Abstract: The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides employing hybridization stringency and/or nuclease digestion for removing failure sequences.

Abstract: The invention is directed to nanopore-based methods for analyzing polymers, such as polynucleotides or proteins, containing optical labels specific for different kinds of monomers. In some embodiments, methods of the invention include steps of (a) translocating a polymer through a nanopore, wherein different kinds of monomers of the polymer are labeled with different optical labels that generate distinguishable optical signals and wherein the nanopore constrains the monomers to move single file through an excitation zone that encompasses a plurality of monomers; (b) detecting a time-ordered set of optical signals from the monomers as the polymer passes they pass through the excitation zone; (c) separating optical signals from different kinds of monomers to form monomer-specific time-ordered sets of optical signals; and (d) determining a sequence of monomers from the monomer-specific time-ordered sets of optical signals from the polymer.

Abstract: The invention is directed to methods for analyzing polymers comprising linear chains of at least two types of monomers, such as polynucleotides, including DNA, RNA, and the like, using nanopores and optical detection. In some embodiments, as few as two different kinds of nucleotide are labeled with different optical labels that generate distinguishable optical signals for the selected kinds of nucleotide in both sense strands and antisense strands of target polynucleotides. Labeled strands are translocated through nanopores where nucleotides of the strands are constrained to pass sequentially through an optical detection region where their labels generate a sequence of optical signals making up an optical signature. In some embodiments, information from optical signatures from both sense and antisense strands are combined to determine complete nucleotide sequences of target polynucleotides.

Abstract: The invention is directed to methods for analyzing polymers comprising linear chains of at least two types of monomers, such as polynucleotides, including DNA, RNA, and the like, using nanopores and optical detection. In some embodiments, as few as two different kinds of nucleotide are labeled with different optical labels that generate distinguishable optical signals for the selected kinds of nucleotide in both sense strands and antisense strands of target polynucleotides. Labeled strands are translocated through nanopores where nucleotides of the strands are constrained to pass sequentially through an optical detection region where their labels generate a sequence of optical signals making up an optical signature. In some embodiments, information from optical signatures from both sense and antisense strands are combined to determine complete nucleotide sequences of target polynucleotides.

Abstract: The invention is directed to nanopore-based methods for analyzing polymers, such as polynucleotides or proteins, containing optical labels specific for different kinds of monomers. In some embodiments, methods of the invention include steps of (a) translocating a polymer through a nanopore, wherein different kinds of monomers of the polymer are labeled with different optical labels that generate distinguishable optical signals and wherein the nanopore constrains the monomers to move single file through an excitation zone that encompasses a plurality of monomers; (b) detecting a time-ordered set of optical signals from the monomers as the polymer passes through the excitation zone; (c) separating optical signals from different kinds of monomers to form monomer-specific time-ordered sets of optical signals; and (d) determining a sequence of monomers from the monomer-specific time-ordered sets of optical signals from the polymer.

Abstract: The invention relates to methods and systems for sequencing and 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.

Abstract: The invention is directed to methods for detecting and quantifying nucleic acid contamination in a tissue sample of an individual containing T cells and/or B cells, which is used for generating a sequence-based clonotype profile. In one aspect, the invention is implemented by measuring the presence and/or level of an endogenous or exogenous nucleic acid tag by which nucleic acid from an intended individual can be distinguished from that of unintended individuals. Endogenous tags include genetic identity markers, such as short tandem repeats, rare clonotypes or the like, and exogenous tags include sequence tags employed to determine clonotype sequences from sequence reads.

Abstract: The invention relates to methods and systems for sequencing and 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.

Abstract: The invention relates to methods and systems for sequencing and 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.

Abstract: The invention relates to methods and systems for sequencing and 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.

Abstract: The invention relates to methods and systems for sequencing and 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.

Abstract: The invention is directed to methods of nucleic acid sequencing that use nanopores to detect and/or measure amounts of compounds, such as products or byproducts of nucleic acid sequencing reactions, and to the determination of a nucleotide sequence using such detection and/or measurement. The detection or measurements may employ products or byproducts having resistive-pulse labels, optical labels, or labels that are capable of generating both optical and resistive-pulse signals. Resistive-pulse labels are molecular labels bound or attached to an analyte which allows detection of the labeled analyte by a change in the electrical properties of a nanopore, such as trans-nanopore resistance. Labels for nanopore detection may also be optical labels, particularly acceptors of acceptor-donor pairs capable of undergoing fluorescent resonance energy transfer (FRET), where the donors are associated with, or label, a nanopore.

Abstract: The invention relates to methods and systems for sequencing and 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.

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 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 relates to methods and systems for sequencing and 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.

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 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.

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 amplifying selected polynucleotides, especially selected subsets of restriction fragments. Generally, methods of the invention are implemented by ligating adaptors containing at least one promoter sequence to such fragments under conditions that promote the formation of closed single stranded or double stranded structures, which are capable of serving as cyclical templates for transcription.