This disclosure provides synthetic nucleic acid oligonucleotides and related methods for analysis of immune repertoires. A disclosed synthetic oligonucleotide contains (a) one or more tags with a sequence that is not contained in a eukaryotic immune repertoire, wherein at least one tag is positioned between a region corresponding to a diversity and joining region (( D)J) and a constant region (C) of an immunoglobulin (IG) or T cell receptor, thereby distinguishing the synthetic oligonucleotide from naturally occurring sequences in the eukaryotic immune repertoire; and wherein the V(D)J region has a predetermined V(D)J clonotype, the synthetic oligonucleotide optionally further comprising a tag proximate to a region corresponding to a eukaryotic variable region (V); and optionally wherein C further comprises one or more priming sites for nucleic acid amplification.
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
2.
METHODS AND COMPOSITIONS FOR THE SIMULTANEOUS IDENTIFICATION AND MAPPING OF DNA METHYLATION
Provided herein is a method for generating a strand of DNA. In some embodiments, this method may comprise: (a) ligating a hairpin adaptor to a double-stranded fragment of DNA to produce a ligation product; (b) enzymatically generating a free 3' end in a double-stranded region of the hairpin adaptor in the ligation product; and (c) extending the free 3' end in a dCTP-free reaction mix that comprises a strand-displacing or nick-translating polymerase, dGTP, dATP, dTTP and modified dCTP to generate a hairpin product that has an original strand and a neosynthesized strand that contains modified Cs.
Homo sapiens, Escherichia coli, Aspergillus oryzae, Momordica charanlia. Pyrococcus furiosus, Cucumis sativusSus scrojdSus scrojd) or (ii) is a non-naturally occurring sequence; and/or an RNA end repair enzyme having an amino acid sequence that (i) corresponds to an amino acid sequence of a species other than the first species (e.g., a bacterial species or a bacteriophage species) or (ii) is a non-naturally occurring sequence.
The present disclosure relates, according to some embodiments, to methods and compositions for preparing polynucleotide libraries enriched for a target sequence from source materials (e.g., samples comprising or constituting biological fluids, tissues, and/or specimens). Methods and compositions may provide efficient enrichment of the targeted polynucleotide. Enrichment may include increasing the relative abundance of the target from the source materials (where it may be present in low abundance) to the produced libraries (where it may be present in higher abundance). In some embodiments, methods include attaching (e.g., ligating, joining or otherwise fusing) an adapter to fragments of interest, nick translation, amplification (e.g., linear amplification), and target sequence selection using, for example, affinity tagging.
The present disclosure relates, according to some embodiments, to immobilized enzyme compositions and methods for cleaving polynucleotide molecules including, for example, double-stranded DNA. Immobilized enzymes may comprise, for example, an enzyme (e.g., a type IIS restriction endonuclease, an RNAP, a capping enzyme), a support (e.g., a magnetic bead), and optionally, a linker disposed between the enzyme and the support. In some embodiments, methods may include contacting an immobilized enzyme with a polynucleotide substrate to form reaction products, separating the immobilized enzyme from the reaction products, and optionally reusing the immobilized enzymes in one or more subsequent reactions, preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter- tagged fragments and, optionally, separated tailing enzyme and/or separated ligase.
Provided herein, among other things, is a method for deaminating a double-stranded nucleic acid. In some embodiments, the method may comprise contacting a double-stranded DNA substrate that comprises cytosines and a double-stranded DNA deaminase having an amino acid sequence that is at least 80% identical to any of SEQ. ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 19, 24, 26, 27, 28, 33, 40, 49, 50, 63, 95, 96, 97, and/or 99 to produce a deamination product that comprises deaminated cytosines. Enzymes and kits for performing the method are also provided.
Provided herein is a composition comprising an enzyme that releases pyrophosphate from a substrate and a dye of Formula 1. A method for detecting pyrophosphate is also provided. A kit comprising a polymerase that releases pyrophosphate by hydrolysis of nucleoside triphosphates during nucleic acid replication, a divalent manganese salt, and the dye are also provided. The present composition, method and kits provide a way to detect and/or quantify substrates or products of enzyme reacted substrates associated with the release pyrophosphate (e.g., nucleic acid amplification reactions and other reactions that hydrolyze ATP) via a distinct color change without substantially affecting the sensitivity and/or specificity of the reaction.
G01N 33/52 - Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper
C12Q 1/48 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
C09B 29/036 - Monoazo dyes prepared by diazotising and coupling characterised by the diazo component from diazotised amines containing a heterocyclic ring the heterocyclic ring containing only nitrogen as hetero atoms
Kits and methods are provided that utilize a mesophilic strand displacing polymerase selected from Bsu DNA polymerase (large fragment) and Klenow in Loop mediated amplification (LAMP) at temperatures in the range of 34°C-52°C. This contrasts with 60°C -65°C required for standard Bst polymerase dependent LAMP. The reduced temperature of the LAMP reaction enables the use of other proteins that are temperature sensitive in a one-step reaction. For example, a Cas protein such as Cas12a may be used with a target nucleic acid specific guide RNA and optionally a reporter oligonucleotide containing a quencher and a fluorophore or lateral flow reagents to determine the presence of pathogens in a sample.
Clostridium butyricumClostridium butyricum) may be synchronized with DNA strand unwinding activity of a helicase (e.g., a nuclease deficient RecBexo- E.coli E.coli ) for a rapid and efficient cleavage of double-stranded DNA targets. Enzymatic properties of CbAgo and different aspects of ds DNA cleavage were thoroughly explored by adapting high-throughput capillary electrophoreses technique for monitoring CbAgo cleavage activity in concurrence with RecBexo-C. The present disclosure shows that in the presence of RecBexo-C., CbAgo can be programmed with guides to cleave any site of interest localized at up to 10 kb distance from the end of linear ds DNA at 37°C temperature. CbAgo /RecBexo-C. can be programmed to generate DNA fragments flanked with unique single- stranded extensions suitable for seamless ligation with compatible DNA fragments. The present disclosure relates further the compositions, methods, systems, and kits for PRC-free assembly of linear DNA molecules by using Cb Ago/RecBexo-C. programmable DNA endonuclease. The results presented here demonstrate that the combination of CbAgo and RecBexo-C. is currently an efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease which can be used to prepare synthetic biology tools that require or benefit from sequence- specific nicking/cleavage of natural DNA at otherwise inaccessible locations.
The present disclosure relates, according to some embodiments, to systems, apparatus, compositions, methods, and workflows that include DNase I variants with desirable properties including, for example, salt tolerance. A DNase I variant, in some embodiments, may have an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, and/or at least 98% identical to SEQ ID NO: 1 and may be identical to SEQ ID NO: 1 at one or more positions selected from the group of positions corresponding to L29, A35, D87, Q88, S94, P103, T108, P121, P132, A135, D145, E161, G172, P190, H208, and A224 of SEQ ID NO:1.
Compositions, methods and kits are provided that describe a novel enzyme family called here a hydroxymethylcytosine carbamoyltransferase that transfers a carbamoyl phosphate substrate onto a hydroxymethylcytosine nucleoside triphosphate or a hydroxymethylcytosine in a nucleic acid. The carbamoyl phosphate substrate may be tagged with a chemically reactive group and optionally a functional group. This enables multiple uses of this enzyme and substrate for detecting nucleic acids with modified nucleotides, enriching for such nucleic acids, sequencing nucleic acids containing modified nucleotides, and for synthesizing oligonucleotides with various labels for various molecular biology applications including stabilizing RNA.
Ordered assembly of large numbers of fragments into a single large DN A have been improved in both frequency and fidelity of the assembled product. This has been achieved by novel compositions and methods that are utilized in a computer system that integrates comprehensive ligation data from multiple sources to provide optimized synthetic overhangs or overhangs from restriction endonuclease cleavage on DIMA fragments for assembly by ligation. Intragenic cut sites are avoided by the use of a novel restriction endonuclease which recognizes 7 nucleotides (bases) and cuts DNA to create 4-base overhangs with the help of a synthetic activator oligonucleotide. Variations in ligation preferences by different ligases provide extra precision in assembly reactions. The use of the improved methods are exemplified by the successful assembly from 52 fragments of a viral genome and also a 52 fragment ordered assembly of a bacteria operon.
The present disclosure relates, according to some embodiments, to methods for preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter- tagged fragments; and/or separating adapter- tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase. In some embodiments, a tailing enzyme and/or a ligase used in library preparation may be immobilized enzymes.
Kits and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The kits and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The kits and methods utilize in various combinations, reversible inhibitors of kit components, thermolabile enzymes, poloxamers, various salts, indicators and one or more Loop-Mediated Isothermal Amplification (LAMP) primer sets for detecting single and/or multiple targets and variants of the targets including SARS-CoV-2 targets and variants thereof in a single reaction. The kits and methods permit detection of the target nucleic with similar sensitivity regardless of the presence of undefined mutations that may enhance the virulence of cells or viruses containing the undefined mutations.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
18.
COMPOSITIONS AND METHODS FOR DETECTING MOLECULAR TARGETS ON CHROMOSOMAL DNA
Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.
Kits and Methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The kits and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The kits and methods utilize in various combinations, reversible inhibitors of kit components, thermolabile enzymes, poloxamers, various salts, indicators and multiple Loop-Mediated Isothermal Amplification (LAMP) primer sets for detecting single and/or multiple targets in a single reaction.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
The present disclosure relates to polymerases (e.g., variants of Family D DNA polymerases) for polynucleotide synthesis, polynucleotide amplification, polynucleotide sequencing, cloning a polynucleotide, or combinations thereof. Variant Family D polymerases have one or more substitutions relative to wild type Family D polymerases (e.g., substitutions impacting substrate selectivity) and may incorporate ribonucleotides and/or modified nucleotides into synthesized or extended strands.
The present disclosure relates to polymerases (e.g., variants of Family D DNA polymerases) for polynucleotide synthesis, polynucleotide amplification, polynucleotide sequencing, cloning a polynucleotide, or combinations thereof. For example, a variant Family D polymerase may have an amino acid sequence that (a) is at least 99%, identical to SEQ ID NO:1 and (b) has a substitution at a position selected from positions corresponding to positions 106-161, 243-267, 326-330, 361-365, 385-397, 441-451, 657-667, 822-829, 919-928, and 940-962, 981-997 of SEQ ID NO:1.
Provided herein are kits, compositions and methods for isolating high molecular weight (HMW) DNA having a desired length in a rapid reproducible manner using beads of at least 200 pm to adsorb DNA from a cell lysate. A device is described for retaining the beads while permitting washing of the DNA bound to the beads and eluting the DNA from the beads. The device permits the DNA eluant released from the beads to exit the device without shearing. The device also permits recycling of the beads.
Methods are provided that, for example, include (a) combining ssDNA containing a modified nucleotide (e.g., a ssDNA with a modified nucleotide proximate to its 5'end) with a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate a first ssDNA fragment having a 3'OΗ and a second ssDNA fragment having the modified nucleotide); wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio (m/m); and (b) cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, a method may comprise (a) combining (i) a ssDNA comprising a modified nucleotide (e.g., proximate to its 5'end) with (ii) a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3'OΗ and a second ssDNA fragment comprising the modified nucleotide) wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio and cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, methods provided herein may include (a) combining (i) a ssDNA (1) immobilized on a substrate and (2) comprising a modified nucleotide with (ii) a ssDNA cleaving enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3'OΗ and a second ssDNA fragment comprising the modified nucleotide); and (b) cleaving the immobilized ssDNA to release the second single stranded DNA fragment from the substrate. At least 95% (m/m) of an ssDNA comprising a modified nucleotide may be cleaved in less than 60 minutes.
Provided herein is a method for chemically capping polynucleotides having a 5' monophosphate. In some embodiments the method may comprise: combining an activated nucleoside 5' mono- or poly-phosphate with a population of polynucleotides that comprises polynucleotides having a 5' monophosphate, to produce a reaction mix; and incubating the reaction mix to produce reaction products that comprise a polynucleotide and a 5'nucleoside cap, linked by a 5' to 5' polyphosphate linkage. The chemical capping method described herein can be incorporated into a variety of cDNA synthesis methods.
Compositions and methods for efficient adapter ligation to small RNAs and other nucleic acids. The composition comprises a 5' exonuclease, a double stranded nicking enzyme, a ligase and partially double-stranded polynucleotides used as adapters.
The present disclosure provides, among other things, a way to amplify and sequence target sequences in a low-input sample. In some embodiments, the method comprises ligating a double-stranded adaptor onto a population of fragments to produce tagged fragments, and linearly amplifying the tagged fragments.
Methods and compositions are provided for optimizing ordered assembly of a plurality of polynucleotide fragments. The optimization involves providing sets of overhang sequences with preferred experimental conditions for high fidelity ordered assembly of polynucleotide fragments by ligation under selected experimental conditions. The methods and compositions provide the use of a computer system with inputs having a plurality of menus and outputs that include a variety of media interfaces. The computer system has access to a ligation frequency database to provide sets of overhang sequences for efficient joining of multiple fragments into the target nucleic acid. In-puts include one or more of the following: numbers and sizes of fragments, optionally a desired target polynucleotide sequence from a database, in which case one output are the recommended polynucleotide fragments for ordered assembly, and selected experimental conditions selected from any or all of ligation protocols and ligation temperature with reaction times, salt concentration in the ligation buffer, choice of ligase and restriction endonuclease and the use of DNA repair enzymes.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
in vitroin vitroin vitro transcription, capped RNA is produced. The methods describe high capping efficiencies at elevated temperatures as well as at standard temperatures for IVT. The RNA polymerase used is a thermostable variant of the T7 RNA polymerase, named polymerase M20, which comprises substitutions at positions 388 and 567, in particular D388E and V567P.
Method comprising incubating a double-stranded nucleic acid having a nick with a nick translating activity, a ligase, and a nucleotide mix comprising at least one modified nucleotide, to generate a product comprising a patch of a newly synthesized strand of a duplex nucleic acid containing a plurality of modified nucleoside monophosphates that are at or adjacent to the site of the nick. The method may also comprise treating the nucleic acid with modified nucleotides with bisulfite or deaminase. In some embodiments, the method may be used to map damaged nucleoside monophosphates in a nucleic acid. Compositions and kits for use in performing the method are also provided.
Provided herein, among other things, are various in vitro methods that involve cleaving dsDNA molecules that comprise a mismatched nucleotide using EndoMS. In some embodiments, the method may comprise ligating a T-tailed double-stranded adapter to A-tailed double-stranded fragments of nucleic acid to produce ligation products that comprise adapter-ligated fragments and double-stranded adapter dimers that comprise a T:T mismatch at the ligation junction and cleaving both strands of the adapter dimers using EndoMS.
Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44°C.
A61K 48/00 - Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
The present invention generally relates to hydro gels and display technologies, especially protein and peptide display technologies. One aspect is generally directed to hydrogel particles comprising an attached nucleic acid and an attached protein. At least a portion of the nucleic acid may encode the protein. The particles may be used for display applications or other assays, e.g., by exposing the particles to certain targets (e.g., cells, other proteins, drugs, or the like) and determining any interactions. For instance, particles exhibiting certain interactions may be separated from other particles, then those particles analyzed to determine the nucleic acids encoding the proteins participating in those interactions. In some cases, the particles may be contained within microfluidic droplets, although such droplets are not required. Hydrogel particles may be particularly useful in certain embodiments due to their ease of preparation, their cell-free nature (e.g., unlike phase display), their porosity or deformability, etc. Other aspects are generally directed to making or using such hydrogel particles, kits involving such particles, or the like.
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
A mutant MMLV reverse transcriptase that may have an improvement in one or more properties is provided. For example, the present reverse transcriptase is believed to be more efficient relative to other commercially available MMLV reverse transcriptase variants, particularly for templates with a higher GC content.
Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES (USA)
THE UNIVERSITY OF TOKYO (Japan)
NEW ENGLAND BIOLABS, INC. (USA)
Inventor
Carlow, Clotilde
Suga, Hiroaki
Yu, Hao
Li, Zhiru
Dranchak, Patricia
Inglese, James
Macarthur, Ryan
Abstract
Disclosed herein are isolated peptides inhibit activity of a cofactor-independent phosphoglycerate mutase. In some examples, the isolated peptide is 6-20 amino acids long and includes the amino acid sequence of any one of SEQ ID NOs: 1-22 or 54, an analog or derivative thereof, or a pharmaceutically acceptable salt or ester thereof. In some examples, the peptide is a cyclic peptide with an N-terminal ring of 6-15 amino acids (for example, 6-10 amino acids) and a C-terminal linear portion of 1-9 amino acids (for example, 3-8 amino acids. Also disclosed h are methods of treating or inhibiting an infection in a subject, including administering to the subject an effective amount of a composition including one of more of the disclosed peptides, or analogs or derivative thereof, or pharmaceutically acceptable salts or esters thereof.
Compositions and methods for performing a template-switching reaction are provided that may include reducing or eliminating concatemerization of the template-switching oligonucleotide (TSO). In some embodiments, the composition may comprise a site-specific nucleic acid cleaving enzyme, a TSO that includes a recognition sequence for a site-specific nucleic acid cleaving enzyme, wherein the TSO has at its 3' end at least one nucleotide that is complementary to one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase, and a site-specific nucleic acid cleaving enzyme that cleaves the TSO at the recognition sequence.
Methods are provided for a rapid, low cost approach to monitoring an amplification reaction. This includes monitoring the progress of isothermal or PCR amplification reactions to completion using pH-sensitive dyes that are either colored or fluorescent. Compositions are described that include a mixture of a DNA polymerase, deoxyribonucleotide triphosphate and Tris buffer in the range of 1.5mM Tris to 5mM Tris or equivalent.
A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.
A method for identifying the location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and optionally reacting a second portion of the sample with a dioxygenase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase that is more efficient at converting methylcytosine to carboxymethylcytosine is also provided.
Methods and compositions are provided for enriching for target sequences from a population of nucleic acids, that includes: combining in solution, a population of nucleic acids and a target isolation probe wherein the target isolation probe comprises an affinity binding domain; permitting a single stranded region of the target isolation probe to hybridize to all or a portion of a target sequence in the population of nucleic acids; selectively immobilizing the hybridized nucleic acids from the population containing the target sequences by associating the target isolation probe with a capture domain and removing unbound material; removing non-target sequences from the 3' end of the target sequence by means of one or more 3' exonucleases thereby generating a blunt ended duplex or a staggered end at the 3' end of the target sequence; optionally ligating a 3' duplex adaptor or a duplex end of a hairpin adaptor to the 3' end of the target sequence and the 5' end of the target isolation probe; extending the 3' end of the target isolation probe to form a blunt end or a staggered end at the 5' end of the target sequence suitable for ligating and ligating an adapter to the 5' end of the target sequence and the 3'extended end of the target isolation probe.
The present invention generally relates to cell-free protein synthesis in microfluidic droplets. Droplets may be used to encapsulate genetic information (DNA/RNA), and through cell-free protein synthesis, provide a linkage of the genetic information with the functional information, e.g., the activity of the expressed protein, and used to convert the functional information into a detectable signal, e.g., to allow sorting of the droplets and retrieve genetic information associated with the drops. In one set of embodiments, microfluidic droplets containing a cell-free protein synthesis system designed to detect protein-protein interaction (e.g., in vitro two-hybrid systems or IVT2H) can be used for high-throughput screening, e.g., of protein binders. This drop-based two-hybrid system may include two (or more) fusion proteins that can bind to each other such that their binding produces a complex that is able to produce a nucleic acid. The nucleic acid may be expressed to produce a protein. In certain embodiments, the protein may be produced within a cell-free system. The protein may be fluorescent or otherwise determinable, such that determination of the protein may be used to allow assays to occur within the droplets, to allow sorting of the droplets to occur, or the like, e.g., as discussed below, for instance, for screening or other applications.
C12N 15/00 - Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
C12N 15/67 - General methods for enhancing the expression
This disclosure provides, among other things, a composition comprising: a 5' exonuclease; a strand-displacing polymerase; and optionally a single strand DNA binding protein and/or a ligase. A method for polynucleotide assembly to form a synthon, as well as a kit for performing the same, are also described.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/62 - DNA sequences coding for fusion proteins
C12N 15/64 - General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
Compositions and methods are provided for storing prokaryotic cells including competent prokaryotic cells at -20°C in a buffer so that the cells are suitable for transformation at 0°C with a foreign molecule.
Methods of capturing N-glycan linked glycomolecules including N-glycans, N-glycopeptides and N-glycoproteins are described. The methods provide substantially unbiased capture of charged and uncharged N-glycans and/or N-glycan linked glycomoleules. Binding reagents for substantially unbiased binding of N-glycans and/or N-glycan linked glycomolecules are also described.
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
C12N 9/04 - Oxidoreductases (1.), e.g. luciferase acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
C12N 9/00 - Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
G01N 33/68 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
The present invention generally relates to systems and methods for determining proteins such as antibodies, or other targets. In one aspect, fusion proteins are exposed to a target, such as an antibody, such that binding of the fusion proteins to the targets produces a complex that is able to produce a nucleic acid. The nucleic acid may be expressed to produce a protein. The protein may be fluorescent or otherwise determinable, such that determination of the protein may be used to determine or identify the target. In some cases, this may be performed within a microfluidic droplet. As an example, a single cell contained within a droplet may produce a target, and determination of the target within the droplet may then be associated with that particular cell.
C12P 21/04 - Cyclic or bridged peptides or polypeptides, e.g. bacitracin
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 40/10 - Libraries containing peptides or polypeptides, or derivatives thereof
C40B 50/08 - Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
Compositions and methods are provided for efficiently preparing a completely deglycosylated antibody where efficiency is measured in relative amounts of reagents in soluble or lyophilized form, and time and temperature of the reaction. Compositions and methods are also provided for separating substantially all N- linked glycans from a glycosylated antibody and for preserving functionality of the antibody. The methods are compatible with glycan labeling and protease digestion without the need for prior purification steps.
C07K 16/00 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies
C07K 16/24 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
Methods and compositions are described for distinguishing target RNA having a 5' diphosphate or triphosphate in a mixture of RNAs. The methods utilized modified labeled nucleotides for capping the 5' end of the RNA. The label on the modified nucleotide is attached to the carbon 3 via the oxygen by replacing the hydrogen ion.
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
Described herein is an inactivated viral particle comprising one or more transcription factor proteins packaged within the particle. A method for using the particle to make induced pluripotent stem cells is also provided.
Compositions and methods are provided for improved reverse transcriptases and their uses in reverse transcription where the improvement may include increased temperature, increased salt, increased activity and/or increased dUTP tolerance.
This disclosure describes, among other things, compositions, kits and methods for identifying 5hmC in eukaryotic genomic DNA. The compositions and methods utilize a PvuRts1I family enzyme for digesting eukaryotic genomic DNA suspected of containing 5hmC or 5fC modified nucleotides providing an end of the DNA with a two base overhang suitable for ligating an adapter at a fixed distance 3' from an 5hmC. Random fragmentation of the genomic DNA can generate a blunt end suitable for attaching a second adapter. The DNA can be sequenced and 5hmC and 5fC residues identified and located at a defined position in the eukaryotic genome. An enrichment step may be used that utilizes a chemoselective agent capable of being selectively added to DNA containing 5hmC. An example is a glucosyltransferase and a glucosyltransferase substrate comprising a chemo-selective group. This DNA can then be enriched by immobilization on a matrix that binds the chemoselective agent added to the DNA containing 5hmC and DNA that does not contain a chemo-selective group is removed by washing. Adapters can be added at one or both ends of the restriction endonuclease cleaved DNA after random fragmentation prior to or after an enrichment step.
Provided herein is a method for reducing amplification of non-template molecules in a nucleic acid sample. In certain embodiments, the method involves adding a helicase to a reaction mixture for non-helicase-dependent amplification of target nucleic acid.
Provided herein in some embodiments is a non-naturally occurring variant of a wild type restriction enzyme defined by SEQ ID NO: 20, wherein the variant has at least a 2 fold increase in cleavage at 5-β glucosylhydroxymethylcytosine (5βghmC) compared with methylcytosine relative to the wild type enzyme. Methods for examining hydroxymethylation of a DNA sample using the variant enzyme are also provided.
Compositions and methods are provided for ligating polynucleotides having a length that is greater than 8 nucleotides on an RNA splint. The ligation reaction provides consistent results in high or low ATP concentrations. The reaction can occur rapidly and is generally at least 10 fold more efficient than T4DNA ligase under optimal conditions for T4DNA ligase and the reaction time is less than 6 hours for example, less than 1 hour.
Nucleic acids comprising β-clucosaminyloxy-5-methylcytosine; compositions, kits and methods of producing the nucleic acids using a glycosyltransferase; and methods of using the nucleic acids are described.
Methods are provided for a rapid, low cost approach to monitoring an amplification reaction. This includes monitoring the progress of isothermal or PCR amplification reactions to completion using pH-sensitive dyes that are either colored or fluorescent. Compositions are described that include a mixture of a DNA polymerase, deoxyribonucleotide triphosphate and a weak buffer of less than 1 mM Tris or equivalent or no buffer.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
Compositions and methods are provided for quantitative detection of amplification products, the methods being suitable for multiplexing. A first oligonucleotide that includes a primer sequence for priming an amplification reaction and also is labeled with a fluorescent label or quencher is mixed with a second oligonucleotide which has a sequence suitable for hybridizing to a portion of the first oligonucleotide and has a fluorescent label if the first oligonucleotide has a quencher or a quencher if the first oligonucleotide has a fluorescent label; and a third nucleotide which includes some or all the primer sequence contained in the first oligonucleotide but is not labeled, the first and third oligonucleotide being combined in a molar ratio of 2.8 to 8.2.
Compositions and methods are provided for inhibiting a polymerase from replicating non target DNA at a temperature below the amplification reaction temperature. The inhibitor is a synthetic nucleic acid which is single stranded but folds to form at least one double stranded region designed to melt at a temperature which is lower than the amplification reaction temperature, and at least one single stranded region where the single stranded region at the 5' end contains at least one uracil or inosine and optionally a sequence at the 3' end contains one or more derivative nucleotide or linkages.
Compositions and methods are provided for discrimination between cytosine and modifications thereof using cytidine deaminases and/or oxygenases. Variants of wild type cytidine deaminases are described which show reduced bias with respect to adjacent nucleotides upstream of the cytosine. The methods provide a rapid and convenient use of enzymes to obtain methylomes.
Compositions of novel polymerase variants and methods of identifying, making and using these novel polymerases are described. The variants have been shown to have advantageous properties such as increased thermostability, deoxyuridine nucleoside triphosphate tolerance, salt tolerance, reaction speed and/or increased reverse transcriptase properties. Uses for these improved enzymes have been demonstrated in isothermal amplification such as LAMP. Enhanced performance resulting from the use of these variants in amplification has been demonstrated both in reaction vessels and in dedicated automated amplification platforms.
Compositions and methods are provided for enhancing enzymatic ligation between nucleic acid fragments that relies on one or more small molecule enhancers having a size of less than 1000 daltons. For example, enhancement of ligation efficiencies are observed for double- stranded nucleic acid fragments that are blunt-ended, have a single nucleotide overhang at the ligation end, or have staggered ends compared to ligation under similar conditions in the absence of the one or more small molecule ligation enhancer. The use of small molecule enhancers for ligating nucleic acids results in an increased number of transformed host cells after transformation with the ligated molecules. This enhancement can be observed with chemically transformed host cells and with host cells transformed by electroporation.
Compositions and methods are described to modify Family B DNA polymerases that contain residual exonuclease activity that interferes with sequencing techniques and with detection of single nucleotide polymorphisms. The compositions are mutant proteins with reduced exonuclease activity compared with presently available "exo-" polymerases, and a sensitive screening assay that enables an assessment of exonuclease activity of any synthetic DNA polymerase.
Compositions relating to a combination of two types of separation matrix; and to variant host cells which contain at least one essential host protein that is fused to an affinity binding tag or has been mutated to replace at least two of a plurality of histidines or basic amino acids are provided. Methods are also provided that relate to isolating a recombinant protein from a lysate.
Methods are provided for ligating a 3' adapter and a 5' adapter to a target polynucleotide so as to avoid adapter dimer formation. Embodiments of the methods include adding a blocking oligonucleotide after the first ligation in which a 3' adapter is ligated to the target polynucleotide so that the blocking oligonucleotide is capable of hybridizing to excess 3' adapter and the ligated 3' adapter. Subsequently, a 5' adapter is ligated to the target polynucleotide thus avoiding adapter dimer formation.
Compositions are provided that include a synthetic oligonucleotide characterized by a double-stranded region, a single-stranded region, a forward primer site, a reverse primer site and one or more cleavage sites therebetween. Methods of use for these compositions include adaptors for the amplification of DNA fragments.
A method is provided for generating a preparation in which more than 70% of the oligonucleotides are adenylated. The method includes reacting an oligonucleotide with an ATP-sensitive ligase where the ligase is characterized by its ability to efficiently generate adenylated oligonucleotides at ATP concentrations at which ligation and circularization of the oligonucleotide is minimal.
Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.
Compositions, methods and a kit are described relating to a novel family of enzymes which preferentially bind to a hydroxymethylated cytosine or a glucosylated hydroxymethylated cytosine and cleave double- stranded DNA at a defined distance 3' of the recognition site to produce a cleavage fragment with a 1-3 base overhang.
Mutations introduced into a restriction endonuclease may alter not only fidelity of cleavage but also specificity. Examples of novel variant endonucleases with altered specificities are described and also variants with high fidelity of cleavage. Examples describe mutations in Btsl that result in at least one of altered cleavage and nicking and high fidelity cleavage.
C12N 15/00 - Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
C12N 9/00 - Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
75.
DETECTION AND QUANTIFICATION OF HYDROXYMETHYLATED NUCLEOTIDES IN A POLYNUCLEOTIDE PREPARATION
Methods and compositions are described for detecting hydroxymethylated nucleotides (hmNs) in a polynucleotide preparation with a view to mapping the location of hmNs in a genome, quantifying the occurrence of hmNs at selected loci and correlating the occurrence of hmNs with gene expression and phenotypic traits. Embodiments describe the use of modifying enzymes together with site-specific endonucleases to detect the hmNs.
Compositions and methods are provided for converting chitin into N-acetylglucosamine, glucosamine and ethanol. The chitin may be used directly from the environment, for example, as occurs in invertebrate cuticles, fungal cells and/or algae. Mutant bacteria were created by knocking out or inactivating one or more genes preferably resulting in the chitin catabolic sensor maintaining an activated state. Methods are further provided for converting the N-acetylglucosamine into ethanol by means of a genetically engineered yeast strain which can be optionally co-cultivated with the Vibrionaceae to produce significant yields of ethanol.
Methods and compositions are provided that relate to a composition that includes a modified maltose-binding protein (MBP) which when fused to a protein results in an increase in binding affinity for maltodextrin compared with the wild type MBP fused to the protein, the modified MBP maintaining enhanced solubility, The modification includes a mutation selected from the group consisting of : F68L, I318V, Q326R, V344M, and T372TTTITITTTLGIEGR387 or consist of two or more mutations selected from the group consisting of : F68L, S146T, A313V, I318V, I318A, Q326R, V344M and T372TTTITITTTLGIEGR387 mutants.
A restriction endonuclease is provided that has been engineered to have a cleavage specificity for a DNA recognition sequence containing a modified nucleotide. Methods for engineering enzymes to cleave DNA containing modified nucleotides at specific sequences are provided.
An enzyme preparation is described that includes a non-specific nuclease and a T7 Endo I mutant in a unit ratio of less than 1:200. This enzyme preparation may be used to generate double-stranded DNA fragments of a size suitable for DNA sequencing. The ends of the fragments can be readily modified as necessary to ligate adaptors or individual nucleotides to one strand of the double-stranded DNA fragments.
Compositions, methods and related uses are provided relating to cleaving modified DNA. For example, a set of DNA fragments obtainable by enzymatic cleavage of a large DNA is described where at least 50% are similarly sized and have a centrally positioned modified nucleotide. In addition, an enzyme preparation is provided that includes one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide. The one or more enzymes are further characterized by an N- terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X)10YXGD. The related uses include creating a methylome, methods of purifying DNA fragments containing a modified nucleotide and diagnostic applications.
Compositions and methods are provided for generating biofuels by fermentation from carbon sources other than glucose using genetically engineered yeast strains. For example, a Saccharomyces strain which is capable of converting glucose to ethanol but not of metabolizing N-acetyl glucosamine is genetically engineered to utilize N-acetyl glucosamine as a nutrient carbon source.
Compositions and methods are provided for amplifying polynucletoidesenzyme reagents for amplification of polynucleotides in the presence of inhibitors from samples containing inhibitors that normally inhibit amplification using an enzyme blend containing a plurality of polymerases. The ability to amplify polynucleotides efficiently in the presence of inhibitors allows the enzyme reagent to be used in both routine amplification and real-time amplification from inhibitor-containing samples.
Compositions and methods are provided that relate to a recombinant protein with DNA polymerase activity in which one or more amino acids are mutated compared with the corresponding wild type protein. The recombinant protein is capable of incorporating one or more modified nucleotides into a nucleic acid substrate with a specific activity greater than 200.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C12P 21/06 - Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Compositions and methods are provided for preserving the thermostability of an endonuclease in the presence of DNA for uses that include DNA repair. The compositions and methods include the presence of zinc ions.
Methods and compositions have been described that relate to a newly identified polypeptide family wherein each member has 0-glycosidase activity and specified sequence characteristics. This family of enzymes can be used for example for cleaving O-linked glycans and for synthesis of neoglycopeptides or neoglycoproteins.
Methods and kits are provided for obtaining small RNAs from a mixture of RNAs of varying sizes such as can be found in a cell lysate or an enzyme-digested RNA. The methods and kits utilize magnetic beads and require the addition of one or more alcohols to bind small RNAs effectively to the beads.
Methods and compositions are provided for detecting small target RNAs where the target RNA may be single-stranded or double-stranded and may be contained in a mixture of RNAs of different types and sizes. The methods and compositions utilize a pl9 fusion protein that is capable of binding double-stranded RNA in a size-specific but sequence-independent manner and is further capable of binding to a matrix such as beads or plastic microwell plates. By labeling the pl9 fusion protein or the target RNA in a polynucleotide duplex either directly or indirectly, low levels of target RNA including microRNAs can be detected from cells. This can be applied to diagnosis of pathological conditions.
Methods and compositions are provided for producing membrane proteins or toxic proteins from recombinant DNA introduced into a prokaryotic host cell by targeting the expressed proteins to the envelope of the host cell. The methods and compositions utilize a protein vehicle fused to a protein of interest. The fusion protein may contain one or more protease cleavage sites to separate the protein of interest from the protein vehicle either in vivo or in vitro. The protein vehicle is characterized by a membrane-targeting peptide and a trans-membrane segment separated by a cytoplasmic amino acid sequence that includes a cytoplasmic affinity-binding domain.
Compositions and methods are provided for enzymes with altered properties that involve a systematic approach to mutagenesis and a screening assay that permits selection of the desired proteins. Embodiments of the method are particularly suited for modifying specific properties of restriction endonucleases such as star activity. The compositions includes restriction endonucleases with reduced star activity as defined by an overall fidelity index improvement factor.
Methods and compositions are provided for creating a binding protein that recognizes a rationally chosen recognition sequence in which a first amino acid has been substituted for a second amino acid using site-directed mutagenesis of a member protein of a set of proteins at an identified position or positions correlated with recognition of a chosen specified target module in the recognition sequence. A system is provided for automating the storage and manipulation of the correlations between positions and types of amino acid residues in the binding protein with specific modules at specified positions in the target recognition sequence and for designing and creating proteins with novel specificities.
G06F 19/00 - Digital computing or data processing equipment or methods, specially adapted for specific applications (specially adapted for specific functions G06F 17/00;data processing systems or methods specially adapted for administrative, commercial, financial, managerial, supervisory or forecasting purposes G06Q;healthcare informatics G16H)
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
Compositions and methods are provided for selection and enrichment of a target gene from a library of polynucleotide sequences such as might be formed from a genome or by random mutagenesis of a genetic sequence. The selection and enrichment occurs in aqueous droplets formed in an emulsion that compartmentalize individual polynucleotides from the library or a plurality of polynucleotides that may include polynucleotides not derived from the library, transcription and translation reagents and optionally additional chemical and enzyme reagents. The selection and enrichment method utilizes a polynucleotide adaptor which when ligated to the polynucleotide fragment enables amplification to occur in the presence of an adaptor specific primer.
A protein is described that has an amino acid sequence characterized by at least 90% sequence identity with SEQ ID NO: 24, the protein being capable of recognizing a sequence consisting of 5'-GCCGAG-3' within the double-stranded DNA and cleaving the substrate predominantly at 21/19 nucleotides from the recognition site. A method is also described that utilizes the protein for creating a DNA tag for use as a unique identifier for paired end sequencing of DNA or serial analysis of gene expression.
Compositions and methods are provided for expression of a toxic protein in a host cell preferably a bacterial host cell where at least one T7 RNA polymerase gene Is contained on the host cell chromosome and one or more genes encoding a T7 RNA polymerase inhibitor is located on an F' plasmid or on the chromosome.
A recombinant BSA (rBSA) that (a) substantially lacks deoxyribonuclease activity as determined by incubating rBSA with linear DNA overnight and gel electrophoresis, (b) lacks animal viruses associated with animal-derived cell growth supplements; and (c) is capable of stabilizing DNA proteins is provided. Methods for making the rBSA and using it to stabilize enzymes are also provided.
Recombinant DNA encoding NruI- and Sbol-like restriction endonucleases and methylases and their amino acid sequences are provided as well as methods for expressing the enzymes in transformed host cells and purifying the enzymes.
C12N 15/00 - Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
C12N 5/10 - Cells modified by introduction of foreign genetic material, e.g. virus-transformed cells
C12N 1/21 - Bacteria; Culture media therefor modified by introduction of foreign genetic material
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
C12N 9/16 - Hydrolases (3.) acting on ester bonds (3.1)
C12Q 1/44 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
C07K 14/00 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
96.
REPAIR OF NUCLEIC ACIDS FOR IMPROVED AMPLIFICATION
Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/o yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a DNA ligase and an effective amount of at least one endonuclease as well as a cofactor selected from NAD+ or ATP.
Methods and compositions are provided for increasing at least one of: (i) binding affinity of a target protein for a maltodextrin substrate and/or (ii) solubility of a target protein. The methods and compositions relate to a modified maltose-binding protein.
The present invention relates to compositions including: (1) isolated DNA encoding the Stul restriction endonuclease and isolated DNA encoding cognate and non-cognate methylase; (2) vectors and cells containing the isolated DNA; and (3) methods for producing the Stul restriction endonuclease.
Specified restriction endonucleases have been characterized for the first time by their amino acid and DNA sequences. These sequences and those with at least 90% identity thereto have been used as probes in sequence similarity analyses to identify sequence matches in a sequence database that corresponds to novel restriction endonucleases or isoschizomers. The sequence similarity analyses includes selecting a positive sequence match from any sequence producing an expectation value of less than or equal to e-02.
Methods are provided for rapid amplification of large DNA including genome amplification. The method utilizes nickases and polymerases but does not utilize primers. Amplification of a large DNA can be achieved within about 10 minutes as determined by detectable bands on gels starting with 25 ng DNA in a volume of 50 騜l. An advantage of these methods of amplification for large DNA is that the sequence is proportionally represented. The methods can further provide amplified DNA in a time-efficient manner. The nick- based amplification can be supplemented by a primer-based amplification for increasing the concentration of particular target sequences. The methods can also be used to provide a fingerprint profile of the DNA.
patents
