Abstract:
The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single- stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, IncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, IncRNA's splice-site variants, translocations, and miRNA.
Abstract:
The present invention relates to a device comprising a biomolecular processor. Each biomolecular processor has one or more bioreactor chambers defined by a solid substrate; a support structure within each bioreactor; a cleaving enzyme immobilized to the support structure and operatively positioned within the bioreactor chamber to cleave monomer or multimer units of a biopolymer molecule operatively engaged by the cleaving enzyme; and one or more time-of-flight channels formed in the solid substrate and fluidically coupled to said one or more bioreactor chambers. Each of the time-of-flight channels have two or more sensors including at least (i) a first sensor contacting the time-of-flight channel proximate to the input end of the channel and (ii) a second sensor contacting the time-of-flight channel proximate to the output end of channel. The present invention further relates to methods of sequencing and identifying biopolymer molecules using the device.
Abstract:
The present invention is directed to a method for identifying one or more of a plurality of target nucleic acid molecules in a sample. This method includes providing a sample potentially containing one or more target nucleic acid molecules and a plurality of oligonucleotide probe sets. Each probe set is characterized by (a) a first oligonucleotide probe, having a target-specific portion and a tunable portion with an endcapped hairpin and (b) a second oligonucleotide probe having a target specific portion and a tunable portion. One of the first and second oligonucleotide probe has an acceptor group and the other of the first and second probe has a donor group. A ligase is provided and blended with the sample and the plurality of oligonucleotide probe sets to form a ligase detection reaction mixture. The mixture is subjected to one or more ligase detection reaction cycles with each cycle comprising a denaturation and hybridization treatment. During the denaturation treatment any hybridized oligonucleotides are separated from the target nucleic acid sequences, and, during the hybridization treatment, the set of oligonucleotide probes hybridize in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligation product. The ligation product contains the tunable portions, the endcapped hairpin, the target-specific portions, the acceptor group, and the donor group. The ligation products are subjected to conditions effective to permit hybridization of the tunable portions of the ligation product to one another to form an internally hybridized ligation product. The fluorescence resonance energy transfer (FRET) between the donor and acceptor groups of the internally hybridized ligation product is detected, thereby indicating the presence of a target nucleic acid molecule in the sample. An alternative embodiment of the present invention involves the use of an initial PCR amplification procedure with primers provided with universal tails. Finally, FRET detection can be carried out through the use of the above-described oligonucleotide probes without any ligation.
Abstract:
The present invention relates to methods and devices for identifying and quantifying, including low abundance, nucleotide base mutations, insertions, deletions, translocations, splice variants, miRNA variants, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, alternative splicings, exon insertions, exon deletions, intron insertions, or other rearrangement at the genome level and/or methylated nucleotide bases.
Abstract:
The present invention is directed to methods for identifying the presence of one or more methylated or unmethylated target nucleotide sequences in a sample that involve a nuclease-ligation reaction. In some embodiments, the ligation products formed in the nuclease-ligation process of the present invention are subsequently amplified using a polymerase chain reaction. The ligated product sequences or extension products thereof are detected, and the presence of one or more methylated or unmethylated target nucleotide sequences in the sample is identified based on the detection.
Abstract:
The present invention is directed to method of using a collection of monomers capable of forming multimers as a fluorescence reporter in different applications such as ligand detection/screening, disease diagnosis, drug discovery or screening, fluorescent labeling and imaging, or other fluorescent methodologies. Each monomer in the collection includes one or more ligand elements useful for binding to a target molecule with a dissociation constant of less than 300 μΜ and a linker element connected to the ligand elements directly or indirectly through a connector. Association of linker elements of different combinations of monomers, with their ligand elements bound to the target molecule to form a multimer, will generate a unique fluorescent signature different from that produced by those monomers either alone or in association with each other in the absence of the target molecule, when subjected to electromagnetic excitement.
Abstract:
The present invention is directed to a monomer useful in preparing therapeutic compounds. The monomer includes one or more pharmacophores which potentially binds to a target molecule with a dissociation constant of less than 300 μM and a linker element connected to the pharmacophore. The linker element has a molecular weight less than 500 daltons, is connected, directly or indirectly through a connector, to the pharmacophore.
Abstract:
The present application is directed to a therapeutically useful compound, comprised of two monomers that are linked to each other through two or more reversible covalent bonds. Each monomer is a polyfunctionalized molecule comprising a bioorthogonal linker element and ligand or pharmacophore, wherein the linker and ligand/pharmacophore are covalently coupled to each other either directly or through an optional connector moiety.
Abstract:
The present application is directed to a therapeutic composition, comprising two precursor compounds (monomers) that are suitable for assembly via two or more reversible covalent bonds. The monomers are polyfunctionalized molecules comprising a bioorthogonal linker element and ligand or pharmacophore, wherein the linker and ligand/pharmacophore are covalently coupled to each other either directly or through an optional connector moiety.
Abstract:
Described herein are silyl monomers capable of forming a biologically useful multimer when in contact with one, two, three or more other monomers in an aqueous media. Such multimer forming associations of monomers may be promoted by the proximal binding of the monomers to their target biomolecule(s). In one aspect, such monomers may be capable of binding to another monomer in an aqueous media (e.g. in vivo) to form a multimer, (e.g. a dimer). Contemplated monomers may include a ligand moiety, a linker element, and a connector element that joins the ligand moiety and the linker element. In an aqueous media, such contemplated monomers may join together via each linker element and may thus be capable of modulating one or more biomolecules substantially simultaneously, e.g., modulate two or more binding domains on a protein or on different proteins.