterized in that a dispersion element is used to separate the light of different wavelengths.
Dependencies are inferred from the claim text.