bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and Application of Peptide Dendrimers As Protein Mimetics
Authors: Tam James P. , Spetzler Jane C.
Published: June 2004

The multiple antigenic peptide (MAP) is a novel approach to preparing peptide immunogens. The MAP consists of an inner core matrix built up of a large layer of Lys residues and a surface of peptide chains attached to the core matrix. Because of its dendrimeric structure, MAP can be very useful as a template for assembling potential peptide surfaces. Two methods for preparing MAP systems are described in this unit. The direct approach for preparing MAP systems is presented in the first two protocols, including the procedure for b‐butyloxycarbonyl (Boc) chemistry and the procedure for 9 ‐fluorenylmethyloxycarbonyl (Fmoc) chemistry. An indirect approach for preparing MAP systems, in which peptide and core matrix are synthesized separately and conjugated by several ligation methods, is then described. The cMAP approach is also executed using either the direct or indirect approach, but requires an additional cyclization step to constrain the peptides after synthesis. The synthesis of cMAP is described, and the preparation of cyclic peptides is illustrated. A support protocol describes the ninhydrin assay to assess the completeness of the coupling reaction. In most cases, MAP systems can be used directly after simple dialysis or desalting. Some immunological studies, however, require purified MAPs. Additional support protocols describe MAP system purification by dialysis and high‐performance gel‐filtration chromatography.
Synthesis of Multiple Peptides on Plastic Pins
Authors: Rodda Stuart J.
Published: June 2004

Scanning protein sequences by bioassay for smaller bioactive peptide sequences requires a source of many peptides homologous with the parent protein sequence. This unit deals with one of the synthetic methods for making such sets of peptides. The strategy of the multiple peptide approach to biological scanning is discussed, along with the synthetic protocols, and the handling of peptides after synthesis, i.e., cleavage, preliminary purification, storage, and analysis. It is specific for the multipin technique using equipment obtained from Chiron Technologies, although some of the approaches are applicable to other multiple synthesis techniques. Procedures for multipin equipment obtained from other suppliers may differ from the procedures described here, and the manufacturer's literature should be consulted. This unit also includes protocols for preparing Fmoc‐amino acid solutions and for acetylating or biotinylating synthesized peptides.
Deprotection of Oligonucleotides and Purification Using Denaturing PAGE
Authors: Ellington Andrew , Chory Joanne
Published: June 2004

The advantages of purification by denaturing polyacrylamide gel electrophoresis (PAGE) are speed, simplicity, and high resolution. Although yields tend to be low (<50% of applied sample), the amount of material recovered is usually far in excess of that required for most molecular biology applications (e.g., cloning and sequencing). Denaturing PAGE can resolve oligonucleotides from 2 to 300 bases, depending on the percentage of polyacrylamide used. The method presented here is thus useful not only for isolating chemically synthesized deoxyribonucleotides but also small RNAs or other single‐stranded polynucleotides.
Liposome‐Mediated Transfection
Authors: Whitt Michael , Buonocore Linda , Rose John K.
Published: June 2004

Using liposomes to deliver DNA into different eukaryotic cell types results in higher efficiency and greater reproducibility than other transfection methods. In this unit, the describes a transient expression system while an involves stable transformation and expression of DNA integrated into the genome of the transfected cell. In both protocols, plasmid DNA derived from either crude (miniprep) or purified (through CsCl) preparations is mixed with a liposome suspension comprised of cationic lipids and applied to monolayer cell cultures.
Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Authors: Kramer Martha F. , Coen Donald M.
Published: June 2004

This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR) and for optimizing this reaction for the sequence and primer set of interest. Important variables that can influence the outcome of PCR include the MgCl2 concentration and the cycling temperatures. Additives that promote polymerase stability and processivity or increase hybridization stringency, and strategies that reduce nonspecific primer‐template interactions, especially prior to the critical first cycle, can greatly improve sensitivity, specificity, and yield. This protocol is designed to optimize the reaction components and conditions in one or two stages. The first stage determines the optimal MgCl2 concentration and screens several enhancing additives. To further improve specificity, sensitivity and yield, the second stage compares methods for optimizing initial specific hybridization to prevent polymerization of misprimed sequences prior to thermal cycling. For initial inhibition of polymerase activity, temperature (i.e., cooling reagents), physical separation (“hot start” method), and reversible antibody binding are compared.
Detection of Cytokine mRNA Expression by PCR
Authors: James Stephen P.
Published: June 2004

Assessment of cytokine expression during an immune response is a critical requirement for studies of basic immune mechanisms and the pathogenesis of disease. As an alternative to measuring cytokine protein levels, analysis of cytokine mRNA may be appropriate, because production of many cytokines is primarily transcriptionally regulated. Therefore, the level of cytokine mRNA present in a sample may serve as a good estimate of the level of cytokine protein present. This unit outlines the amplification of IL‐2 mRNA from clinical biopsy specimens by the polymerase chain reaction (PCR), but the approach can be readily modified to study any cytokine RNA of interest in biopsies or small samples. In the , cDNA is synthesized from the sample RNA by reverse transcriptase; to control for experimental variation in both the reverse transcription and PCR amplification steps, an internal control RNA is included in the reaction. Together, the reverse‐transcribed cDNAs are amplified by PCR.
Evaluating Human T Cell Receptor Gene Expression by PCR
Authors: Gussoni Emanuela , Panzara Michael A. , Steinman Lawrence
Published: June 2004

This unit describes the use of PCR to characterize and quantify rearranged transcripts from specific T cell receptor variable gene families in human tissue and peripheral blood lymphocytes. The strategy outlined in this unit has been extensively used on different sources of human tissue including brain, spinal cord, and skeletal muscle. A protocol is provided to clone and sequence PCR‐amplified cDNA transcripts to study the junctional diversity of the expressed genes. A support protocol describes a method for reverse transcribing total RNA to make the cDNA required by the other protocols.
Analysis of Inositol Phospholipid Turnover During Lymphocyte Activation
Authors: Imboden John B. , Shoback Dolores M. , Inokuchi Sharon
Published: June 2004

Receptor‐mediated activation of phospholipase C (PLC) leads to the hydrolysis of membrane inositol phospholipids, generating diacylglycerol (DAG) and water‐soluble inositol phosphates. This signaling mechanism is used by antigen receptors on T and B cells that have been implicated as mediators of receptor‐induced influx of extracellular Ca2+. This unit provides protocols that describe the resolution of InsP by Dowex anion‐exchange chromatography. This technique provides a reliable means of separating inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, but does not resolve isomers of these. An describes the separation of inositol phosphates by anion‐exchange HPLC. A protocol for resolution of inositol phospholipids by thin‐layer chromatography (TLC) is also provided.
Antiphosphotyrosine Blotting
Authors: Siegel Jeffrey N.
Published: June 2004

Antiphosphotyrosine blotting is a technique for detecting tyrosine‐phosphorylated substrates by the use of antibodies that recognize these residues on a wide variety of proteins. This unit describes conditions for cell lysis and immunoprecipitation of proteins with an antiphosphotyrosine antibody, followed by electrophoretic separation, immunoblotting, and color detection of the blotted proteins. This combination of steps provides particularly sensitive conditions for the detection of tyrosine‐phosphorylated substrates, but also gives good results for any protein transferred to nitrocellulose, including whole‐cell lysates or proteins immunoprecipitated with another antibody. Although the alkaline phosphatase color‐detection system has the advantage of providing superior resolution and higher sensitivity without the use of any radioactivity, the 125I‐labeled Staphylococcus protein A detection system is described for use in conjunction with the blotting protocol.
Using Protein Kinase and Protein Phosphatase Inhibitors to Dissect Protein Phosphorylation Pathways
Authors: Karaszkiewicz James W. , Henrich Curtis J.
Published: June 2004

It is often desirable to attempt to measure and to modulate the activity of the protein kinases and phosphatases responsible for a given phenotypic change. The protocols in this unit describe how to assay a variety of protein kinase activities in complex mixtures, such as whole cell lysates, in order to screen compounds as potential inhibitors of these activities. The effects of known or potential inhibitors can be evaluated by monitoring phenotypic changes in intact cells following treatment with the inhibitor. The activities of the major serine/threonine‐specific protein phosphatases in most mammalian cells can be distinguished using a selective inhibitor, okadaic acid. Support protocols describe preparation of cell extracts needed for the kinase assays, and of32P‐labeled phosphorylase used in the inhibition assays.
TRAF‐Mediated TNFR‐Family Signaling
Authors: Arron Joseph R. , Walsh Matthew C. , Choi Yongwon
Published: June 2004

The tumor necrosis factor (TNF) superfamily consists of a wide variety of proteins, some cell‐bound and others secreted, that regulate many cellular processes. In particular, TNF‐family proteins regulate the life and death not only of tumor cells, but of activated cells of the immune system. This overview discusses the six known mammalian TRAF proteins, of which TRAF1, 2, 3, 5, and 6 have been shown to interact directly or indirectly with members of the TNFR superfamily. Structural features of TRAF proteins are described along with a discussion of TRAF‐interacting proteins and the signaling pathways activated by TRAF proteins. Finally, a discussion is provided on the phenotypes of TRAF‐knockout mice.
Alternative Pathway Evaluation
Authors: Giclas Patricia C.
Published: June 2004

The alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.
Assays for Membrane Complement Receptors
Authors: O'Rear Lynda D. , Ross Gordon D.
Published: June 2004

Leukocytes express several types of receptors for activated products of the complement (C) system. This unit describes assays for a subset of these receptors: those specific for fixed fragments of C3 (CR1, CR2, CR3, and CR4). Although the assays have been designed for tests of human C receptors, they can often be carried out with rodent (e.g., mouse, rat, or guinea pig) cells by substitution of rodent‐specific reagents. Two basic protocols are presented for rosette assay of CR1, CR2, CR3, and CR4 on adherent and nonadherent cells. Isolated leukocytes may be tested for rosette formation in suspension or adherent to a surface. Nonadherent cells such as lymphocytes, NK cells, or cells from an undifferentiated leukocyte cell line must be tested in suspension. The unit also contains three support protocols for formation and quantitation of sheep erythrocyte‐C3 complexes for use in the two basic protocols.
Assays for the Mannan‐Binding Lectin Pathway
Authors: Gadjeva Mihaela , Thiel Steffen , Jensenius Jens C.
Published: June 2004

This unit contains protocols that can be used to measure mannan‐binding lectin (MBL) levels and MBL pathway activity in human plasma or serum. Using a modification of the conventional ELISA, the detection reagent used in the methods described (e.g., an antibody or streptavidin) is labeled with Eu3+ instead of an enzyme. The sensitizing reagent used in these assays employ the use of anti‐MBL antibody or mannan. The unit also includes an MBL/MASP complex activity assay to evaluate MBL activity in serum or plasma samples, which allows for the measurement of the specific activity of the MBL pathway.
Measurement of Bacterial Ingestion and Killing by Macrophages
Authors: Campbell Priscilla A. , Canono Beth P. , Drevets Douglas A.
Published: June 2004

This unit presents fairly simple assays for measuring the binding of bacteria to macrophages, internalization of bacteria (also called ingestion or phagocytosis), and bacterial killing by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this: a washing procedure and a more stringent method in which cells are sedimented through sucrose. In addition, it is important to distinguish those bacteria truly ingested by a macrophage from those that are bound to, but not internalized by, the cell. A simple but effective way to do this is described in an alternate protocol. The unit also presents two ways to measure the ability of a macrophage to kill bacteria it has internalized. The first is a straightforward assay in which bacterial colonies are enumerated before and after a killing period; a subsequent colony count will indicate whether the bacteria grew within or were killed by the macrophage. The second protocol describes a way to measure bacterial viability based on bacterial metabolism, in which the ability of bacterial dehydrogenases to mediate the reduction of a tetrazolium salt to purple formazan is monitored by measuring absorbance spectrophotometrically.
Animal Models of IgA Nephropathy
Authors: Emancipator Steven N.
Published: June 2004

IgA nephropathy (IgAN) is a form of immune complex glomerulonephritis that occurs spontaneously in humans. This unit describes the induction of active disease in inbred mice, utilizing inert proteins or a common viral pathogen as the inciting antigen. An alternate protocol is offered for the induction of disease in rats by noninfectious protein antigens. Support protocols are presented for the evaluation of the extent of disease, for preparation of infectious and inactivated suspensions of viral stock, and for quantification of the virus.
Autoimmune Ovarian Disease Induced by Immunization with Zona Pellucida (ZP3) Peptide
Authors: Tung Kenneth , Agersborg Sally , Bagavant Harini , Garza Kristine , Wei Kevin
Published: June 2004

Autoimmune ovarian disease is a known cause of human premature ovarian failure. An experimental murine model can be created by immunization with a peptide of ZP3, an ovary‐specific glycoprotein. Mice injected with the ZP3 peptide develop histological evidence of ovarian inflammation (oophoritis), as well as antibody to the zona pellucida and T cell responses to the peptide. This unit describes the immunologic properties of the ZP3 peptides, the method of active autoimmune oophoritis induction, and the evaluation and semiquantitation of ovarian pathology. Surgical manipulations of the ovaries have been instrumental in the novel application of this autoimmune model; thus the most useful operations are also detailed. Surgical procedures are included for adult oophrectomy, neonatal ovariectomy, and implantation of ovarian grafts into mice without endogenous ovaries.
Affinity‐Based Biosensors for Biomolecular Interaction Analysis
Authors: Gunnarsson Kerstin
Published: June 2004

This overview presents an introduction to affinity‐based biosensors, most notably the BIACORE. These biosensors detect molecular interactions by immobilizing one of the interactants (the ligand or target molecule) on the surface of a sensor chip. A solution containing the other interactant is then applied to the chip. Binding of analyte to the surface results in a response proportional to the mass bound. A discussion of sensor chips and practical applications is also provided.
Animal Models of Mycobacteria Infection
Authors: Orme Ian M. , Roberts Alan D.
Published: June 2004

This unit describes the infection of mice and guinea pigs with mycobacteria via the aerogenic route, or of mice via the intravenous route, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other opportunistic Mycobacterial species.
Helicobacter Animal Models
Authors: Nedrud John G. , Blanchard Thomas G.
Published: June 2004

In this unit, protocols for growing Helicobacter organisms on plates or in liquid cultures are presented, followed by protocols for infecting mice with Helicobacter felis and H. pylori and for infecting ferrets with H. mustelae. Also, a procedure is described for adapting an H. pylori isolate to growth in mice. Support protocols describe methods for quantifying numbers of Helicobacter organisms, and how to create a growth curve for Helicobacter cultures. One important technique in investigating Helicobacter infection is assaying the disease processes that occur in the stomach, and a protocol is provided for preparing tissue sections for this purpose. It is also important to confirm that organisms recovered from tissue samples are, in fact, Helicobacter species, and a support protocol describes morphological and biochemical tests for this purpose. Helicobacter bacteria produce large amounts of the enzyme urease, and a support protocol describes how to perform a rapid urease test on animal‐tissue biopsies. Assays of Helicobacter‐specific immune responses require appropriate antigens, and preparation of both Helicobacter lysates and outer‐membrane proteins are detailed for use in these assays.
Influenza Virus
Authors: Cottey Robert , Rowe Cheryl A. , Bender Bradley S.
Published: June 2004

This unit contains several methods for infecting mice with influenza virus. It also includes protocols needed to propagate influenza virus in hen eggs, quantitate virus titers (in tissue culture medium and in influenza‐infected mouse serum), and adopt human isolates of influenza for growth in mice. Methods for measuring the 50% mouse lethal dose are also included. Finally, protocols for generating anti‐influenza cytotoxic T lymphocytes (CTL) from splenocyte precursors and harvesting pulmonary CTL following respiratory tract challenge of mice with influenza virus are provided.
Mouse 4T1 Breast Tumor Model
Authors: Pulaski Beth A. , Ostrand‐Rosenberg Suzanne
Published: June 2004

The 4T1 mammary carcinoma is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone The 4T1 tumor has several characteristics that make it a suitable experimental animal model for human mammary cancer. First, tumor cells are easily transplanted into the mammary gland so that the primary tumor grows in the anatomically correct site, as described in this unit. Second, as in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor. Also, the progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to that of human mammary cancer. In this unit, a protocol describes surgical removal of the primary tumor, so that metastatic disease can be studied in an animal setting comparable to the clinical situation where the primary tumor is surgically removed, and metastatic foci remain intact. Another advantage of 4T1 is its resistance to 6‐thioguanine. This property enables precise quantitation of metastatic cells, even when they are disseminated and at sub‐microscopic levels in distant organs, as described here.
Nomenclature of the Human T Cell Receptor Genes
Authors: Lefranc Marie‐Paule
Published: June 2004

The human T cell receptors (TcR) α‐β and γ‐δ are the products of four sets of genes on two chromosomes: T cell receptors α (TRA) and δ (TRD) on chromosome 14 at 14q11.2, T cell receptor β (TRB) on chromosome 7 at 7q35, and T cell receptor γ (TRG) on chromosome 7 at 7p15‐p14. This appendix presents tabulated lists of the human TcR α, β, γ, and δ genes named in accordance with the International ImMunoGeneTics database and approved by the Human Genome Organization Nomenclature Committee in 1999. Two additional tables list corresponding nomenclatures for these genes.
Nomenclature and Serology of HLA Class I and Class II Alleles
Authors: McCluskey James , Kanaan Carmel , Diviney Mary
Published: June 2004

This overview presents nomenclature and serology information on human leucocyte antigens, or HLA molecules, which are encoded by a cluster of genes linked on the short arm of chromosome 6. This region is known as the major histocompatibility coclass II molecules based upon their structure, tissue distribution, and source of peptide antigen, as well as upon their interactions with T cell subsets.
Monitoring Cell Growth
Authors: Strober Warren
Published: June 2004

This appendix provides two protocols for monitoring cell growth. Counting cells using a hemacytometer is tedious but it allows one to effectively distinguish live cells from dead cells (using Trypan Blue exclusion). In addition, this procedure is less subject to errors due to cell clumping or heterogeneity of cell size. The use of an electronic cell counter is quicker and easier than counting cells using a hemacytometer. However, an electronic cell counter as currently constructed does not distinguish live from dead cells in a reliable fashion and is subject to error due to the presence of cell clumps. Overall, the electronic cell counter is best reserved for repetitive and rapid counting of fresh peripheral blood cells and should be used with caution when counting cell populations derived from tissues.
Labeling Cells in Microtiter Plates for Determination of [3H]Thymidine Uptake
Authors: Shevach Ethan M.
Published: June 2004

A number of protocols in Current Protocols in Immunology use as their end‐point the determination of cell proliferation by determining the incorporation of [3H]thymidine into cellular DNA. This appendix presents a protocol in which the radioactive label is added during the last 4 to 24 hr of the culture. A semiautomated cell harvesting apparatus is then used to lyse the cells with water and precipitate the labeled DNA on glass fiber filters. The filter pads are then dried and counted by standard liquid scintillation counting techniques in a scintillation counter.
Cryopreservation of Cells
Authors: Yokoyama Wayne M.
Published: June 2004

Laboratories interested in working with in vitro cultured cells must be able to freeze and thaw cell lines. Such capabilities are necessary to maintain the constancy of the cells since prolonged culture may result in phenotypic and genotypic changes. In addition, cell freezing minimizes problems with contamination as well as the overall expense of maintaining cell lines for long periods. Finally, freezing of cells provides a method of making cell lines available to other laboratories since frozen cells are readily shipped to distant points. This appendix describes how cell lines can be frozen for long‐term storage and thawed for growth in a tissue culture vessel.
Collection of Clinical and Epidemiological Data for Linkage Studies
Authors: Farrer Lindsay
Published: June 2004

Accurate assessment of the genetic basis for a disorder is necessary to determine if linkage studies will be meaningful. This unit provides an overview of key concepts and presents guidelines for collecting diagnostic, epidemiological, and genealogical data from potential study participants. The commentary discusses methods for evaluating evidence of genetic versus environmental influences in a disease and for assessing individual pedigrees.
Model‐Free Tests for Genetic Linkage
Authors: Amos Christopher I.
Published: June 2004

This unit covers statistical methods of linkage analysis that do not require assuming a detailed genetic model as is required for standard lod score analysis. The unit has been updated to include the latest methods in sib‐pair analysis as well as relative‐ pair analysis, including the NPL method used in GENEHUNTER and GENEHUNTER PLUS.
Analysis of Gene‐Gene Interactions
Authors: Moore Jason H.
Published: June 2004

The goal of this unit is to introduce gene‐gene interactions or epistasis as a significant complicating factor in the search for disease susceptibility genes. This unit begins with an overview of gene‐gene interactions and why they are likely to be common; then, it reviews several statistical and computational methods for detecting and characterizing genes whose effects are dependent on other genes. The focus of this unit is genetic association studies of discrete and quantitative traits since most of the methods for detecting gene‐gene interactions have been developed specifically for these study designs.
Construction of Small‐Insert Libraries from Genomic DNA
Authors: Hudson Thomas J.
Published: June 2004

Construction of small‐insert genomic libraries involves ligation of a size‐selected insert to vector DNA. In this unit, vector DNA is prepared by digestion with a restriction endonuclease followed by phosphatase treatment to dephosphorylate the vector ends. Insert DNA is prepared by complete digestion of DNA with a compatible 4‐base‐cutting restriction endonuclease (e.g., AluI, HaeIII, RsaI, or Sau3A), as such frequently cutting endonucleases give the highest fragment number in the desired range. Alternatively, fragments may be generated by sonication of genomic DNA. Once the fragments have been generated, they are size selected on an agarose gel. The portion of the gel containing DNA of the desired size is excised and the DNA released from the agarose by digestion with b‐agarase. Insert and vector DNAs are then ligated with T4 DNA ligase and used to transform competent Escherichia coli cells to create a small‐insert library.
Overview of Somatic Cell Hybrid Mapping
Authors: Wasmuth John J.
Published: June 2004

This unit provides an introduction to the various types of hybrid panels in use and reviews the status of commercially available panels.
Chromosome Banding Techniques
Authors: Schreck Rhona R. , Distèche Christine M.
Published: June 2004

Chromosome banding techniques produce a series of consistent landmarks along the length of metaphase chromosomes that allow for both recognition of individual chromosomes within a genome and identification of specific segments of individual chromosomes. These landmarks facilitate assessment of chromosome normalcy, identification of sites of chromosome breaks and alterations, and location of specific genes. This unit covers these basic banding techniques (Q‐banding, G‐banding, and R‐banding), which produce virtually identical patterns of bands along the length of human chromosomes, although the bands and polymorphic regions highlighted may differ with each technique. These techniques highlight reproducible landmarks along the length of the chromosome and specialized staining techniques can be used to highlight particular regions of chromosomes, such as heterochromatic and repeated‐sequence segments. These specialized techniques, nucleolar organizer region (NOR) staining, centromeric heterochromatin staining (C‐banding), methylated satellite DNA staining (distamycin‐DAPI banding), and replication banding are also presented in this unit.
Morphology Antibody Chromosome Technique for Determining Phenotype and Genotype of the Same Cell
Authors: Knuutila Sakari
Published: June 2004

The morphology antibody chromosome (MAC) technique is a powerful combination of methods that permits analysis of both phenotype and genotype on a single interphase or mitotic cell as a basis for lineage analysis of neoplastic and normal cells. This unit describes MAC with sequential phenotypic analysis using antibody and an alkaline phosphatase anti‐alkaline phosphatase (APAAP) complex and genotypic analysis using in situ hybridization with either enzymatic or fluorescence detection. Alternate methods for phenotypic analysis are also described, which include the use of horseradish peroxidase‐conjugated antibodies, fluorochrome‐conjugated antibodies, May‐Grunwald‐Giemsa cytochemical staining, and Sudan black B cytochemical staining. An additional protocol describes G‐ and C‐banding as alternatives to in situ hybridization (ISH) for genotyping MAC specimens. Support protocols describe methods for preparing specimenscytospin preparations, in situ cultures, paraffin‐embedded or cryostat sections, and blood and bone marrow smears. describes a procedure for chromosome painting of previously GTG‐banded slides.
Principles and Applications of PRINS in Cytogenetics
Authors: Koch Jørn
Published: June 2004

PRINS (PRimed IN Situ labeling) is a technique that traditionally has been used for the detection of tandemly repeated target sequences in chromosomes and nuclei (“in situ”) by oligonucleotide probes. With a simple short procedure rendering virtually 100% of potential targets labeled, such sequences can be detected down to a target size of a few hundred bases. More recently it has been found that single‐copy sequences can also be detected at high sensitivity, if signals are enhanced with the Tyramide system. This unit provides protocols for basic PRINS or dideoxy‐PRINS (ddPRINS), and for combinatorial labeling (combining PRINS with FISH). These protocols are also applicable after immunostaining, provided that the antibody has been “secured” at the site by fixation in (para)formaldehyde.
Generating Subclones from Large‐Insert Genomic Clones
Authors: Monaco Anthony P. , Larin Zoia
Published: June 2004

This unit provides a simple protocol for generating a partial‐digest sublibrary of yeast DNA containing a YAC of interest. The starting material is high‐molecular‐weight chromosomal DNA embedded in agarose plugs. Many genome equivalents of these YAC subclones in bacteriophage or cosmid vectors can be plated and screened by hybridization with total human DNA to identify clones that originate from the human portion of the YAC. The human‐positive clones can be picked into 96‐well microtiter plates for spotting on membranes. Once stored in ordered arrays, the YAC subclones can be constructed into contigs using an end‐probe or Alu‐PCR hybridization strategy, or a gel fingerprinting technique.This unit provides a simple protocol for generating a partial‐digest sublibrary of yeast DNA containing a YAC of interest.
Sequence Databases: Integrated Information Retrieval and Data Submission
Authors: Weisemann Jane M. , Boguski Mark S. , Ouellette B.F. Francis
Published: June 2004

This unit describes the NCBI's Entrez database browser. Entrez integrates DNA and protein sequence data, threedimensional structures, and taxonomic information with its associated abstracts and citations contained in PubMed (MEDLINE). It is possible to search the Entrez information space using conventional search queries (authors, gene names, map location) as well as by bibliographic associations (articles that are related to one another) and sequence homology. Also described are the procedures for submission of new data, updates, and corrections to the sequence databases.
Detection of Mutations by Denaturing Gradient Gel Electrophoresis
Authors: Børresen‐Dale Anne‐Lise , Hovig Eivind , Smith‐Sørensen Birgitte
Published: June 2004

This unit describes the procedure for determining the melting profile for a given PCR‐amplified sequence by perpendicular denaturing gradient gel electrophoresis (DGGE) and, using that information, for developing a screening assay based on either parallel DGGE, CDGE (Constant Denaturant Gel Electrophoresis), or TTGE (Temporal /Temperature Gradient Electrophoresis). Four support protocols describe techniques for pouring perpendicular and parallel denaturing gradient gels, constant denaturant gels, and temporal temperature gradient gels.
Detection of Mutations by Fluorescence‐Assisted Mismatch Analysis (FAMA)
Authors: Tosi Mario , Verpy Elisabeth , Meo Tommaso
Published: June 2004

Fluorescence‐assisted mismatch analysis (FAMA) methodology uses bifluorescent double‐stranded DNA substrates to maximize the reliability and sensitivity of mutation‐scanning procedures that rely on cleavage of mismatches using chemical. This unit presents a nested PCR procedure to fluorescently label each DNA strand, followed by chemical cleavage to detect the point mutations. Fluorescent end labeling with strand‐specific fluorophores, electrophoretic separation of cleavage products in an automated Perkin‐Elmer ABI 373 or 377 DNA sequencer and analysis using the Perkin‐Elmer GENESCAN software expands sensitivity by highlighting weak signals through superimposition of strand‐specific fluorescence electropherograms for different samples.
Human Mutation Databases
Authors: Horaitis Ourania , Cotton Richard G.H.
Published: June 2004

The first part of this unit compares general and locus‐specific mutation databases. The second section deals with submitting data. The third part provides guidance for accessing mutation data. The final section offers advice on database construction.
Overview of Clinical Cytogenetics
Authors: Korf Bruce R.
Published: June 2004

This unit provides an introduction to clinical cytogenetics. It opens with indications for prenatal and postnatal chromosome analysis, followed by a brief discussion of the applications of fluorescence in situ hybridization (FISH). It suggests tissue sources for prenatal and postnatal analysis, and closes with a review of numerical and structural chromosome abnormalities. This unit provides an introduction to clinical cytogenetics
Diagnosis of Microdeletion Syndromes by Fluorescence in situ Hybridization (FISH)
Authors: Shaffer Lisa G.
Published: June 2004

This unit opens with an overview of microdeletions and methods for their detection. It goes on to describe a vast array of autosomal microdeletion syndromes, X‐linked microdeletion syndromes, and microduplication syndromes. The final portion of the unit offers guidance for detecting such syndromes with Fluorescence in situ Hybridization (FISH).
Serial Analysis of Gene Expression (SAGE)
Authors: Blackshaw Seth , Kim Jae B. , Croix Brad St. , Polyak Kornelia
Published: June 2004

This unit provides a protocol for performing serial analysis of gene expression (SAGE). SAGE involves the generation of short fragments of DNA, or tags, from a defined point in the sequence of all cDNAs in the sample analyzed. This short tag, because of its presence in a defined point in the sequence, is typically sufficient to uniquely identify every transcript in the sample. SAGE allows one to generate a comprehensive profile of gene expression in any sample desired from as little as 100,000 cells or 1 ug of total RNA. SAGE generates absolute, rather than relative, measurements of RNA abundance levels, and this fact allows an investigator to readily and reliably compare data to those produced by other laboratories, making the SAGE data set increasingly useful as more data is generated and shared.
Production of High‐Titer Lentiviral Vectors
Authors: Zufferey Romain , Trono Didier
Published: June 2004

Conventional retroviral vectors are useful for gene delivery because of their large cloning capacity and ability to integrate into the chromosome of the target cell. However, their inability to transduce nondividing cells limits their utility. In contrast, lentiviruses, a retrovirus subfamily, can infect both non‐replicating and dividing cells. This unit describes the production of human immunodeficiency virus‐derived vectors by transient transfection of 293T cells. In addition, the multiple safeguards engineered into the latest version of HIV‐based vectors are described.Conventional retroviral vectors are useful for gene delivery because of their large cloning capacity and ability to integrate.
Gene Delivery Using Helper Virus–Free HSV‐1 Amplicon Vectors
Authors: Fraefel Cornel
Published: June 2004

Herpes simplex virus type 1 (HSV‐1)‐based amplicon vectors contain only ˜1% of the viral genome. Consequently, replication and packaging of amplicons depend on helper functions provided either by replication‐defective mutants of HSV‐1 (helper viruses) or by replication‐competent, but packaging‐defective, HSV‐1 genomes. Sets of cosmids that overlap and represent the entire HSV‐1 genome can form, via homologous recombination, circular replication‐competent viral genomes, which give rise to infectious virus progeny. If the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected amplicon DNA. Resulting stocks of packaged amplicon vectors are free of contaminating helper virus. The basic protocol describes the cotransfection of amplicon and cosmid DNA into 2‐2 cells and the harvesting of packaged vector particles. Support protocols describe preparing cosmid DNA and methods for determining the titers of amplicon stocks.
Gene Transfer to Arteries
Authors: Akyürek Levent M. , San Hong , Nabel Elizabeth G. , Singh Ripudamanjit , Simari Robert D.
Published: June 2004

This unit provides a set of protocols for introducing recombinant genes into normal, injured, and atherosclerotic arteries. The protocols include animal preparation, surgical techniques, and delivery systems. Protocols describe gene delivery to normal, injured, and stented porcine iliofemoral arteries, employing a double balloon infusion catheter to deliver the vector. Another basic protocol describes gene delivery to atherosclerotic arteries using a hyperlipidemic double‐injury rabbit model, and requires surgical exposure of the artery and instillation of the gene vector via a catheter. Additional protocols describe gene delivery to normal and injured murine carotid and femoral arteries. An describes a percutaneous method for arterial gene delivery. These protocols may be adapted to deliver genes to either injured or noninjured atherosclerotic arteries.
Human Hematopoietic Cell Culture, Transduction, and Analyses
Authors: Nolta Jan A. , Kohn Donald B.
Published: June 2004

This unit provides methods to introduce genes into human hematopoietic progenitor cells. The describes isolating CD34+ cells, transducing these cells with a retroviral vector in the presence of a stromal cell layer, assaying the efficiency of transduction, and establishing long‐term cultures. One support protocol describes how colonies can be plated from long‐term cultures. An alternate procedure describes the use of fibronectin‐coated plates as a substitute for the stromal cell layer in transduction. Support protocols describe methods for maintenance and supernatant collection from vector‐producing fibroblasts (VPF), screening medium components for the ability to support hematopoietic cell growth, and establishing colonies from long‐term cultures. Other protocols provide PCR‐based methods to analyze individual colonies for transduction. Methods are given to analyze cells harvested from long‐term cultures. Additional protocols describe freezing and thawing hematopoietic cells.This unit provides methods to introduce genes into human hematopoietic progenitor cells.
Overview of Human Repetitive DNA Sequences
Authors: Doggett Norman A.
Published: June 2004

This appendix contains brief descriptions of the most abundant classes of repetitive DNA in the human genome. The chromosomal distribution of these classes of repeats are shown for human chromosome 16.
Human and Mouse Gene Nomenclature
Authors: Wain Hester , Povey Sue , Maltais Lois
Published: June 2004

Standard genetic nomenclature is necessary to help researchers, clinicians, and the public to access data on their genes of interest, and to communicate in a globally understood language of approved gene symbols. In both human and mouse, one unique symbol (acronym/abbreviation) and one name are assigned for each gene. Co‐ordination between human and mouse gene nomenclature is a successful endeavor, due in part to the historical interaction between the two nomenclature committee groups. This interaction grew out of the Human Gene Mapping (HGM) Workshops. This appendix discusses development and organization of gene nomenclature, how to find a gene and how to name a new gene.
Automation and Robotics for Genetic Analysis
Authors: Smith James H. , Madan Deepika , Salhaney Jay , Engelstein Marcy
Published: June 2004

This guide to laboratory robotics covers a wide variety of methods amenable to automation including mapping, genotyping, barcoding and data handling, template preparation, reaction setup, colony and plaque picking, and more.
Establishment of Permanent Cell Lines by Epstein‐Barr Virus Transformation
Authors: Gilbert John
Published: June 2004

This basic protocol presents Epstein‐Barr virus (EBV) transformation of B cells to create a permanent cell line to provide cells for genetic and DNA analysis. Virus stocks are prepared from cultures of an infected marmoset cell line in the first support protocol. Transformed cells may be analyzed immediately or frozen for future use as described in the second support protocol.
Basic Statistics
Authors: Moore Jason H. , Thornton Tricia A. , Ritchie Marylyn D.
Published: June 2004

Statistics is a relatively new science with most of the important developments occurring within the last 100 years. The goal of this unit is to summarize some of the philosophical ideas behind statistics and to provide a general framework in the form of flowcharts for deciding which statistical test is appropriate for a given dataset and scientific question. Also provided is a list of references and software for commonly used statistical methods.
Proper Alignment and Adjustment of the Light Microscope
Authors: Salmon Edward D. , Canman Julie C.
Published: June 2004

The light microscope is a basic tool for biologists, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase‐contrast, differential interference contrast, and fluorescence epi‐illumination), and how it should be maintained as required to obtain maximum image‐forming capacity and resolution. The principles of microscopy and step‐by‐step alignment and adjustment procedures are described in this unit.
[4+2] Cycloaddition of ortho‐Quinone Methides Promoted by Ionic Liquids: an Efficient and Mild Protocol for the Synthesis of Tetrahydropyranobenzopyrans
Authors: Yadav J. S. , Subba Reddy B. V. , Sadashiv K. , Padmavani B.
Published: June 2004

The stereoselective synthesis of trans‐annelated pyrano[3,2‐c]benzopyrans has been achieved by intramolecular [4+2] cycloaddition of o‐benzoquinone methides that are generated in situ from o‐hydroxybenzaldehydes and unsaturated alcohols using an air‐ and moisture‐stable ionic liquid, i.e., 1‐butyl‐3‐methylimidazolium tetrafluoroborate [bmim]BF4 under mild and neutral conditions.
N‐Methylprolinol Catalysed Asymmetric Baylis−Hillman Reaction
Authors: Radha Krishna Palakodety , Kannan V. , Narasimha Reddy P. V.
Published: June 2004

N‐methylprolinol is used as a chiral base catalyst for the Baylis–Hillman reaction to obtain the adducts in good yields with moderate to good enantioselectivities in 1,4‐dioxane:water (1 : 1, v/v) under ambient conditions.
Oxidation of p‐Cresol to p‐Hydroxybenzaldehyde with Molecular Oxygen in the Presence of CuMn‐Oxide Heterogeneous Catalyst
Authors: Wang Feng , Yang Guanyu , Zhang Wei , Wu Wenhai , Xu Jie
Published: June 2004

A high‐yield synthesis of p‐hydroxybenzaldehyde from p‐cresol and molecular oxygen was achieved over a CuMn‐oxide supported carbon catalyst. The reaction parameters such as pressure, stirring speed, reaction temperature, solvent, and the amount of sodium hydroxide in the reaction media were optimized. As a result, a high conversion of p‐cresol (99%) and a high selectivity to p‐hydroxybenzaldehyde (96%) were realized at the same time. Catalyst separation and recycling tests clearly showed that the reaction proceeded on the heterogeneous catalyst but not on dissolved species.
Oxidation of Amino Diols Mediated by Homogeneous and Heterogeneous TEMPO
Authors: Testa Maria Luisa , Ciriminna Rosaria , Hajji Chakib , Zaballos Garcia Elena , Ciclosi Marco , Sepulveda Arques Jose , Pagliaro Mario
Published: June 2004

The conversion of amino diols to aminohydroxy acids by oxidation of the primary hydroxy group mediated by homogeneous and heterogeneous TEMPO (2,2,6,6‐tetramethylpiperidine 1‐oxyl radical) is reported. The synthesis uses NaOCl as primary oxidant and TEMPO, either dissolved in the homogeneous phase or entrapped in a sol‐gel matrix, as catalytic mediator. Homogeneous TEMPO is suitable for the oxidation of aliphatic methylamino diols, while the hybrid organic‐inorganic silica sol‐gel catalysts are more selective mediators for the oxidation of benzylic amino diols like the potent antibiotic chloramphenicol which, under homogeneous conditions, are unselectively oxidized to benzoic acids.
Sunflower oil hydrogenation on Pd/C in SC propane in a continuous recycle reactor
Authors: Ramírez E. , Recasens F. , Fernández M. , Larrayoz M. A.
Published: June 2004

Fluid‐phase, continuous hydrogenations of sunflower oil on 2% Pd/C were carried out in an internal recycle, radial‐flow, packed‐bed microreactor (50 cm3) using propane as supercritical‐fluid solvent. Temperature (428–488 K), oil liquid hourly space velocity (LHSV = 30–70), H2 mol composition (2–10%), and stirrer speed (52–262 rad/s) were changed according to a four‐variable, two‐level, central composite design to predict the effect of process variables on the iodine value (IV) and on trans fatty acid content (trans C18:1). Feed and product were well above the condensation conditions so that a single fluid phase was present (according to recent calculations by Pereda et al.). The total system pressure, the molar oil concentration and the catalyst mass were held constant at 20 MPa, 1 mol %, and 0.1085 g, respectively. An empirical quadratic‐form response‐surface model is shown to fit the results, and shows the regions where a potential CSTR process could be operated to obtain a certain outlet iodine value and a minimum trans C18:1 content. For the time‐on stream values used here catalyst deactivation effects were not observed. In an extension of the results, a kinetic analysis of the steady‐state CSTR reaction rate data allows determination of the kinetic constants, and their temperature dependency, for the multiple reactions of hydrogenation–isomerization network involving triglyceride species. The kinetic formalism, proposed earlier for vegetable oil hydrogenations, was used. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1545–1555, 2004