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 invention provides methods and compositions for analyzing nucleic acid sequences. In some aspects, the methods utilize clonal objects, such as DNA balls, that have been captured on beads. Using the methods described here, compositions are fabricated wherein a bead and one clonal object are affinity bound or hybridized to each other through an affinity binding patch or hybridization patch on the surface of the bead. The invention also provides a population of beads having affinity bound or hybridized clonal objects at a ratio of 1:1. The invention additionally provides methods for amplifying a target nucleic acid molecule utilizing the compositions described herein.
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. According to certain methods, detecting a small nucleic acid target comprises the target displacing the displaceable sequence of a detector complex to form a detector probe-small nucleic acid target duplex, illuminating the duplex with light of an appropriate wavelength, and determining the presence of a detectable fluorescent signal or the change in a detectable signal.
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 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 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 invention relates to the detection of target sequences. The present description discloses compositions and methods involving analog nucleic acids, such as PNA and L-DNA, for the detection of nucleic acids. Additionally, hybrid detectable markers are provided.
Abstract:
The present invention provides methods and compositions for analyzing nucleic acid sequences. In some aspects, the methods utilize clonal objects, such as DNA balls, that have been captured on beads. Using the methods described here, compositions are fabricated wherein a bead and one clonal object are affinity bound or hybridized to each other through an affinity binding patch or hybridization patch on the surface of the bead. The invention also provides a population of beads having affinity bound or hybridized clonal objects at a ratio of 1:1. The invention additionally provides methods for amplifying a target nucleic acid molecule utilizing the compositions described herein.
Abstract:
Provided herein are compositions, materials, methods and kits for immobilizing a template polynucleotide in a first orientation, and immobilizing a complementary sequence of the template polynucleotide in an orientation that is flipped compared to the orientation of the template polynucleotide. Provided herein are adaptive oligonucleotides that can be used in various nucleic acid manipulations to generate immobilized complement polynucleotides that are flipped in orientation compared to the orientation of the immobilized template polynucleotides.
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.