Compositions and methods for amplifying selected polynucleotides, including DNA and RNA, particularly in multiplex amplification reactions using common primers amplification. Generally, methods of the invention employ multiple steps such as template-specific hybridization, a linear amplification, partial degradation of nucleic acid, and ligation. At the end of the process the sequences of selected polynucleotides are flanked by the common sequences which can be used for exponential amplification using common primers. In some aspects the polynucleotides are associated with a barcode and the presence of the barcode is detected to measure the amount of the polynucleotide.

Compositions and methods for amplifying selected polynucleotides, including DNA and RNA, particularly in multiplex amplification reactions using common primers amplification. Generally, methods of the invention employ multiple steps such as template-specific hybridization, a linear amplification, partial degradation of nucleic acid, and ligation. At the end of the process the sequences of selected polynucleotides are flanked by the common sequences which can be used for exponential amplification using common primers. In some aspects the polynucleotides are associated with a barcode and the presence of the barcode is detected to measure the amount of the polynucleotide.

Compositions and methods for amplifying selected polynucleotides, including DNA and RNA, particularly in multiplex amplification reactions using common primers amplification. Generally, methods of the invention employ multiple steps such as template-specific hybridization, a linear amplification, partial degradation of nucleic acid, and ligation. At the end of the process the sequences of selected polynucleotides are flanked by the common sequences which can be used for exponential amplification using common primers. In some aspects the polynucleotides are associated with a barcode and the presence of the barcode is detected to measure the amount of the polynucleotide.

Compositions and methods for amplifying selected polynucleotides, including DNA and RNA, particularly in multiplex amplification reactions using common primers amplification. Generally, methods of the invention employ multiple steps such as template-specific hybridization, a linear amplification, partial degradation of nucleic acid, and ligation. At the end of the process the sequences of selected polynucleotides are flanked by the common sequences which can be used for exponential amplification using common primers. In some aspects the polynucleotides are associated with a barcode and the presence of the barcode is detected to measure the amount of the polynucleotide.

This disclosure relates to compositions and methods for single-step, multi-stage amplification reactions that combine many stages of sample preparation process in a single tube reaction. The disclosed technology provides a mean of performing multiplexed nested PCR in a single vessel, without any need of purification steps, and is based on the use of three sets of primers: a pair of outer primers, a pair of inner primers that are nested within the pair of outer primers, and tail primers that are complementary to tails on the inner primers. By adjusting the temperature conditions, annealing temperatures of the primers, number of amplification cycles, and the concentrations of the outer, inner, and tail primers, it is possible to carry out multiplexed nested PCR in a single vessel.

This disclosure relates to compositions and methods for single-step, multi-stage amplification reactions that combine many stages of sample preparation process in a single tube reaction. The disclosed technology provides a mean of performing multiplexed nested PCR in a single vessel, without any need of purification steps, and is based on the use of three sets of primers: a pair of outer primers, a pair of inner primers that are nested within the pair of outer primers, and tail primers that are complementary to tails on the inner primers. By adjusting the temperature conditions, annealing temperatures of the primers, number of amplification cycles, and the concentrations of the outer, inner, and tail primers, it is possible to carry out multiplexed nested PCR in a single vessel.

A reaction mixture for providing a reaction batch for performing a quantitative real-time PCR contains at least one target DNA, which at least in parts corresponds to the DNA section being quantified, at least one reference DNA of defined sequence and in a defined amount, at least two different fluorescent probes of different sequence which generate a signal at different wavelengths, primers, deoxynucleotides and a DNA polymerase. The target DNA and the reference DNA have the same primer binding sites and different probe binding sites. At least one of the fluorescent probes is intended for binding to a section of the target DNA outside the primer binding sites in the amplicon, and at least one of the fluorescent probes is intended for binding to a section of the reference DNA outside the primer binding sites in the amplicon.

A reaction mixture for providing a reaction batch for performing a quantitative real-time PCR contains at least one target DNA, which at least in parts corresponds to the DNA section being quantified, at least one reference DNA of defined sequence and in a defined amount, at least two different fluorescent probes of different sequence which generate a signal at different wavelengths, primers, deoxynucleotides and a DNA polymerase. The target DNA and the reference DNA have the same primer binding sites and different probe binding sites. At least one of the fluorescent probes is intended for binding to a section of the target DNA outside the primer binding sites in the amplicon, and at least one of the fluorescent probes is intended for binding to a section of the reference DNA outside the primer binding sites in the amplicon.

This invention relates to a method of sequencing a nucleic acid molecule, in particular the method comprising: (a) providing the nucleic acid immobilised on a surface; (b) forming a single-stranded gap section by partially duplexing the nucleic acid such that the single-stranded gap section is flanked by duplex sections, wherein the sequence to be sequenced is a sequence of the single-stranded gap section; (c) providing a set of at least four fluorescently labelled oligonucleotide probes, (d) detecting binding, or absence thereof, of the fluorescently labelled oligonucleotide probes with the gap section of the immobilised nucleic acid, wherein the identity of the interrogated nucleotide of the gap section of the immobilised nucleic acid is identified as the complementary base of the nucleotide X of the fluorescently labelled oligonucleotide probe that has the highest incidence of binding; (e) repeating steps (c) and (d) for interrogating subsequent nucleotide positions of the gap section of the immobilised nucleic acid until sufficient nucleotides of the gap section have been identified to be able to determine a sequence. Also provided are methods of single-molecule phenotyping and sequencing; methods of single-molecule phenotyping and identification of a molecule tagged with nucleic acid; an immobilised nucleic acid molecule; and a composition comprising oligonucleotide probes.

This invention relates to a method of sequencing a nucleic acid molecule, in particular the method comprising: (a) providing the nucleic acid immobilised on a surface; (b) forming a single-stranded gap section by partially duplexing the nucleic acid such that the single-stranded gap section is flanked by duplex sections, wherein the sequence to be sequenced is a sequence of the single-stranded gap section; (c) providing a set of at least four fluorescently labelled oligonucleotide probes, (d) detecting binding, or absence thereof, of the fluorescently labelled oligonucleotide probes with the gap section of the immobilised nucleic acid, wherein the identity of the interrogated nucleotide of the gap section of the immobilised nucleic acid is identified as the complementary base of the nucleotide X of the fluorescently labelled oligonucleotide probe that has the highest incidence of binding; (e) repeating steps (c) and (d) for interrogating subsequent nucleotide positions of the gap section of the immobilised nucleic acid until sufficient nucleotides of the gap section have been identified to be able to determine a sequence. Also provided are methods of single-molecule phenotyping and sequencing; methods of single-molecule phenotyping and identification of a molecule tagged with nucleic acid; an immobilised nucleic acid molecule; and a composition comprising oligonucleotide probes.