Showing papers in "Current protocols in molecular biology in 2010"

Computer control of microscopes using µManager.

Abstract: With the advent of digital cameras and motorization of mechanical components, computer control of microscopes has become increasingly important. Software for microscope image acquisition should not only be easy to use, but also enable and encourage novel approaches. The open-source software package µManager aims to fulfill those goals. This unit provides step-by-step protocols describing how to get started working with µManager, as well as some starting points for advanced use of the software.

Galaxy: A Web‐Based Genome Analysis Tool for Experimentalists

Abstract: High-throughput data production has revolutionized molecular biology. However, massive increases in data generation capacity require analysis approaches that are more sophisticated, and often very computationally intensive. Thus, making sense of high-throughput data requires informatics support. Galaxy (http://galaxyproject.org) is a software system that provides this support through a framework that gives experimentalists simple interfaces to powerful tools, while automatically managing the computational details. Galaxy is distributed both as a publicly available Web service, which provides tools for the analysis of genomic, comparative genomic, and functional genomic data, or a downloadable package that can be deployed in individual laboratories. Either way, it allows experimentalists without informatics or programming expertise to perform complex large-scale analysis with just a Web browser.

RNA-Seq: A Method for Comprehensive Transcriptome Analysis

Abstract: A recently developed technique called RNA Sequencing (RNA-Seq) uses massively parallel sequencing to allow transcriptome analyses of genomes at a far higher resolution than is available with Sanger sequencing- and microarray-based methods. In the RNA-Seq method, complementary DNAs (cDNAs) generated from the RNA of interest are directly sequenced using next-generation sequencing technologies. The reads obtained from this can then be aligned to a reference genome in order to construct a whole-genome transcriptome map. RNA-Seq has been used successfully to precisely quantify transcript levels, confirm or revise previously annotated 5' and 3' ends of genes, and map exon/intron boundaries. This unit describes protocols for performing RNA-Seq using the Illumina sequencing platform.

Bisulfite Sequencing of DNA

Abstract: Exact positions of 5-methylcytosine (m(5)C) on a single strand of DNA can be determined by bisulfite genomic sequencing (BGS). Treatment with bisulfite ion preferentially deaminates unmethylated cytosines, which are then converted to uracil upon desulfonation. Amplifying regions of interest from deaminated DNA and sequencing products cloned from amplicons permits determination of methylation at single-nucleotide resolution along single DNA molecules, which is not possible with other methylation analysis techniques. This unit describes a BGS technique suitable for most DNA sources, including formaldehyde-fixed tissue. Considerations for experimental design and common sources of error are discussed.

Single Molecule Sequencing with a HeliScope Genetic Analysis System

Abstract: Helicos™ Single Molecule Sequencing (SMS) provides a unique view of genome biology through direct sequencing of cellular nucleic acids in an unbiased manner, providing both accurate quantitation and sequence information. Sample preparation does not require ligation or PCR amplification, avoiding the GC-content and size biases observed in other technologies. DNA is simply sheared, tailed with poly A, and hybridized to a flow cell surface containing oligo-dT for sequencing-by-synthesis of billions of molecules in parallel. This process also requires far less material than other technologies. Gene expression measurements can be done using 1st-strand cDNA-based methods (RNA- Seq) or using a novel approach that allows direct hybridization and sequencing of cellular RNA for the most direct quantitation possible. A diverse array of applications have been successfully performed including genome sequencing for accurate variant detection, ChIP-Seq using picogram quantities of DNA, copy number variation studies from both fresh tumor tissue and FFPE tissue samples, sequencing of ancient and degraded DNAs, small RNA studies leading to the identification of new classes of RNAs and the direct capture and sequencing of RNA from cell quantities as few as 250 cells. Because most next generation sequencing technologies require amplification and a specific size range of target molecules, DNAs not meeting those criteria cannot be sequenced in a reliable manner. Single-molecule sequencing does not suffer from those limitations as no amplification is necessary and degraded or modified molecules can be used directly as templates. Principles and methods for using the Helicos® Genetic Analysis System will be discussed.

Chromatin interaction analysis using paired-end tag sequencing.

Abstract: Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET) is a technique developed for large-scale, de novo analysis of higher-order chromatin structures Cells are treated with formaldehyde to cross-link chromatin interactions, DNA segments bound by protein factors are enriched by chromatin immunoprecipitation, and interacting DNA fragments are then captured by proximity ligation The Paired-End Tag (PET) strategy is applied to the construction of ChIA-PET libraries, which are sequenced by high-throughput next-generation sequencing technologies Finally, raw PET sequences are subjected to bioinformatics analysis, resulting in a genome-wide map of binding sites and chromatin interactions mediated by the protein factor under study This unit describes ChIA-PET for genome-wide analysis of chromatin interactions in mammalian cells, with the application of Roche/454 and Illumina sequencing technologies

ChIP-Seq: a method for global identification of regulatory elements in the genome.

Abstract: This unit describes ChIP-Seq methodology, which involves chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (Seq), and enables the genome-wide identification of binding sites of transcription factors (TFs) and other DNA-binding proteins. The process is initiated by cross-linking DNA and DNA-bound proteins. Subsequently, chromatin is isolated from nuclei and subjected to sonication. An antibody against a specific TF or DNA-binding protein is then used to immunoprecipitate specific DNA-TF complexes. ChIP DNA is purified, sequencing adapters are ligated, and 30- to 35-nucleotide (nt) sequence reads are generated. The sequence of the DNA fragments is mapped back to the reference genome for determination of the binding sites.

Serine/threonine protein phosphatase assays.

Abstract: Methods for assaying serine/threonine protein phosphatases are discussed. Three commonly used protocols are presented that employ either colorimetric or radiometric assays. These methods can be used to assay a variety of preparations of serine/threonine phosphatases, from crude lysates to purified proteins. Strategies for the application of a particular protocol for a particular purpose are discussed. The assays can be used in either a high-throughput mode where simple comparison of activities can be done, or in specific assays where kinetic data can be derived.

Genome-Wide Location Analysis by Pull Down of In Vivo Biotinylated Transcription Factors

Abstract: Recent development of methods for genome-wide identification of transcription factor binding sites by chromatin immunoprecipitation (ChIP) has led to novel insights into transcriptional regulation and greater understanding of the function of individual transcription factors. ChIP requires highly specific antibody against the transcriptional regulator of interest, and availability of suitable antibodies is a significant impediment to broader application of this approach. This limitation can be circumvented by tagging the transcriptional regulator of interest with a short bio epitope which is specifically biotinylated by the E. coli enzyme BirA. The biotinylated transcription factor can then be selectively pulled down on streptavidin beads under stringent conditions. This unit provides a detailed protocol for genome-wide location analysis of in vivo biotinylated transcription factors by streptavidin pull-down followed by high-throughput sequencing (bioChIP-seq).

Installation of Site‐Specific Methylation into Histones Using Methyl Lysine Analogs

Abstract: Chromatin structure is influenced by post-translational modifications on histones, the principal basic protein component of chromatin. In order to study one of these modifications, lysine methylation, in the context of reconstituted chromatin, this unit describes the installation of analogs of methyl lysine residues into recombinant histones. The modification site is specified by mutating the lysine of interest to cysteine. The mutant histones are expressed and purified, and the cysteine residue alkylated to produce N-methyl aminoethylcysteine, an isosteric analog of methyl lysine. Using different alkylating reagents, it is possible to install analogs of mono-, di-, or trimethyl lysine. While these analogs are not identical to methyl lysine residues, they show similar biochemical properties to their natural counterparts. The ease of synthesis of methyl lysine analog (MLA) histones, especially on a large scale, makes them particularly useful reagents for studying the effects of histone lysine methylation on chromatin structure, biophysics and biochemistry.

Normalizing cDNA libraries.

Abstract: The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses.

Analysis of protein tyrosine phosphatases and substrates.

Abstract: Protein tyrosine phosphorylation is a reversible post-translational modification that is essential for life in eukaryotic cells. The combinatorial action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs) determines the net level of cellular tyrosine phosphorylation. This unit discusses methods to determine the level of protein tyrosine phosphatase activity and methods for discovering novel substrates for protein tyrosine phosphatases.

Visualization of Kinase Activity with FRET‐Based Activity Biosensors

Abstract: Genetically encodable FRET-based kinase activity reporters (KARs) enable real-time monitoring of kinase activity dynamics in living cells with high spatiotemporal resolution. This unit describes a general protocol for utilizing KARs to visualize kinase activity in living mammalian cells with fluorescence microscopy. Curr. Protoc. Mol. Biol. 91:18.15.1-18.15.9. © 2010 by John Wiley & Sons, Inc. Keywords: kinase activity reporter; FRET; live-cell imaging

Methylation-sensitive single-molecule analysis of chromatin structure.

Abstract: Methylation-sensitive single-molecule analysis of chromatin structure is a high-resolution method for studying nucleosome positioning As described in this unit, this method allows for the analysis of the chromatin structure of unmethylated CpG islands or in vitro–remodeled nucleosomes by treatment with the CpG-specific DNA methyltransferase SssI (MSssI), followed by bisulfite sequencing of individual progeny DNA molecules Unlike nuclease-based approaches, this method allows each molecule to be viewed as an individual entity instead of an average population Curr Protoc Mol Biol 89:21171-211716 © 2010 by John Wiley & Sons, Inc Keywords: chromatin remodeling; nucleosome positioning; methylation footprint

Extraction of Proteins from Formalin‐Fixed, Paraffin‐Embedded Tissue Using the Qproteome Extraction Technique and Preparation of Tryptic Peptides for Liquid Chromatography/Mass Spectrometry Analysis

Abstract: This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin-fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the tryptic peptides and number of identified proteins. Since FFPE tissues are generally available in clinical practice, this method can be used to analyze biomarkers in different pathological situations (e.g., healthy vs. diseased). The method can also be used for protein extraction from fresh-frozen tissue. Curr. Protoc. Mol. Biol. 90:10.27.1-10.27.12. © 2010 by John Wiley & Sons, Inc. Keywords: formalin-fixed tissue; mass spectrometry; proteomics; protein extraction

Analysis of serine-threonine kinase specificity using arrayed positional scanning peptide libraries.

Abstract: Protein kinases vary substantially in their consensus phosphorylation motifs, the residues that are either preferred or deselected by the kinase at specific positions surrounding the phosphorylation site. The protocol described here is used to rapidly determine phosphorylation motifs for serine-threonine kinases. The procedure involves screening an arrayed combinatorial peptide library consisting of 198 biotinylated substrates. Peptides are phosphorylated by the kinase of interest in the presence of radiolabeled ATP and then captured on streptavidin membrane. The membrane is subsequently washed, dried and exposed to a phosphor screen to visualize and quantify incorporation of radiolabel into the peptides. The phosphorylation motif is thereby derived from the relative extent of phosphorylation of each peptide in the array.

Drug-induced permeabilization of S. cerevisiae.

Abstract: Budding yeast are not permeable to many drugs This unit provides a protocol in which polygodial is used to permeabilize the cell membrane, thereby allowing budding yeast cells to be treated with drugs that otherwise would be ineffective Curr Protoc Mol Biol 92:132B1-132B4 © 2010 by John Wiley & Sons, Inc Keywords: permeabilization; Alpha-amantin; DRB; S cerevisiae

Fluorescent Peptide Assays for Protein Kinases

Abstract: Protein kinases are enzymes that regulate many cellular events in eukaryotic cells, such as cell cycle progression, transcription, metabolism and apoptosis. Protein kinases each have a conserved ATP binding site, as well as one or more substrate-binding site(s) that exhibit recognition features for a protein substrate. By thus bringing ATP and a substrate into close proximity, each protein kinase can modify its substrate by transferring the γ-phosphate of the ATP molecule to a serine, threonine or tyrosine residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated, dependent on the phosphorylated versus dephosphorylated forms of the substrate.

Isolation of proteins by heat-induced extraction from formalin-fixed, paraffin-embedded tissue and preparation of tryptic peptides for mass spectrometric analysis.

Abstract: This unit describes a method of isolating of proteins from formalin-fixed and paraffin-embedded (FFPE) tissue for mass spectrometry analysis. Heat-induced antigen retrieval is the basis of the protein extraction strategy presented in this protocol. This protocol may be used to identify nuclear, cytosolic, and membrane proteins from FFPE tissues extracted from tissue blocks or slides. Curr. Protoc. Mol. Biol. 90:10.26.1-10.26.7. © 2010 by John Wiley & Sons, Inc. Keywords: peptide extraction; formalin-fixed and paraffin-embedded tissue; mass spectrometry; protein identification

Mapping Networks of Protein-Mediated Physical Interactions Between Chromatin Elements

Abstract: Understanding of transcriptional regulation has advanced in recent years in part due to development of technologies which allow determination of physical proximities between interacting chromatin regions at a resolution beyond that offered by conventional microscopy techniques. However, these methods do not specifically identify the protein component(s) that might mediate such interactions. This unit provides a detailed protocol for Combined 3C-ChIP-Cloning (6C) technology, which combines multiple techniques to simultaneously identify physical proximities between chromatin elements as well as the proteins that mediate these interactions. The unit further explores how the 6C assay can be combined with other techniques for a complete, cell-type-specific mapping of all inter- and intrachromosomal interactions mediated by specific proteins. Thus, the 6C assay provides a useful tool to address the role of specific proteins in nuclear organization and to advance our understanding about the relation of chromatin higher-order organization and transcriptional regulation.