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 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 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, 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 for amplifying a plurality of target nucleic acids. In some embodiments, a first oligo-dT-universal primer comprising a 3? oligo-dT portion and a first 5? universal portion is used to reverse transcribe a plurality of 3? poly-A tail-containing nucleic acids. A poly-A tail is added to the 3? end of the first strand products to form a two-tailed reaction product. The two-tailed reaction product is amplified in a PCR, wherein the PCR comprises the first oligo-dT-universal primer, and a second oligo-dT-universal primer, wherein the second oligo-dT-universal primer comprises a 3? oligo-dT portion and a second 5? universal portion, wherein the second 5? universal portion of the second oligo-dT-universal primer comprises a different sequence than the first 5? universal portion of the first oligo-dT-universal primer. The present teachings also provide compositions and kits for amplifying target nucleic acids containing monomorphic tails.

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 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 invention relates to methods and compositions for the detection of targets in a sample.

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 novel methods, compositions, and kits for analyzing mature micro RNAs (miRNAs). By taking advantage of the observation that most mature miRNAs in cells are tightly associated with RISCs, the present teachings provide approaches for studying mature miRNAs without the complications of additional nucleic acids. For example, in some embodiments the present teachings provide a method of purifying mature miRNAs comprising heating a sample to form a lysate, and, degrading the additional nucleic acids. The resulting mixture lacks the additional nucleic acids, and contains mature miRNAs associated with RISCs. Liberating the mature miRNAs from RISCs, for example by a protease, a detergent, and/or heat, can result in a pure collection of mature miRNAs.

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 relate to methods, compositions, and kits for detecting one or more target polynucleotide sequences in a sample, and methods compositions and kits for forming concatameric ligation products. In some embodiments of the present teachings, oligonucleotides are hybridized to complementary target polynucleotides and are ligated together to form a concatameric ligation product. In some embodiments of the present teachings, the concatameric ligation product can be amplified, and the identity and quantity of the target polynucleotides determined based on sequence introduced in the ligation reaction. Some embodiments of the present teachings provide methods for removing unligated probes from the reaction mixture. Some embodiments of the present teachings provide for highly multiplexed detection, identification, and quantification of a plurality of target polynucleotides using a variety of analytical procedures.

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, and kits for detecting the presence or absence of (or quantifying) target polynucleotide sequences in at least one sample using encoding and decoding reactions. When a particular target polynucleotide is present in a sample, a reaction product is formed in the encoding reaction that includes addressable primer portions. At least one label and at least one address primer is employed in the decoding amplification reaction thereby providing a detectable signal value depending upon whether a sequence is present or absent. In some embodiments universal bases and reduced libraries of probes can be employed for highly multiplexed analysis of target polynucleotides.

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 for amplifying a plurality of target nucleic acids. In some embodiments, a first oligo-dT-universal primer comprising a 3? oligo-dT portion and a first 5? universal portion is used to reverse transcribe a plurality of 3? poly-A tail-containing nucleic acids. A poly-A tail is added to the 3? end of the first strand products to form a two-tailed reaction product. The two-tailed reaction product is amplified in a PCR, wherein the PCR comprises the first oligo-dT-universal primer, and a second oligo-dT-universal primer, wherein the second oligo-dT-universal primer comprises a 3? oligo-dT portion and a second 5? universal portion, wherein the second 5? universal portion of the second oligo-dT-universal primer comprises a different sequence than the first 5? universal portion of the first oligo-dT-universal primer. The present teachings also provide compositions and kits for amplifying target nucleic acids containing monomorphic tails.