Showing papers on "Nucleic acid methods published in 2007"

The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?

Abstract: Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.

Methods and means for nucleic acid sequencing

Abstract: The present invention provides a nucleic acid sequencing method. The method comprises enriching a nucleic acid sample for target nucleic acids, where the nucleic acid sample is enriched through at least a first round of hybridization selection and amplification, and a second round of hybridization selection and amplification. The enriched nucleic acids are in a form convenient for sequencing with the Cantaloupe sequencing technology, which employs shotgun sequencing by hybridization (SBH) of immobilized rolling circle amplicons.

Methods for generating amplified nucleic acid arrays

Abstract: The present invention relates to methods for generating an array of amplified nucleic acid sequences. The methods can utilize amplicons that form nucleic acid balls that can be arrayed on a solid support. The invention additionally provides methods for obtaining targeted nucleic acid sequences.

Nucleic acid and amino acid sequences relating to Staphylococcus epidermidis for diagnostics and therapeutics

Abstract: The invention provides isolated polypeptide and nucleic acid sequences derived from Staphylococcus epidermidis that are useful in diagnosis and therapy of pathological conditions; antibodies against the polypeptides; and methods for the production of the polypeptides. The invention also provides methods for the detection, prevention and treatment of pathological conditions resulting from bacterial infection.

μParaflo™ Biochip for Nucleic Acid and Protein Analysis

Abstract: We describe in this chapter the use of oligonucleotide or peptide microarrays (arrays) based on microfluidic chips. Specifically, three major applications are presented: (1) microRNA/small RNA detection using a microRNA detection chip, (2) protein binding and function analysis using epitope, kinase substrate, or phosphopeptide chips, and (3) protein-binding analysis using oligonucleotide chips. These diverse categories of customizable arrays are based on the same biochip platform featuring a significant amount of flexibility in the sequence design to suit a wide range of research needs. The protocols of the array applications play a critical role in obtaining high quality and reliable results. Given the comprehensive and complex nature of the array experiments, the details presented in this chapter is intended merely as a useful information source of reference or a starting point for many researchers who are interested in genome- or proteome-scale studies of proteins and nucleic acids and their interactions.

Methods and compositions for the amplification, detection and quantification of nucleic acid from a sample

Abstract: Methods and kits for the amplification, detection and quantification of a nucleic acid from a sample are disclosed The methods of the invention may be used in a wide range of applications, including, but not limited to, the detection and quantification of fetal nucleic acid from maternal plasma, the detection and quantification of circulating nucleic acids from neoplasms (malignant or non-malignant), accurate pooling analysis for low frequency alleles, or any other application requinng sensitive quantitative analysis of nucleic acids

Use of Single-Stranded Nucleic Acid Binding Proteins in Sequencing

Abstract: The invention provides methods for stabilizing a nucleic acid sequencing reaction. Generally, methods of the invention include exposing a target nucleic acid to a single-stranded nucleic acid binding protein and performing a sequencing reaction.

Methods and compositions for the extraction and amplification of nucleic acid from a sample

Abstract: Provided herein are methods, compositions and kits to extract and relatively enrich by physical separation or amplification short base pair nucleic acid in the presence of a high background of genomic material (e.g., host or maternal nucleic acids).

Insights from Crystallographic Studies into the Structural and Pairing Properties of Nucleic Acid Analogs and Chemically Modified DNA and RNA Oligonucleotides

Abstract: Chemically modified nucleic acids function as model systems for native DNA and RNA; as chemical probes in diagnostics or the analysis of protein-nucleic acid interactions and in high-throughput genomics and drug target validation; as potential antigene-, antisense-, or RNAi-based drugs; and as tools for structure determination (i.e., crystallographic phasing), just to name a few. Biophysical and structural investigations of chemically modified DNAs and RNAs, particularly of nucleic acid analogs with more significant alterations to the well-known base-sugar-phosphate framework (i.e., peptide or hexopyranose nucleic acids), can also provide insights into the properties of the natural nucleic acids that are beyond the reach of studies focusing on DNA or RNA alone. In this review we summarize results from crystallographic analyses of chemically modified DNAs and RNAs that are primarily of interest in the context of the discovery and development of oligonucleotide-based therapeutics. In addition, we re-examine recent structural data on nucleic acid analogs that are investigated as part of a systematic effort to rationalize nature's choice of pentose in the genetic system.

Liposomal Formulations for Nucleic Acid Delivery

Abstract: 9.1 Liposomes for the Delivery of Nucleic Acid Drugs.............................................................237 9.2 Liposome Constituents .........................................................................................................239 9.2.1 Cationic Lipids .........................................................................................................239 9.2.2 The Role of Helper Lipids in Promoting Intracellular Delivery ..............................240 9.2.3 PEG–Lipids...............................................................................................................241 9.2.4 Active Targeting........................................................................................................242 9.3 Methods of Encapsulating Nucleic Acids ............................................................................242 9.3.1 Passive Nucleic Acid Encapsulation.........................................................................243 9.3.2 The Ethanol Drop (SALP) Method of Nucleic Acid Encapsulation........................247 9.3.3 Encapsulation of Nucleic Acid in Ethanol-Destabilized Liposomes .......................247 9.3.4 The Reverse-Phase Evaporation Method of Nucleic Acid Encapsulation ...............248 9.3.5 The Spontaneous Vesicle Formation by Ethanol Dilution (SNALP) Method of Nucleic Acid Encapsulation .....................................................................................249 9.4 Analytical Methods...............................................................................................................251 9.4.1 Measuring Particle Size ............................................................................................251 9.4.2 Zeta Potential ............................................................................................................253 9.4.3 Encapsulation............................................................................................................253 9.5 Pharmacology of Liposomal NA..........................................................................................254 9.5.1 Pharmacokinetics and Biodistribution of Liposomal NA Following Systemic Administration ..........................................................................................................254 9.5.2 Toxicity of Liposomal NA Formulations .................................................................256 9.5.3 Immune Stimulation .................................................................................................258 9.5.4 Immunogenicity........................................................................................................259 9.5.5 The Efficacy of Liposomally Formulated NA Drugs...............................................260 References ......................................................................................................................................262

Histidine-lysine peptides as carriers of nucleic acids.

Abstract: With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

Nucleobase Modifications in Peptide Nucleic Acids

Abstract: Peptide nucleic acid (PNA) is an oligonucleotide mimic originally designed upon a repeating N-(2-aminoethyl)glycine polyamide backbone to which nucleobase heterocycles are attached through a methylene carbonyl linkage to the alpha-amino group These molecules possess remarkable hybridization properties with DNA or RNA forming complexes with high stability and with excellent sequence discrimination despite the substantial structural divergence from natural nucleic acids Since the disclosure of PNA, a vibrant research community with interest in the chemistry and applications of polyamide-based nucleic acid analogs has developed This has led to the synthesis and evaluation of a wide variety of modified polyamide nucleic acids The focus of this report is a comprehensive review of nucleobase modifications in aminoethylglycine (aeg) PNA with reference, where appropriate, to the same modification in DNA or RNA

Electronic sensing for nucleic acid sequencing

Abstract: Methods for sequencing nucleic acids are presented. Sequencing is accomplished through the detection of a redox active species that is indicative of nucleotide incorporation. In embodiments of the invention, an electrochemical signal indicative of nucleotide incorporation is amplified through cycling before it is detected. Arrays are provided that are capable of massively parallel nucleic acid sequence determination.

Purification and Concentration of DNA from Aqueous Solutions

Abstract: This unit presents basic procedures for manipulating solutions of single- or double-stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated. The Basic Protocol, using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether, respectively. An alternative to these methods is nucleic acid purification using glass beads, and this technique is also presented. These protocols may also be used for purifying RNA. The final two alternate protocols are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low-molecular-weight oligonucleotides and triphosphates.

Vaccines and immunotherapeutics using codon-optimized IL-15 and methods for using the same

Abstract: Nucleic acid molecules that encode IL-15 or fragments thereof, which express protein at a higher level than nucleic acid molecules with native coding sequences for IL-15 are disclosed. Nucleic acid molecules with additional modifications such as the absence of coding sequences for IL-15 signal sequences and/or the absence of IL-15 untranslated sequences and/or inclusion of non-IL-15 signal sequences are also disclosed. Vectors, including plasmids and viral vectors, comprising such nucleic acid molecules; and to host cells comprising such nucleic acid molecules are disclosed as well as methods of using such nucleic acid molecules alone or in combination with nucleic acid sequences encoding immunogens which are part of the nucleic acid molecules and/or part of a different nucleic acid molecule. Recombinant vaccines and live attenuated pathogens encoding fusion proteins, and methods of using the same, are disclosed.

Synthetic nucleic acid molecule compositions and methods of preparation

Abstract: A method to prepare synthetic nucleic acid molecules having reduced inappropriate or unintended transcriptional characteristics when expressed in a particular host cell.

Reagents for labelling nucleic acids and uses thereof

Abstract: The present invention relates to labelling kits containing novel non-natural nucleotide monomers and to methods of making and using such compounds. The invention further relates to a method of detecting the presence of a nucleic acid, e.g., RNA, of interest in a sample, the method having the following steps: providing the sample; ligating a nucleic acid of interest with a labelling reagent according to the instant invention; providing a nucleic acid array having probes directed to the nucleic acid of interest; hybridizing the labelled nucleic acid fragments to said nucleic acid array; and determining the extent of hybridization to said probes to determine the presence of the nucleic acid of interest.

Assays for measuring nucleic acid binding proteins and enzyme activities

Abstract: Processes for measuring DNA or RNA binding proteins, specific nucleic acids, as well as enzyme activities using labeled nucleic acids of labeled protein/peptide molecules are provided.

Nucleic acid library design and assembly

Abstract: Aspects of the invention relate to the design and synthesis of nucleic acid libraries. Certain embodiments relate to the design and synthesis of nucleic acid libraries that express polypeptides. In some embodiments, the invention provides methods for analyzing polypeptide sequences and identifying those that may confer undesirable properties in vivo (e.g., poor solubility, high immunogenicity, low stability, etc.). In some embodiments, the invention provides methods for synthesizing a library of nucleic acids having predetermined sequences (e.g., a library of nucleic acids that encode polypeptides having related sequences with predetermined sequence variations).

Compositions and methods for oligonucleotide formulations

Abstract: The present invention relates generally to imrnunostimulatory nucleic acids, compositions thereof and methods of using the imrnunostimulatory nucleic acids. In particular the invention relates to palindrome-containing imrnunostimulatory nucleic acids and the use of these nucleic acids in treating disease.

Combined extension and ligation for nucleic acid assembly

Abstract: Certain aspects of the present invention provide methods for assembling nucleic acid molecules. Some embodiments involve analyzing nucleic acid sequences and determining appropriate assembly strategies based on the presence or absence of sequence features that are known or predicted to interfere with extension-based and/or ligation-based assembly techniques. Aspects of the invention also provide kits, compositions, devices, and systems for assembling synthetic nucleic acids using polymerase-based techniques, ligase-based techniques, or combinations thereof.

Methods of enriching for and identifying polymorphisms

Abstract: The invention encompasses methods for enriching for and identifying a polymorphism within a nucleic acid sample either by separating a subset of a nucleic acid sample or by selectively replicating a subset of a nucleic acid sample such that the polymorphism is contained within a nucleic acid population with reduced complexity, and then identifying the polymorphism within the enriched nucleic acid sample. Methods also are disclosed for enriching for and identifying a polymorphism by contacting a nucleic acid sample that includes a subset of nucleic acid molecules having a sequence that binds to a sequence-specific binding activity with a molecule having a sequence-specific binding activity under conditions which permit specific binding, such that the subset of nucleic acid molecules bound to the activity is enriched for nucleic acid molecules having the sequence recognized by the sequence-specific binding activity, and detecting a polymorphism with respect to a reference sequence in the subset of nucleic acid molecules.

Anti-chemokine (ccl2, ccl3, ccl5, cxcl11, cxcl12) single-domain antibodies

Abstract: The present invention relates to amino acid sequences that are directed against chemokines, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences. The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides; to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host ceils and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes.

Detectable nucleic acid tag

Abstract: Provided herein are nucleic acid tags that are linked to, or capable of linking to, a protein of interest In particular, the nucleic acid tags are oligonucleotides comprising a reporter function and a protein tagging function Also provided herein, are nucleic acid tag compositions, kits and methods of use thereof

Nucleic acid purification method

Abstract: Disclosed is a process for separating and/or purifying a nucleic acid by elution of the nucleic acid from anion exchange resins under conditions of high salt concentration and the presence in the eluant of an additive comprising guanidine, or a guanidine-like derivative. The process allows high recovery of nucleic acids from anion exchange resins without impairing the nucleic acid stability as compared with conventional ion exchange chromatographic procedures.

Methods for nucleic acid mapping and identification of fine-structural-variations in nucleic acids and utilities

Abstract: The present invention relates generally to methods for high-throughput analysis of fine structural variations in nucleic acids. In particular, the present invention relates to novel strategies, vector and vector components to produce pairs of linked-nucleic acid tags, wherein constituent members of a linked nucleic acid tag-pair are of a user defined separation distance, and/or are markers of nucleic acid positions that demarcate adjacent cleavage sites for one or more different restriction endonucleases along the length of a target nucleic acid molecule.

A plastic microchip for nucleic acid purification

Abstract: A microchip for purifying nucleic acids from bacterial pathogens was designed and fabricated in plastic. The fabricated plastic microchips were tested for their ability to purify nucleic acids from the bacteria Listeria monocytogenes (L. monocytogenes), Escherichia coli (E. coli), and Salmonella typhimurium (S. typhimurium). These chips were constructed using rapid and low-cost plastic fabrication techniques including hot embossing and plastic casting. Silicon molds fabricated by photolithography and dry etching were used for chip prototyping. Zeonor plastic (poly (cycloolefin) resin) and epoxy microchips were fabricated using hot embossing and plastic casting, respectively. A low temperature sputtering technique was used to coat a layer of silicon dioxide onto the channel region for nucleic acid binding in chaotropic salt solutions. The purification channels contain an array of features to increase the surface area for DNA binding and purification. DNA was quantified with PicoGreen fluorescent dye and the quality of the material as a substrate for polymerase chain reaction (PCR) was tested using target specific primers. DNA could be recovered from the microchip and detected using PCR from a minimum of 106 of L. monocytogenes, E. coli, and S. typhimurium cells, respectively. With the simplicity of the plastic chip’s fabrication and DNA purification, our microchip makes it ideal for a miniaturized DNA testing system.

T-structure invasive cleavage assays, consistent nucleic acid dispensing, and low level target nucleic acid detection

Abstract: The present invention relates to systems, methods and kits for low-level detection of nucleic acids, detecting at least two different viral sequences in a single reaction vessel, and increasing the dynamic range of detection of a viral target nucleic acid in a sample. The present invention also relates to T-structure invasive cleavage assays, as well as T-structure related target dependent non-target amplification methods and compositions. The present invention further relates to methods, compositions, devices and systems for consistent nucleic acid dispensing onto surfaces.