The invention relates to a process and a device for analysing single molecules, particularly to the parallel analysis of a plurality of single molecules. It is suitable for detecting interactions, e.g. binding between single molecules and/or reactions, e.g. elongation or degradation of single molecules. Particularly, the process of the invention relates to the sequencing of single nucleic acid molecules. The single molecule to be analysed is present in free form, i.e. dissolved or suspended in a liquid medium, within a reaction space formed around the sample spot. According to the present invention, an electrical field is applied across the reaction space, whereby a concentration of single molecules, at the sample spots is effected.

The invention relates to a process and a device for analysing single molecules, particularly to the parallel analysis of a plurality of single molecules. It is suitable for detecting interactions, e.g. binding between single molecules and/or reactions, e.g. elongation or degradation of single molecules. Particularly, the process of the invention relates to the sequencing of single nucleic acid molecules. The single molecule to be analysed is present in free form, i.e. dissolved or suspended in a liquid medium, within a reaction space formed around the sample spot. According to the present invention, an electrical field is applied across the reaction space, whereby a concentration of single molecules, at the sample spots is effected.

The invention relates to a method for the detection of the occurrence of initiation of replication events in genomic DNA in a eukaryotic cell, involving contacting said eukaryotic cell comprising said genomic DNA with a first nucleotide probe, under conditions enabling in situ hybridization of said first nucleotide probe with a target region in the DNA genome, wherein said target region comprises a nucleic acid sequence which has no identified corresponding annealing RNA in a metabolically active cell and therefore remains RNA-free during transcription and replication of said DNA genome and detecting said first nucleotide probe hybridized to said DNA. Further detection of at least one RNA molecule can be achieved. The invention also relates to a nucleic acid molecule suitable for use as a probe, hybridizing with a target region in a eukaryotic genomic DNA, and comprising a nucleic acid sequence which has no identified corresponding annealing RNA in the metabolically active cell containing said eukaryotic genomic DNA and therefore remains RNA-free during transcription and replication of said DNA genome. The invention also encompasses kit(s) for carrying out in situ hybridization and use of the method(s), nucleic acid molecule(s) or kit(s) of the invention in the detection of mitochondrial disease(s), neoplasic diseases(s) or cancer(s), or in the testing of the cytotoxicity of organic or chemical compounds, especially drugs, on eukaryotic cells.

The invention relates to a method for the detection of the occurrence of initiation of replication events in genomic DNA in a eukaryotic cell, involving contacting said eukaryotic cell comprising said genomic DNA with a first nucleotide probe, under conditions enabling in situ hybridization of said first nucleotide probe with a target region in the DNA genome, wherein said target region comprises a nucleic acid sequence which has no identified corresponding annealing RNA in a metabolically active cell and therefore remains RNA-free during transcription and replication of said DNA genome and detecting said first nucleotide probe hybridized to said DNA. Further detection of at least one RNA molecule can be achieved. The invention also relates to a nucleic acid molecule suitable for use as a probe, hybridizing with a target region in a eukaryotic genomic DNA, and comprising a nucleic acid sequence which has no identified corresponding annealing RNA in the metabolically active cell containing said eukaryotic genomic DNA and therefore remains RNA-free during transcription and replication of said DNA genome. The invention also encompasses kit(s) for carrying out in situ hybridization and use of the method(s), nucleic acid molecule(s) or kit(s) of the invention in the detection of mitochondrial disease(s), neoplasic diseases(s) or cancer(s), or in the testing of the cytotoxicity of organic or chemical compounds, especially drugs, on eukaryotic cells.

Embodiments of present disclosure are directed to methods for amplifying nucleic acid, comprising two steps: a first step of preparing a reaction mixture comprising the target nucleic acid and a second step of processing the reaction mixture in a thermocycler. During a first phase of the processing step, the thermocycler may be configured to heat the reaction mixture to a first temperature and cool the reaction mixture to a second temperature repeatedly for a first plurality of cycles. During the first phase, fluorescence probes do not anneal to template strands and do not emit fluorescence signals. During a second phase of the processing step, the thermocycler may heat the reaction mixture to a third temperature and cool the reaction mixture to a fourth temperature repeatedly for a second plurality of cycles. During the second phase, fluorescence probes anneal to the template strands and are degraded by DNA polymerase to emit fluorescence signals for detection and/or quantification of the target nucleic acid. Methods for amplifying nucleic acid in accordance with the disclosure may be employed for nucleic acid amplification and detection in clinical and research settings.

Embodiments of present disclosure are directed to methods for amplifying nucleic acid, comprising two steps: a first step of preparing a reaction mixture comprising the target nucleic acid and a second step of processing the reaction mixture in a thermocycler. During a first phase of the processing step, the thermocycler may be configured to heat the reaction mixture to a first temperature and cool the reaction mixture to a second temperature repeatedly for a first plurality of cycles. During the first phase, fluorescence probes do not anneal to template strands and do not emit fluorescence signals. During a second phase of the processing step, the thermocycler may heat the reaction mixture to a third temperature and cool the reaction mixture to a fourth temperature repeatedly for a second plurality of cycles. During the second phase, fluorescence probes anneal to the template strands and are degraded by DNA polymerase to emit fluorescence signals for detection and/or quantification of the target nucleic acid. Methods for amplifying nucleic acid in accordance with the disclosure may be employed for nucleic acid amplification and detection in clinical and research settings.

A method is used for the qualitative evaluation of real-time PCR data, where a time/PCR amplification plot of an associated sample is classified as a negative plot or as a positive plot. The method involves providing a real-time PCR amplification plot to be classified, plotting at least 20 successive amplitude values of corresponding successive PCR cycle indices of the sample. Next, a quality metric is determined, on the basis of the at least one amplitude value. A first criterion is determined by a comparison of the quality metric with a first standard value. A sequence of values is then determined, which indicates a gradient of the PCR amplification plot to be classified, and a second criterion is determined as to whether the sequence of values exceeds a second standard value. Finally, the real-time PCR amplification plot is classified as a positive plot if all the criteria given above are satisfied.

A method is used for the qualitative evaluation of real-time PCR data, where a time/PCR amplification plot of an associated sample is classified as a negative plot or as a positive plot. The method involves providing a real-time PCR amplification plot to be classified, plotting at least 20 successive amplitude values of corresponding successive PCR cycle indices of the sample. Next, a quality metric is determined, on the basis of the at least one amplitude value. A first criterion is determined by a comparison of the quality metric with a first standard value. A sequence of values is then determined, which indicates a gradient of the PCR amplification plot to be classified, and a second criterion is determined as to whether the sequence of values exceeds a second standard value. Finally, the real-time PCR amplification plot is classified as a positive plot if all the criteria given above are satisfied.

A nucleic acid testing device includes: a stage on which is placed a tissue section to which a solution has been added, in which the solution contains a labeling substance of a target nucleic acid and an amplification reagent for the target nucleic acid; a temperature adjuster that adjusts the temperature of the tissue section on the stage; a temperature controller that controls the temperature adjuster to advance nucleic acid amplification reaction in the tissue section; an intensity detector that detects label intensity in the tissue section over time; and a storage unit that stores detection information generated by the intensity detector.

A nucleic acid testing device includes: a stage on which is placed a tissue section to which a solution has been added, in which the solution contains a labeling substance of a target nucleic acid and an amplification reagent for the target nucleic acid; a temperature adjuster that adjusts the temperature of the tissue section on the stage; a temperature controller that controls the temperature adjuster to advance nucleic acid amplification reaction in the tissue section; an intensity detector that detects label intensity in the tissue section over time; and a storage unit that stores detection information generated by the intensity detector.