The present invention relates to methods of imaging template hybridisation for estimating cluster numbers prior to solid phase amplification and sequencing. More particularly, an initial round of imaging is carried out at the single molecule template hybridisation stage which allows a general estimation of cluster numbers prior to clusters being formed. Amplification of the signal allows single molecule imaging to be carried out using standard sequencing imaging apparatus.

Disclosed are compositions and a method relating to amplifying and detecting nucleic acids.

Disclosed are compositions and a method relating to amplifying and detecting nucleic acids.

The present invention provides methods for immobilizing target nucleic acids on a solid support utilizing combinatorial capture probe pairs. These pairs contain first and second capture oligonucleotides that each comprise a target binding region, a capture region and a stem region positioned between the target binding and capture regions. The target binding regions comprise nucleic acid sequences that allow them to hybridize to adjacent regions on the target nucleic acid. The stem regions have nucleic acid sequences that are complementary to each other and the capture regions each comprise a sequence that when positioned adjacent to one another produce a combined nucleic acid sequence that is complementary to a portion of an oligonucleotide bound to a solid support. When the first and second capture oligonucleotides are annealed to the target nucleic acid, the stem regions are brought together allowing them to hybridize, which in turn brings the capture regions together to produce a combined nucleic acid sequence. This combined nucleic acid sequence is then able to hybridize to the oligonucleotide bound to the solid support thereby immobilizing the target nucleic acid.

The present invention provides methods for immobilizing target nucleic acids on a solid support utilizing combinatorial capture probe pairs. These pairs contain first and second capture oligonucleotides that each comprise a target binding region, a capture region and a stem region positioned between the target binding and capture regions. The target binding regions comprise nucleic acid sequences that allow them to hybridize to adjacent regions on the target nucleic acid. The stem regions have nucleic acid sequences that are complementary to each other and the capture regions each comprise a sequence that when positioned adjacent to one another produce a combined nucleic acid sequence that is complementary to a portion of an oligonucleotide bound to a solid support. When the first and second capture oligonucleotides are annealed to the target nucleic acid, the stem regions are brought together allowing them to hybridize, which in turn brings the capture regions together to produce a combined nucleic acid sequence. This combined nucleic acid sequence is then able to hybridize to the oligonucleotide bound to the solid support thereby immobilizing the target nucleic acid.

Provided is a linker element and a method of using the linker element to construct a sequencing library, wherein the linker element consists of a linker A and a linker B, the linker A is obtained through the complementary pairing of a long nucleic acid strand and a short nucleic acid strand, the 5′ end of the long strand has a phosphoric acid modification, and the 3′ end of the short strand has an enclosed modification, with enzyme sites in the short strand; and the linker B is a nucleic acid single strand, and the 3′ end thereof can be in a complementary pairing with the 5′ end of the long strand of the linker A. Using the linker element of the present invention for constructing a sequencing library ensures the linking directionality of the linkers while solving the problems of fragment interlinking, linker self-linking and low linking efficiency, and reducing the purification reaction between steps, shortening the linking time and reducing costs.

Provided is a linker element and a method of using the linker element to construct a sequencing library, wherein the linker element consists of a linker A and a linker B, the linker A is obtained through the complementary pairing of a long nucleic acid strand and a short nucleic acid strand, the 5′ end of the long strand has a phosphoric acid modification, and the 3′ end of the short strand has an enclosed modification, with enzyme sites in the short strand; and the linker B is a nucleic acid single strand, and the 3′ end thereof can be in a complementary pairing with the 5′ end of the long strand of the linker A. Using the linker element of the present invention for constructing a sequencing library ensures the linking directionality of the linkers while solving the problems of fragment interlinking, linker self-linking and low linking efficiency, and reducing the purification reaction between steps, shortening the linking time and reducing costs.

The invention provides a version of fluorescent in situ hybridization (FISH) in which all the steps are performed at physiological temperatures, i.e., body temperature, to detect and identify pathogenic bacteria in clinical samples. Methods of the invention use species-specific fluorescent probes to label clinically important infectious bacteria. A sample such as a urine sample is loaded into a cartridge, fluorescently labeled, and imaged with a microscope. Labelled bacteria are pulled down onto an imaging surface and a dye cushion is used to keep unbound probes off of the imaging surface. A microscopic image of the surface shows whether and in what quantities the infectious bacteria are present in the clinical sample.

The invention provides a version of fluorescent in situ hybridization (FISH) in which all the steps are performed at physiological temperatures, i.e., body temperature, to detect and identify pathogenic bacteria in clinical samples. Methods of the invention use species-specific fluorescent probes to label clinically important infectious bacteria. A sample such as a urine sample is loaded into a cartridge, fluorescently labeled, and imaged with a microscope. Labelled bacteria are pulled down onto an imaging surface and a dye cushion is used to keep unbound probes off of the imaging surface. A microscopic image of the surface shows whether and in what quantities the infectious bacteria are present in the clinical sample.

The invention relates to a method for determining levels of interactions between biomolecules, such as proteins, in a sample, comprising providing a first and a second information carrying (IC) oligonucleotide, wherein the first and second IC oligonucleotide are attached, covalently or non-covalently, to a first and a second affinity reagent, such as antibodies, that have the capacity to bind to a first and a second biomolecule, wherein the first and second IC oligonucleotide each comprises at least one single-stranded stretch that is complementary to a part of another oligonucleotide, thereby, upon hybridisation of the at least one single-stranded stretch in at least one of the first and second IC oligonucleotides to its complementary part of another oligonucleotide, enabling measurement of the relative proportion of interacting and non-interacting first and second biomolecules in the sample at a single cell or single molecular level. Further, the invention relates to kits including necessary oligonucleotides and reagents to carry out the method of the invention.