Abstract: The invention provides a method of determining ratios of target DNA molecules in a sample. A digital readout of the target DNA molecules is provided by converting ratios of target DNA molecules into equivalent ratios of amplifiable tags, which are, in turn, converted into ratios of color-coded micelles in an emulsion reaction. The micelles may be detected and counted by various methods, including by flow cytometers or slide-based imaging devices. The invention is useful for detection of relative expression levels of selected genes, gene copy number polymorphisms, allelic imbalance, relative levels of iRNAs, and related phenomena of scientific and medical interest.

Abstract: The invention provides a method of generating nested sets of double stranded DNA (dsDNA) circles that may be used as size ladders in nucleic acid separations and as templates in DNA sequencing operations. In one aspect, the invention provides methods for generating nested sets of double stranded DNA circles in a self-sustaining enzymatic reaction comprising the activities of at least one endonuclease, at least one single stranded exonuclease, and at least one ligase. In another embodiment, such nested sets are generated from linear dsDNA fragments having ligatable terminators that are self-ligated to form corresponding dsDNA circles.

Abstract: The invention provides methods and compositions for counting molecules in a sample, wherein each molecule is labeled with a unique oligonucleotide tag. Such tags are amplified and identified rather than the molecules themselves; that is, the problem of counting molecules is converted into the problem of counting tags. In one aspect of the invention, molecules to be counted are labeled by sampling. That is, conjugates are formed between the molecules to be counted and oligonucleotide tags of a very large set, or repertoire. After conjugation, a sample of conjugates is taken that is sufficiently small so that substantially every molecule has a unique oligonucleotide tag. Counting of different tags may be accomplished in a variety of ways. In one aspect, different tags may be counted by carrying out a series of sorting steps to generate successively less complex mixtures in which tags are enumerated using length-encoded “metric” tags.

Abstract: The invention provides methods and compositions for selectively amplifying one or more target polynucleotides in a sample. In one aspect, a plurality of selection oligonucleotides are provided that are capable of simultaneously annealing to separate regions of a target polynucleotide to form a complex that is enzymatically converted into a closed double stranded DNA circle that incorporates the sequence region between the two separate regions. Sequences that fail to form such complexes may be removed by nuclease digestion and the sequences of the remaining DNA circles may be amplified by a variety of techniques, such as rolling circle replication after nicking, PCR amplification after linearization, or the like.

Abstract: The invention provides methods and compositions for detecting the presence of, or for measuring amounts of, molecular targets, particularly molecular complexes comprising two or more proteins, such as receptor complexes of cell surface membranes. In one aspect of the invention, reagent pairs are provided that comprise a reactive species generator that specifically binds to a first region of a molecular target and one or more signaling reagents that each specifically bind to one or more second regions that do not overlap with the first region. In one aspect, each signaling reagent comprises a binding compound specific for a molecular target, such as a component of the molecular complex, and attached to the binding compound, a nanoparticle having one or more leuco dyes attached. In one embodiment, such nanoparticles are releasable bound to the binding compound, so that the nanoparticles may be released and isolated for analysis in a controlled manner as part of an assay protocol.

Abstract: The invention provides methods and compositions for detecting the presence of, or for measuring amounts of, molecular complexes, particularly complexes comprising two or more proteins, such as receptor complexes of cell surface membranes. In one aspect of the invention, reagent pairs are provided that comprise a cleaving probe that specifically binds to at least one component of a molecular complex and one or more signaling reagents that specifically bind to one or more components of the molecular complex, at least one of which is different from the component to which the cleaving probe is attached. Each signaling reagent comprises a binding compound specific for a component of the molecular complex and a signaling polynucleotide attached thereto by a cleavable linkage.

Abstract: The invention provides methods, kits and materials for determinining simultaneously signature sequences of a population of tagged polynucleotides. Size ladders of polynucleotide fragments are generated from the population of tagged polynucleotides that contain a plurality of size classes. After the size classes are separated, tags of the separated fragment are copied and labeled according to the identity of one or more bases at the ends of the fragments. In a preferred embodiment, the labeled tags are then specifically hybridized to plurality of identical microarrays of tag complements such that the tags from different size classes are hybridized to separate microarrays. Signature sequences are determined by signals generated at hybridization sites having the same address on each of the plurality of microarrays.

Abstract: The invention provides methods and compositions for hybridization-based assays that employ oligonucleotide tags, wherein probes specific for the same target polynucleotide are labeled with a plurality of different oligonucleotide tags. When probes are used in conjunction with a microarray, or like, readout platform, containing hybridization sites of tag complements, assay of a target polynucleotide results in a signal being generated from any of a plurality hybridization sites with predetermined addresses and the number of such sites generating a signal is proportional to the relative amount of the target polynucleotide in a population, test sample, or reaction volume, as the case may be. The invention provides methods and compositions for measuring amounts of selected target polynucleotides in a sample and for providing a digital readout of such amounts.

Abstract: The invention provides a method for constructing a high resolution physical map of a polynucleotide. In accordance with the invention, nucleotide sequences are determined at the ends of restriction fragments produced by a plurality of digestions with a plurality of combinations of restriction endonucleases so that a pair of nucleotide sequences is obtained for each restriction fragment. A physical map of the polynucleotide is constructed by ordering the pairs of sequences by matching the identical sequences among the pairs.