Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.

Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.

The present invention relates to methods for determining endonuclease activity in a sample. In particular, the present invention relates to a method for determining viable pathogenic bacteria in a sample based on patterns of endonuclease activity.

A method includes (i) providing an RNA polynucleotide; (ii) modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form; (iii) optionally performing a reverse transcription of the RNA polynucleotide; (iv) cleaving the stem-loop segment of the annealed polynucleotide to yield a 3′ A overhang; (v) connecting an adaptor polynucleotide complex associated with an RNA translocase enzyme and at least one cholesterol tether segment to the polynucleotide obtained in step (iv); (vi) contacting the modified RNA polynucleotide obtained in step (v) with a transmembrane pore such that the RNA translocase controls the movement of the RNA polynucleotide through the transmembrane pore and the cholesterol tether anchors the RNA polynucleotide in the vicinity of the transmembrane pore; and (vii) taking one or more measurements during the movement of the RNA polynucleotide through the transmembrane pore Other features are also disclosed.

A method includes (i) providing an RNA polynucleotide; (ii) modifying the RNA polynucleotide by annealing and ligating a polynucleotide comprising a 3′ terminal random multimer segment and having a stem-loop form; (iii) optionally performing a reverse transcription of the RNA polynucleotide; (iv) cleaving the stem-loop segment of the annealed polynucleotide to yield a 3′ A overhang; (v) connecting an adaptor polynucleotide complex associated with an RNA translocase enzyme and at least one cholesterol tether segment to the polynucleotide obtained in step (iv); (vi) contacting the modified RNA polynucleotide obtained in step (v) with a transmembrane pore such that the RNA translocase controls the movement of the RNA polynucleotide through the transmembrane pore and the cholesterol tether anchors the RNA polynucleotide in the vicinity of the transmembrane pore; and (vii) taking one or more measurements during the movement of the RNA polynucleotide through the transmembrane pore Other features are also disclosed.

A method for detecting different analytes includes mixing different analytes with sensing probes, wherein at least some of the sensing probes are specific to respective ones of the analytes. The analytes respectively are captured by the sensing probes that are specific to those analytes. Fluorophores respectively are coupled to sensing probes that captured respective analytes. The sensing probes are mixed with beads, wherein the beads are specific to respective ones of the sensing probes, and wherein the beads include different codes identifying the analytes to which those sensing probes are specific. The sensing probes respectively are coupled to beads that are specific to those sensing probes. The beads are identified that are coupled to the sensing probes that captured analytes using at least fluorescence from the fluorophores coupled to those sensing probes. The analytes that are captured are identified.

A method for detecting different analytes includes mixing different analytes with sensing probes, wherein at least some of the sensing probes are specific to respective ones of the analytes. The analytes respectively are captured by the sensing probes that are specific to those analytes. Fluorophores respectively are coupled to sensing probes that captured respective analytes. The sensing probes are mixed with beads, wherein the beads are specific to respective ones of the sensing probes, and wherein the beads include different codes identifying the analytes to which those sensing probes are specific. The sensing probes respectively are coupled to beads that are specific to those sensing probes. The beads are identified that are coupled to the sensing probes that captured analytes using at least fluorescence from the fluorophores coupled to those sensing probes. The analytes that are captured are identified.

The present invention relates to a method of detecting a target nucleic acid on the basis of rolling circle amplification (RCA), and more specifically, to a method of detecting a target nucleic acid, the method in which a target nucleic acid (a nucleic acid having a target nucleic acid sequence), when present, forms a circular template with a template for performing an amplification reaction, wherein during the amplification reaction, a restriction enzyme is added to further induce a new RCA reaction, thus increasing the reaction rate and sensitivity, and to an RCA composition for implementing the method. The method of detecting a target nucleic acid according to the present invention, by detecting a barcode sequence predefined according to the type of the target nucleic acid, enables multiple detections of the presence of the target nucleic acid without sequencing, is inexpensive for not using costly enzymes, such as CRISPR, can detect barcode sequences, and can utilize various existing nucleic acid detection systems, and thus, can be useful in the detection of gene mutations.

The present invention relates to a method of detecting a target nucleic acid on the basis of rolling circle amplification (RCA), and more specifically, to a method of detecting a target nucleic acid, the method in which a target nucleic acid (a nucleic acid having a target nucleic acid sequence), when present, forms a circular template with a template for performing an amplification reaction, wherein during the amplification reaction, a restriction enzyme is added to further induce a new RCA reaction, thus increasing the reaction rate and sensitivity, and to an RCA composition for implementing the method. The method of detecting a target nucleic acid according to the present invention, by detecting a barcode sequence predefined according to the type of the target nucleic acid, enables multiple detections of the presence of the target nucleic acid without sequencing, is inexpensive for not using costly enzymes, such as CRISPR, can detect barcode sequences, and can utilize various existing nucleic acid detection systems, and thus, can be useful in the detection of gene mutations.

A method of sequencing nucleic acids is provided using DNA origami as a barcode for a nucleic acid probe.