Abstract: The present teachings provide methods, compositions, and kits for performing primer extension reactions on at least two target polynucleotides in the same reaction mixture. In some embodiments, a reverse transcription reaction is performed on a first target polynucleotide with a hot start primer comprising a self-complementary stem and a loop, and extension products form at high temperatures but extension products form less so at low temperatures since the self-complementary stem of the hot start primer prevents hybridization of the target specific region to the target. However, non-hot start primers with free target specific regions can hybridize to their corresponding targets at the low temperature and extension can happen at the low temperature.

Abstract: The present disclosure relates to the amplification of target nucleic acid sequences for various sequencing and/or identification techniques. This can be accomplished via the use of target primers. The use of these target primers, as described herein, allows for the reduction in the amplification of undesired hybridization events (such as primer dimerization) while allowing for the amplification of the target nucleic acid sequences.

Abstract: The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3? target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3? target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

Abstract: The present teachings are generally directed to methods for normalizing at least one species of small nucleic acid that is present in a population of small nucleic acid species, wherein the relative concentration of at least one small nucleic acid species is substantially greater than the relative concentration of at least one other small nucleic acid species in the population. At least one small nucleic acid species is normalized using a multiplicity of primers comprising degenerate sequences. In some embodiments, a small nucleic acid species is identified by inserting at least part of an extension product from a normalized population into a vector and subsequently sequencing the insert. In some embodiments, a small nucleic acid species is identified by determining the sequence of at least part of an extension product.

Abstract: The present disclosure relates to the amplification of target nucleic acid sequences for various sequencing and/or identification techniques. This can be accomplished via the use of target primers and isothermal multiple strand displacement (MDA) processes. The use of these target primers and MDA, as described herein, allows for, for example, the reduction in the amplification of undesired hybridization events (such as primer dimerization and the “jackpot mutation” effect of PCR) while allowing for the amplification of the target nucleic acid sequences.

Abstract: The present teachings provide methods and compositions for sequencing one or more target nucleic acids. High levels of multiplexing are provided by the use of an emulsion PCR comprising primer-immobilized beads. The resulting reaction products can be sequenced by any of a variety of mobility-dependent analytical techniques, such as mass spectrometry. In some embodiments, a first collection of amplification products on a first collection of beads are transferred to a second collection of beads. In some embodiments, a first collection of amplification products on a first collection of beads is amplified in a rolling circle amplification reaction. The present teachings also provide compositions, kits, and devices for performing and sequencing the products of the emulsion amplification reactions as described herein.

Abstract: The present disclosure relates to the amplification of target nucleic acid sequences for various sequencing and/or identification techniques. The use of these primers, as described herein, allows for the reduction in the amplification of nonspecific hybridization events (such as primer dimerization) while allowing for the amplification of the target nucleic acid sequences.

Abstract: The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3? target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3? target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

Abstract: The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3? target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3? target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

Abstract: The present teachings provide methods, compositions, and kits for synthesizing and sequencing nucleic acids. In some embodiments, elaborated nucleotide phosphorothiolate compounds are employed along with efficient cleaving reactions. Improved sequencing efficiency is achieved by the rapid polymerase-mediated incorporation of elaborated nucleotide phosphorothiolate compounds. Increased sequencing efficiency is also achieved by the ability of the cleaving reactions to restore the incorporated nucleotides to their natural structure prior to subsequent elongation.

Abstract: The present disclosure relates to the amplification of target nucleic acid sequences. This can be accomplished via the use of various primers. The use of these primers, as described herein, results in nucleic acid structures that can reduce the amplification of nonspecific hybridization events (such as primer dimerization) while allowing the amplification of the target nucleic acid sequences.

Abstract: The present teachings provide methods and compositions for sequencing one or more target nucleic acids. High levels of multiplexing are provided by the use of an emulsion PCR comprising primer-immobilized beads. The resulting reaction products can be sequenced by any of a variety of mobility-dependent analytical techniques, such as mass spectrometry. In some embodiments, a first collection of amplification products on a first collection of beads are transferred to a second collection of beads. In some embodiments, a first collection of amplification products on a first collection of beads is amplified in a rolling circle amplification reaction. The present teachings also provide compositions, kits, and devices for performing and sequencing the products of the emulsion amplification reactions as described herein.

Abstract: The present teachings provide novel methods, compositions, and kits for detecting siRNA-containing RISCs. In some embodiments, modified siRNA constructs are employed that contain an anti-sense strand and a sense strand, wherein the anti-sense strand comprises a 3? end, wherein the 3? end comprises a fluorophore, and wherein the sense strand comprises a 5? end, wherein the 5? end comprises a quencher. Following transfection, uptake of the anti-sense strand by RISC liberates the fluorescent signal, allowing for detection of siRNA-containing RISCs.

Abstract: The present teachings provide methods, compositions, and kits for performing primer extension reactions on at least two target polynucleotides in the same reaction mixture. In some embodiments, a reverse transcription reaction is performed on a first target polynucleotide with a hot start primer comprising a self-complementary stem and a loop, and extension products form at high temperatures but extension products form less so at low temperatures since the self-complementary stem of the hot start primer prevents hybridization of the target specific region to the target. However, non-hot start primers with free target specific regions can hybridize to their corresponding targets at the low temperature and extension can happen at the low temperature.

Abstract: The present teachings provide methods, compositions, and kits for performing primer extension reactions. In some embodiments, a reverse transcription reaction is performed on a target polynucleotide with a hot start primer comprising a blunt-ended self-complementary stem, and a loop, and extension products form at high temperatures but reduce extension product formation at low temperatures.

Abstract: The present teachings provide methods, compositions, and kits for reverse transcribing and amplifying small nucleic acids such as micro RNAs. High levels of multiplexing are provided by the use of a zip-coded stem-loop reverse transcription primer, along with a PCR-based pre-amplification reaction that comprises a zip-coded forward primer. Detector probes in downstream decoding PCRs can take advantage of the zip-code introduced by the stem-loop reverse transcription primer. In some embodiments, further amplification is achieved by cycling the reverse transcription reaction. The present teachings also provide compositions and kits useful for performing the reverse transcription and amplification reactions described herein.

Abstract: The present teachings provide methods, compositions, and kits for reverse transcribing and amplifying small nucleic acids such as micro RNAs. In some embodiments, the present teachings provide methods of forming micro RNA signatures from single cells, including stem cells. In some embodiments, the present teachings provide methods for determining the identity and/or purity of cells. The present employ performing a multiplexed reverse transcription reaction comprising stem-loop reverse transcription primers, which optionally undergoes temperature cycling, followed by a multiplexed PCR-based pre-amplification reaction, and a subsequently a plurality of lower-plex decoding PCRs.

Abstract: The present teachings are directed to compositions, methods, and kits for detecting and quantitating small nucleic acid molecules, including small DNA molecules and small RNA molecules. The detector probes of the current teachings, including single-loop detector probes, double-loop detector probes, and bimolecular detector probes, are designed to selectively hybridize with a corresponding small nucleic acid target and to produce, under appropriate conditions, a detectable signal or a detectably different signal. The detector complexes of the current teachings comprise a detector probe comprising a first reporter group and a displaceable sequence comprising a second reporter group, wherein the displaceable sequence is hybridized to the detector probe.

Abstract: The present invention relates to the detection of target sequences. Detection can be achieved through the use of ID-tag complexes. These ID-tag complexes are relatively stable in the absence of a target sequence. In the presence of a target sequence, the complexes dissociate and form new complexes or duplexes, which can be purified or eliminated and detected on an ID-tag system.

Abstract: The present teachings are generally directed to methods for normalizing at least one species of small nucleic acid that is present in a population of small nucleic acid species, wherein the relative concentration of at least one small nucleic acid species is substantially greater than the relative concentration of at least one other small nucleic acid species in the population. At least one small nucleic acid species is normalized using a multiplicity of primers comprising degenerate sequences. In some embodiments, a small nucleic acid species is identified by inserting at least part of an extension product from a normalized population into a vector and subsequently sequencing the insert. In some embodiments, a small nucleic acid species is identified by determining the sequence of at least part of an extension product.