bluuebunny/crossref_metadata_2025 huggingface dataset:
Choosing and Preparing Antigen‐Presenting Cells
Authors: Harding Clifford V.
Published: June 2004

The first issue in many antigen‐processing experiments is to choose and obtain appropriate antigen‐presenting cells (APCs). Macrophages have characteristics that dictate their use in certain antigen‐processing experiments, so this unit describes preparation of activated macrophages, which involves eliciting peritoneal macrophages from mice by infection with Listeria monocytogenes. This method is often preferred because the resulting macrophages are more consistently activated to a high degree, but macrophages can also be obtained (as described here) by sequential injection of L. moncytogenes and proteose peptone, by induction with concanavalin A (Con A), or by isolation from bone marrow. Lipopolysaccharide‐stimulated B lymphoblasts can also be used as APCs. Protocols are included for preparation of L. moncytogenes stocks and preparation of L929‐conditioned medium which is used for culturing bone marrow‐derived macrophages.
Bacteriophage Library Construction and Selection of Recombinant Antibodies
Authors: Galanis Maria , Irving Robert A. , Hudson Peter J.
Published: June 2004

This unit describes the use of E. coli and bacteriophages to display a diverse library of antibody fragments equivalent in complexity to the mammalian immune repertoire, and subsequent screening of the library for antibody fragments with specific binding affinities. The methods are also used for affinity enhancement (maturation), through the display and selection of improved affinity mutants derived from a single parent antibody. This unit discusses the following key components needed in library construction technology: a repertoire of antibody genes, typically amplified by polymerase chain reaction (PCR) technology; construction of scFv genes by PCR assembly; a method for producing a stable library, using bacteriophage that can both display individual antibodies on the viral surface and carry the gene encoding the antibody; a method of growing phage for selection; a method of selecting the highest‐affinity antibody from the phage library; a method for monitoring progress of phage selection; an affinity‐enhancement strategy for improving and manipulating the selected antibody; and expression of affinity‐enhanced antibodies.
MHC‐Peptide Tetramers to Visualize Antigen‐Specific T Cells
Authors: Altman John D. , Davis Mark M.
Published: June 2004

Mature T lymphocytes of the CD8 or CD4 classes bear αβ T cell receptors (TCR) that are specific for a molecular complex consisting of a major histocompatibility complex class I or II (MHC class I or II) molecule bound to a unique self or foreign peptide. Until recently, methods for monitoring the T cell immune response to a viral or tumor antigen were restricted primarily to functional assays based on limiting dilution analysis, because the lack of specific molecular reagents to identify clonal T cells obviated approaches to identify and enumerate specific T cells. Development of efficient methods to express and refold MHC class I molecules with synthetic peptides coincided with identification of specific protein sequences that provide the substrate for enzymatic biotinylation. This combination has led to the development of a straightforward method for generating synthetic TCR ligands, making them tetravalent to provide increased avidity, and labeling them through a streptavidin moiety with useful fluorescent tags such as fluorescein or phycoerythrin. This unit describes the preparation of MHC class I/peptide tetramers in detail, including bacterial expression and refolding of the MHC class I light chain, β2‐microglobulin (β2m), as well as the formation of a complex consisting of the MHC class I heavy chain of interest, β2m, and a chosen peptide.
Binding of Biotinylated Peptides to MHC Class II Proteins on Cell Surfaces
Authors: Rothbard Jonathan B. , Busch Robert
Published: June 2004

This unit describes a simple, reproducible, and semiquantitative assay for measuring ligand binding to cell surface receptors. This approach is useful as a quick screen for peptide binding to MHC class II proteins that avoids the need to purify the MHC class II molecules and that uses small numbers of cells. The basic protocol describes procedures for incubating cells expressing the MHC molecule of interest with a biotinylated peptide, washing off excess peptide, and detecting the bound peptides by staining with fluorescently labeled avidin. Bound fluorescence is then quantitated by flow cytometry. An alternate protocol provides a more sensitive method of measuring the peptide‐receptor complexes, and is useful when receptor density or binding site availability become limiting factors in the basic protocol. The alternate protocol uses an avidin/anti‐avidin/avidin sandwich, which has been found to increase sensitivity substantially without sacrificing specificity. Support protocols are provided for biotinylation of synthetic peptides in solution and for preparation of peptide stock solutions for use in the binding assays.
Detection of Low‐Affinity Ligand‐Receptor Interactions at the Cell Surface with Fluorescent Microspheres
Authors: Brown Marion H.
Published: June 2004

A method of producing highly avid multivalent ligand binding reagents for detecting low‐affinity interactions at the cell surface is described in this unit. The principle is to immobilize multiple copies of extracellular regions of cell‐surface molecules on plastic fluorescent beads, to present the coated beads to cells, and to analyze binding in a quantitative manner by flow cytometry. The method of attaching proteins to the beads is designed to maximize display of the ligand‐binding region. The approach is applicable to immobilization of fusion proteins on a variety of beads, and can also be adapted for use with native proteins. A protocol for the biotinylation of MAbs is also presented.
Measurement of MHC/Peptide Interactions by Gel Filtration
Authors: Sidney John , Southwood Scott , Oseroff Carla , del Guercio Marie‐France , Sette Alessandro , Grey Howard M.
Published: June 2004

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules. The establishment of an MHC/peptide binding assay, and its subsequent use in determining the MHC binding capacities of peptide ligands, requires sufficient stocks of purified MHC and both labeled and unlabeled peptides. Accordingly, this unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. A support protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high‐affinity, assay‐specific, radiolabeled ligands and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a 2‐day incubation, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Two methods for separation by size‐exclusion gel‐filtration chromatography are described, as is data analysis.
Measurement of the Kinetics of Biomolecular Interactions Using the IAsys Resonant Mirror Biosensor
Authors: George Andrew J. T.
Published: June 2004

In this unit, the resonant mirror biosensor or IAsys, produced by Affinity Sensors, is discussed for the measurement of the kinetics of biomolecular interactions. All biosensors operate using a common principle‐‐that binding events occurring on the sensing surface of the biosensor are converted by a transducer into an electronic signal which can then be processed by a computer to yield information on the amount of binding. Where biosensors vary is in their configuration and the nature of the transducer. Both the IAsys and BIACORE series of machines use optical techniques to detect the binding event, but the IAsys uses a resonant mirror device as the transducer, while the BIACORE uses surface plasmon resonance.When using biosensors the experiment can be divided into various phases, which are separately in this unit. The phases involve (1) the immobilization of the ligand onto the sensing surface; (2) addition of the ligate to the sensing surface, and following its association to the ligand, including the kinetics of binding; (3) washing of free ligate from chamber and following the dissociation of the ligand‐ligate complex; (4) regeneration of the surface; repetition of phases 2 and 3; and (5) data analysis. A protocol is also included for optimization of ligand pH for immobilization to the carboxymethylated dextran surfaces described in this unit.
Measuring Protein‐Protein Interactions by Equilibrium Sedimentation
Authors: Schuck Peter , Braswell Emory H.
Published: June 2004

This commentary unit provides a basic introduction to the principles and practice of sedimentaiton equilibrium analytical ultracentrifugation for the study of reversible protein interactions. Equilibrium sedimentation is one of the most effective methods for the detection and characterization of protein interactions and can be applied to the measurement of self association, heterologous association, binding stoichiometry, and the determination of association constants. The unit includes an extensive discussion of the instrumentation required to carry out equilibrium analytical ultracentrifugation and to perform data analysis. A strategic planning section gives the reader several experimental scenarios and conditions for using equilibrium sedimentation.
Binding of Small Peptides to Immobilized Antibodies: Kinetic Analysis by Surface Plasmon Resonance
Authors: Andreu David , Gomes Paula
Published: June 2004

This unit describes a method for screening small viral peptides as specific antigens using a surface plasmon resonance (SPR) biosensor. The basic protocol in this unit is suited for direct single‐step SPR analysis of small ligand‐large receptor interactions, where small peptides are used as analytes (injected in the continuous buffer flow) and monoclonal antibodies (MAbs) are immobilized on the SPR sensor chip surface. An alternate protocol is included for situations where kinetic analysis is not possible and uses a surface competition assay to indirectly measure the kinetics of small analyte binding.
Peptide Induction of Surface Expression of Class I MHC
Authors: Hansen Ted , Myers Nancy
Published: June 2004

This unit describes a method for comparing the relative binding of different peptides to the same MHC class I (MHC‐I) molecule using live cells. Live cells expressing suboptimally loaded MHC‐I proteins are incubated with medium containing diluted amounts of synthetic peptides to be tested for binding to class I. After overnight incubation with peptide, surface class I expression is monitored by flow cytometry using an allele‐specific MAb. Relative binding affinity of peptide reliably correlates with the amount of surface induction of the class I molecule to which it specifically binds. The mechanistic basis of this assay is that surface MHC‐I molecules become conformationally unstable shortly after peptide dissociation. However, the binding of an exogenous peptide can stabilize the surface class I molecule, prevent conformational instability, and thus increase class I surface expression in an allele‐specific manner.
Schistosomiasis
Authors: Lewis Fred
Published: June 2004

The trematode parasites in the family Schistosomatidae (phylum Platyhelminthes) infect a wide range of vertebrates. Three species of the genus Schistosoma are of major medical importance. This unit deals exclusively with the parasite Schistosoma mansoni, which is the species most frequently maintained in the laboratory. Among the far‐ranging investigations in the immunology of schistosomiasis are studies in vaccine development, immunopathology of granulomatous inflammation and fibrosis, eosinophil function, and in vivo regulation of TH1 and TH2 responses. This unit describes maintenance and collection procedures for various stages of the schistosome that have immunologic interest, including infection of mice with cercariae, collection of cercariae, preparation of in vitro‐derived schistosomules and in vivo‐derived schistosomules, and collection of adult worms and eggs. Included also are techniques for preparing soluble egg antigen (SEA), one of the more commonly used schistosome antigenic preparations. A discussion is given of the basic steps that are important in maintaining the snail intermediate host, and infecting the snails with schistosome miracidia. The unit deals exclusively with the parasite Schistosoma mansoni, which is the species most frequently maintained in the laboratory. Since part of the life cycle of all schistosomes involves a snail host, a description of proper maintenance for the snails is provided. Often, problems in experiments can be traced back to improper snail and parasite maintenance, or lack of attention to detail during mammalian exposure to the infective stage (cercaria) of the parasite.
Experimental Asexual Blood Stage Malaria Immunity
Authors: Amante Fiona H. , Good Michael F.
Published: June 2004

This unit describes three methods for investigating the immune response to murine malaria parasites. Immunization protocols using recombinant fragments of the merozoite surface protein‐1 of Plasmodium yoelii, whole blood stage malaria parasites, and live infection with parasitized red blood cells from a Plasmodium‐infected donor are provided. Methods for chemotherapeutic drug care of Plasmodium‐infected mice and for inducing malaria by adoptive transfer of antigen‐specific T cells are included. Finally, support protocols describe methods for growing, maintaining, and cryopreserving murine asexual blood stage malaria parasites and for preparing blood stage antigen for use in ELISAs.
Animal Models for Candidiasis
Authors: Romani Luigina
Published: June 2004

As little is known about the yeast/host interaction in Candida species other than C. albicans, this unit details the materials and experimental procedures required for development of experimental C.albicans infections, such as GI and disseminated infections in infant Swiss White mice and in adult immunocompromised BALB/c mice, primary acute disseminated infection in BALB/c and DBA/2 mice, and vaginal infection in CBA/J mice and in Wistar rats. Detailed materials and methods for C. albicans growth and detection are also described.
A Mouse Model for Cytomegalovirus Infection
Authors: Brune Wolfram , Hengel Hartmut , Koszinowski Ulrich H.
Published: June 2004

This unit describes procedures for infecting newborn and adult mice with murine cytomegalovirus (mCMV). Methods are included for propagating mCMV in cell cultures and for preparing a more virulent form of mCMV from salivary glands of infected mice. A plaque‐forming cell (PFC) assay is provided for measuring mCMV titers of infected tissues or virus stocks. In addition, a method is described for preparing the murine embryonic fibroblasts used for propagating mCMV and for the PFC assay.
Animal Models Using Lymphocytic Choriomeningitis Virus
Authors: von Herrath Matthias , Whitton J. Lindsay
Published: June 2004

This unit includes protocols for inducing systemic infection and persistent infection of mice with lymphocytic choriomeningitis virus (LCMV). Methods used to measure T cell responses to LCMV are then described. A protocol to assess anti‐LCMV immunity in vivo is also included. Support protocols for preparing LCMV stocks and measuring LCMV titers using a plaque assay are also included. Finally, a support protocol for detecting anti‐LCMV antibodies by ELISA is presented.
Immunohistochemistry
Authors: Hofman Florence
Published: June 2004

This unit describes several methods for localizing specific antigens in various tissue and cell preparations using immunohistochemistry (IHC). Protocols describe preparation of suitable material for IHC including fresh, unfixed, frozen tissue specimens; unfixed cells, either freshly isolated or from suspension or adherent cultures, or fixed, paraffin‐embedded tissue sections. By careful selection of reagents, it is possible to detect two antigens simultaneously. For antigens that are sensitive to fixative, it may be necessary to unmask the antigen by a new technique called “antigen retrieval”. If there is cross‐reactivity between the secondary antibody and antigens present in the target cells or tissue, the secondary antibody can be preabsorbed. The different IHC protocols are represented schematically and summarized in a table, which also lists advantages and disadvantages of each approach. Causes of background staining and ways to eliminate it are also discussed.
Commonly Used Mouse Strains
Authors: Kruisbeek Ada M.
Published: June 2004

This appendix lists commonly used mouse strains and selected cell‐surface molecules expressed by these strains. H‐2 is the gene locus residing on chromosome 17 of the mouse which encodes major histocompatibility proteins. Only those H‐2 loci involved in immunologic interactions frequently cited in this manual are listed here‐‐i.e., Aa‐, Ab‐, Ea‐, and Eb‐‐region‐encoded MHC class II molecules, and K‐and
D‐region‐encoded MHC class I molecules. In addition to H‐2 loci, others are listed that encode immunologically important proteins. Congenic strains listed in this appendix represent inbred strains that were derived from their origin (BALB/c, C57Bl/10, and C3H) by selective matings, such that they differ from that originating strain at only one independently segregating genetic locus. The locus of choice in these cases was H‐2; thus, these strains differ from their strain of origin only in their H‐2 complex, but not in any other loci. During the production of the congenic lines, several strains were derived that had undergone intra‐H‐2 recombination events. These strains are listed here and have been invaluable in defining the major roles of various MHC glycoproteins.
Commonly Used Detergents
Authors: Coligan John E.
Published: June 2004

This appendix lists reference Epstein‐Barr virus (EBV)‐transformed B lymphoblastoid cell lines (B‐LCL) and the HLA types expressed by these cell lines. These B‐LCL are useful for studies of cellular immunity (e.g., restriction of antigen‐specific responses), biochemical characterization of histocompatibility molecules, and as controls for DNA‐based typing methods.
Murine Models of Immunodeficiency and Autoimmune Disease
Authors: Cohen Philip
Published: June 2004

Genetically determined murine immunodeficiency states are useful for understanding the function of specific immune‐system genes and cellpopulations. In addition, certain immunodeficient strains may be exploited as hosts for foreign tumors or immune cells. The more commonly used immunodeficiency models are described in this appendix. Not included are strains better known for primary neurological or neuromuscular abnormalities or for defective osteoclast function. Many of the recently described immune‐deficient “knockout” strains are described, including cytokine and cytokine receptor knockout strains. The most widely studied murine strains for autoimmune disease and experimental autoreactivity are also listed.
HLA Type of EBV‐Transformed Human B Cell Lines
Authors: Hurley Carolyn Katovich , Johnson Armead H.
Published: June 2004

This appendix lists reference Epstein‐Barr virus (EBV)‐transformed B lymphoblastoid cell lines (B‐LCL) and the HLA types expressed by these cell lines. These B‐LCL are useful for studies of cellular immunity (e.g., restriction of antigen‐specific responses), biochemical characterization of histocompatibility molecules, and as controls for DNA‐based typing methods.
Web Sites of Interest to Immunologists
Authors: Lefranc Marie‐Paule
Published: June 2004

The astonishing increase in the amount of biologically relevant data on the World Wide Web (WWW) enables immunologists to immediately access databases; scan literature; use tools and software; find information on collections, educational resources, and research groups; and exploit that information for scientific projects and clinical trials. This appendix presents a selection of the links available at the international ImMunoGeneTics (IMGT) database. Most of the sections such as Databases, Immunology themes, Journals, Societies, and Tools, provide direct access to Web sites. Three other sections (Molecular Biology Servers, Resources, and Universities and Institutes) offer their own lists of useful links.
Mouse MHC Genes and Products
Authors: Trowsdale John , Campbell R. Duncan
Published: June 2004

John Trowsdale, Cambridge University, Cambridge, United Kingdom; and R. Duncan Campbell, MRC Immunochemistry Unit, Oxford, United Kingdom
Listing, Location, Binding Motifs, and Expression of Nonclassical Class I and Related Genes and Molecules
Authors: Forman James , Lindahl Kirsten Fischer
Published: June 2004

The tables presented in this appendix list nonclassical class I or related genes/molecules arranged by the chromosomal region where they are encoded. This includes genes that fall into the Ib region of the murine major histocompatibility complex (MHC) which includes H2‐Q, ‐T, and ‐M, as well as CD1, which lies outside the MHC region. A final table includes genes/molecules that are encoded in diverse regions. They are included in this section because they are either class I related in that their heavy chain is related to classical class I and/or they are associated with ion given is for the C57BL/6 (B6) strain unless otherwise noted.
Receptors Involved in the Regulation of Murine NK Cells
Authors: Washington A. Valance , McVicar Daniel W.
Published: June 2004

This appendix presents a table of receptors found on murine NK cells. Information is given regarding the specific type of cell(s) in which the particular receptor is found, the chromosomal location, and other names and characteristics including cytoplasmic tyrosine motifs. Only documented receptors and receptor groups are presented, focusing on surface markers found on murine NK cells that regulate the balance between status quo and activation. This appendix also highlights a definite need in the field: the identification of the ligands to these receptors. Less than half of the receptors presented in this table have identified ligands. While the receptors represent advances in understanding the natural killer cell, each unidentified ligand indicates that there is still a long road ahead.
Heat‐Shock Proteins
Authors: Li Zihai , Srivastava Pramod
Published: June 2004

Heat‐shock proteins (HSPs), or stress proteins, are highly conserved and present in all organisms and in all cells of all organisms. Selected HSPs, also known as chaperones, play crucial roles in folding/unfolding of proteins, assembly of multiprotein complexes, transport/sorting of proteins into correct subcellular compartments, cell‐cycle control and signaling, and protection of cells against stress/apoptosis. More recently, HSPs have been implicated in antigen presentation with the role of chaperoning and transferring antigenic peptides to the class I and class II molecules of the major histocompatibility complexes. In addition, extracellular HSPs can stimulate professional antigen‐presenting cells of the immune system, such as macrophages and dendritic cells. HSPs constitute a large family of proteins that are often classified based on their molecular weight: hsp10, hsp40, hsp60, hsp70, hsp90, etc. This unit contains a table that lists common HSPs and summarizes their characteristics including (a) name, (b) subcellular localization, (c) known function, (d) chromosome assignment, (e) brief comments, and (f) references.
Equipment
Authors: Donovan John , Brown Patricia
Published: June 2004

Special equipment and utensils are itemized in the materials list of each protocol. Here, no attempt has been made to list all items required for each procedure, but rather only those items that might not be readily available in the laboratory, have specific specifications, or require special preparation. This appendix lists standard items found in the modern immunology laboratory, i.e., items used extensively in this manual and thus generally not included in the individual materials lists. For a full inventory of equipment and utensils used in a modern immunology laboratory, it will be useful to review both the listings here and the materials lists accompanying each unit (or those units of interest).
Wright‐Giemsa and Nonspecific Esterase Staining of Cells
Authors: Strober Warren
Published: June 2004

This appendix provides two protocols for staining cells. The stains used in the Wright‐Giemsa protocol, the Romanowsky stains, are a mixture of methylene blue (and other closely related thiazine dyes) and eosin. The staining protocol is a two‐stage method that allows for a more intense staining of the nuclei than would be possible if the Wright and Giemsa stains were mixed together. In the second protocol, nonspecific esterase stain are used to identify cell types containing esterases that have a characteristic ability to split esters under particular conditions. In the staining method given here, the substrate, a‐naphthyl butyrate, is incubated with cells under conditions in which esterases present in monocytes/macrophages split the substrate to yield an intermediate that can be coupled with a substance, hexazotized pararosaniline, to yield a colored precipitate. Thus, this staining reaction can be used to identify monocytes/macrophages in cell preparations.
Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures
Authors: Fitch Frank W. , Gajewski Thomas F. , Yokoyama Wayne M.
Published: June 2004

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R.
Published: June 2004

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers using molecular biology techniques. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques are presented in this appendix. These three procedures cover a range from 5 to 10 ng DNA/ml to 50mg DNA/ml.
Preparing Frozen Bacterial Stocks
Authors: Morrison Sherie L.
Published: June 2004

After bacteria containing plasmids of interest have been produced, it is frequently convenient to store them for later use. This appendix provides instruction for storing bacteria in such a way that they typically remain viable for years.
Gram Staining
Authors: Coico Richard
Published: June 2004

Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram‐positive and Gram‐negative bacteria on the basis of differential staining with a crystal violet‐iodine complex and a safranin counterstain. The cell walls of Gram‐positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram‐negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.
Monoclonal Antibodies to Mouse Cell‐Surface Antigens
Authors: Alaverdi Noosheen
Published: June 2004

The cluster of differentiation (CD) nomenclature, originally devised by the International Workshop on Human Leukocyte Differentiation Antigens (HLDA) to classify leukocyte surface antigens, has become more accepted by researchers in nonhuman animal models. The CD homologues from other species are usually identified by multiple studies, including biochemical, functional analysis, cloning, and immunological assays. This unit compiles a complete (as of August, 2001) list of monoclonal antibodies (mAb) to the mouse CD antigens and several non‐CD surface antigens that are commonly used as phenotypic and functional markers in the mouse system. There are many reagents recognizing polymorphic epitopes of the mouse MHC and different subunits of TCR; for simplicity, only a few mAbs to the framework determinants of these molecules are listed here.
Genetic Mapping in the Mouse
Authors: Pavan William J.
Published: June 2004

Several molecular genetic approaches are available for identifying the location of a conserved human DNA sequence in the mouse genome. The choice of approach depends on the reagents available and the precise information that the investigator would like to determine. This unit presents a method for determining the location of the murine homologue of a human gene using linkage analysis with available mouse interspecific backcrosses. Restriction fragment length polymorphism (RFLP) analysis is used to map human gene fragments in the mouse genome. The approaches described in this unit are analogous to those used for linkage analysis in human pedigrees, except the mouse crosses can and have been designed so only two alleles are segregating for each locus and the phase of every chromosome is known.
Pedigree Selection and Information Content
Authors: Hauser Elizabeth R. , Weeks Daniel E.
Published: June 2004

This unit covers the important criteria and methods for appropriately selecting pedigrees for linkage analysis. It includes methods for determining the power of a dataset to detect genetic loci for both model‐based (lod score) and model‐free (affected sib‐pair, affected‐relative pair) analyses.This unit covers the important criteria and methods for appropriately selecting pedigrees for linkage analysis.
Strategies for Genotype Generation
Authors: Gilbert John , Vance Jeffrey
Published: June 2004

This unit describes methods for genotyping in families and individuals. Systematic step‐by‐step approaches are presented for setting up a study and carrying out the subsequent analyses. The discussion is divided into two parts. The Strategic Approach section provides an outline for the general approach to be used when beginning a new family or individual study, including guidelines for sample and data handling, cytogenetic analysis, choice of screening tests, and organization of DNA for genotyping. The commentary discusses some of these issues in more detail, providing information on molecular methods for genotyping, principally based on use of the polymerase chain reaction (PCR), in combination with simple sequence length polymorphisms (SSLPs) and, more recently, single nucleotide polymorphisms (SNPs). This discussion is followed by an outline of various genotyping techniques used in positional cloning and candidate gene analysis. Finally, potential new methods for automated mass genotyping are briefly discussed.
Analysis of Genetic Linkage Data for Mendelian Traits
Authors: Pericak‐Vance Margaret A.
Published: June 2004

This unit describes linkage analysis, an invaluable tool in mapping human disease genes. Linkage analysis is one of several methods used to map genes. Specifically, in linkage analysis cosegregation of two or more genes (traits) is examined in a family unit to determine if they segregate independently according to Mendel's laws or if they do not segregate independently because of their close physical proximity. The procedures outlined in this unit are not always straightforward, and a number of caveats are presented in the commentary regarding possible complications and confounding factors that may arise. Specific examples are given, but it is not possible to cover all possible scenarios or variables. The intention is to enable the reader to master basic principles used in the linkage approach and thus be in a position to undertake, with proper consultation, a linkage‐analysis study. This unit deals specifically with lod score analysis in Mendelian disorders.
Single‐Sperm Typing
Authors: Lien Sigbjorn , Szyda Joanna , Leeflang Esther P. , Hubert Rene , Zhang Lin , Schmitt Karin , Arnheim Norman
Published: June 2004

This unit presents protocols for sperm isolation using two different methods, amplification of simple sequence‐length polymorphisms (SSLP) and/or single nucleotide polymorphisms (SNP) from single cells or whole genome‐amplified single cells using primer extension preamplification (PEP), and discusses the statistical analysis of sperm‐typing recombination data. Newer methods for studying recombination over very short distances (a few kilobases) using total sperm DNA and allele‐specific PCR are also discussed.
PCR Methods of Genotyping
Authors: Hudson Thomas J. , Clark Chris D. , Gschwend Michele , Justice‐Higgins Erica
Published: June 2004

Two protocols discuss labeling PCR products with radioactive labels. The PCR products are then analyzed on a denaturing polyacrylamide gel and visualized by direct autoradiography. The resulting band pattern is used to define the SSLP genotype of the individual. Another method of genotyping uses a simple silver staining technique to detect PCR‐amplified SSLPs electrophoresed in denaturing polyacrylamide gels. In a fourth protocol, multiple unlabeled PCR products from one individual are pooled, separated on a denaturing polyacrylamide gel, transferred to a nylon membrane, and sequentially hybridized to nonradioactive end‐labeled probes derived from the primers used to amplify each SSLP marker. The probes are detected on film using chemiluminescence. Support protocols describe the preparation of an M13 sequence ladder size standard and digoxigenin labeling of the probe, both of which are required for the chemiluminescent method.
Single‐Nucleotide Polymorphism Genotyping Using Microarrays
Authors: Patil Nila , Nouri Nassim , McAllister Linda , Matsukaki Hajime , Ryder Thomas
Published: June 2004

This unit provides a set of protocols for high throughput SNP genotyping using oligonucleotide microarrays. The basic protocol utilizes allele‐specific hybridization in which the microarray provides both the allele discrimination and read‐out. In the alternate approach, the allele discrimination is accomplished in solution and then the array is used to provide the read‐out of the genotyping reaction.Single nucleotide polymorphisms (SNPs) offer several advantages over other genetic markers including their high density in the.
Statistical Analysis of Radiation Hybrid Data
Authors: Chakravarti Aravinda
Published: June 2004

This unit opens with a comparison of radiation hybrid and somatic cell hybrid analyses. Definitions, assumptions, and a mathematical model for radiation hybrid analysis are presented. In addition, parametric and nonparametric methods of data analysis are presented.
Use of Commercially Available Radiation Hybrid Panels
Authors: Dechairo Bryan M. , Carey Alisoun H.
Published: June 2004

Several panels are available for puchase, and this unit provides update information on the use of the three commercially available panels and on the interpretation of mapping results using the Internet.Radiation hybrid panels continue to serve as integral biological reagents in physical mapping projects and positional cloning
In Situ Hybridization to Metaphase Chromosomes and Interphase Nuclei
Authors: Knoll Joan H. M. , Lichter Peter
Published: June 2004

In situ hybridization is used to determine the chromosomal map location and the relative order of genes and DNA sequences within a chromosomal band. It can also be used to detect aneuploidy, gene amplification, and subtle chromosomal rearrangements. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in the first basic protocol. Two support protocols are provided to amplify weak fluorescent signals obtained in FISH. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described in alternate protocols. Nonisotopic labeling of DNA probes by nick translation is described in a support protocol. The order of closely spaced FISH probes along chromosomes in interphase nuclei can be determined. A basic protocol for isotopic in situ hybridization (IISH) with 3H is provided followed by a support protocol for preparation of autoradiographic emulsion.
Chromosome Microdissection
Authors: Meltzer Paul , Bittner Michael
Published: June 2004

This unit describes chromosome microdissection methodologies that are useful in both clinical and research laboratories. In combination with FISH, region‐specific probes can be made to determine the origin of marker chromosomes. Another application of the microdissection technology is in the generation of regionspecific cDNA libraries, known as microdissection‐mediated cDNA capture.
Mitotic Chromosome Preparations from Mouse Cells for Karyotyping
Authors: Akeson Ellen C. , Davisson Muriel T.
Published: June 2004

This unit contains protocols for the preparation of mitotic chromosomes from mouse peripheral blood, Giemsa banding of those chromosomes, and classification into a karyotype, including recognition of some common pitfalls of misidentification and information for determining aberrant chromosomes. The methods described can be used to identify visible chromosomal rearrangements and their precise cytological breakpoints in the living mouse. In conjunction with fluorescent in situ hybridization (FISH), the metaphase spreads can also be used for the linear placement of loci on a chromosome and for determining the insertion site(s) of a foreign transgene.
Single‐Nucleotide Sequence Discrimination In Situ Using Padlock Probes
Authors: Nilsson Mats , Landegren Ulf , Antson Dan‐Oscar
Published: June 2004

DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation. This mechanism has been exploited to distinguish DNA sequence variants in situ using so‐called padlock probes. Padlock probes are linear oligonucleotides with target‐complementary sequences at both ends, and an on‐target‐complementary segment in between. The end sequences are brought next to each other upon hybridization to the target DNA sequence, and if the ends are perfectly matched to the target sequence, they can be joined by a DNA ligase. Padlock probes detect target sequences with very high specificity, because both probe segments must hybridize to the target for circularization to occur. This unit presents a protocol for discrimination between closely similar DNA sequences in situ using padlock probes. A discussion of methods for greatly amplifying the signal from circularized probes is also included.DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation.
Strategies for Large‐Insert Cloning and Analysis
Authors: Moir Donald T. , Smith Douglas R.
Published: June 2004

This revised introduction provides an overview of several new methods and resources available to researchers involved in generating and screening large insert clones. Emphasis has been placed on the use of bacterial artificial chromosome clones. The table of available libraries has been updated and focused on the proven, widelyused libraries of YAC and BAC clones. Finally, the table of physical mapping databases has been expanded to include web sites for each of the individual human chromosomes, where available, as well as the wholegenome efforts with radiation hybrid and STS/EST mapping. A rapid convenient degenerate primervector primer method for obtaining PCR fragments representing the ends of P1 clone inserts has been added to Unit here. In addition, Unit here has been expanded to include a method for building BAC contigs by sequencing BAC insert ends.This revised introduction provides an overview of several new methods and resources available to researchers involved in gener This revised introduction provides an overview of several new methods and resources available to researchers involved in gener
Construction of Bacteriophage P1 Libraries with Large Inserts
Authors: Sternberg Nat , Shepherd Nancy S.
Published: June 2004

The bacteriophage P1 cloning system was originally developed as an alternative to YAC and cosmid systems for cloning high‐molecular‐weight genomic DNA. This unit details the preparation of the bacteriophage P1 library. Three support protocols provide the raw materials for the basic procedure, including the vector (pAd10sacBII), the mammalian DNA inserts, and the two packaging extracts that contain the viral proteins necessary to construct a P1 bacteriophage incorporating the vector and insert. A fourth support protocol describes how to induce replication of the plasmids cloned in the basic protocol, isolate the cloned DNA, and analyze the final products.
Screening Large‐Insert Libraries by Hybridization
Authors: Ross (gridding: YAC, BAC, and PAC colony hybridization) Mark T. , LaBrie (BAC/PAC colony hybridization and analysis) Sam , McPherson (overgo probes and hybridization) John , Stanton Vincent P.
Published: June 2004

Three types of hybridization screening are discussed in this unit colony hybridization, DNA hybridization, and YAC Alu‐PCR‐product hybridization. The first protocol describes methods for arraying large‐insert clones. Subsequent protocols discuss hybridizing YAC and BAC/PAC colony blots. An alternate protocol provides a guide for preparing and hybridizing with, high‐specific‐activity oligonucleotide probes called “overgo” probes. Support protocols indicate the procedures for identifying the plate and well address of positive clones by manual and software‐assisted methods. Hybridization of Alu‐PCR‐product probes to Alu‐PCR products of YAC clones are provided. An alternate protocol describes a method for preparing DNA from individual YACs and for producing and using Alu‐PCR product blots from individual YACs. Additional support protocols provide guidelines for preparing Alu‐PCR probes from cosmids, YACs, BACs/PACs, and somatic cell hybrids, and describe the use of 96‐well plates to radiolabel and spin‐column‐purify large numbers of probes. A method for YAC walking is also provided.Three types of hybridization screening are discussed in this unit colony hybridization, DNA hybridization, and YAC Alu‐PCR.
Purification and Characterization of YACs Containing Large Inserts
Authors: Gemmill Robert M. , Bolin Richard , Strauss William M. , Pavan William
Published: June 2004

This unit provides protocols for characterizing DNA segments cloned in YACs and for purifying YACs from yeast chromosomes. The first basic protocol describes Southern blotting and partial‐digest restriction analysis of YACs. These methods are useful for determining the size and complexity of the cloned insert DNA, the presence and location of particular restriction sites or sequences, and even the species of origin of the insert DNA (indicated by hybridization to species‐specific repetitive elements such as Alu repeats). The second basic protocol describes gel purification of YACs for use in procedures requiring pure YAC DNA, such as mammalian‐cell transformation and subcloning into smaller insert vectors. The third basic protocol details characterizing and analyzing YACs: in vivo fragmentation via homologous recombination with specialized fragmentation vectors containing specific probe sequences or repetitive elements, followed by Southern blotting with YAC‐ and human‐derived probes.
Constructing Contigs from Large‐Insert Clones
Authors: Foote Simon , Marshall Vikki , Munroe David J. , Segre Julia A.
Published: June 2004

This unit describes three approaches that are widely used to define alignments between overlapping clones bearing large‐insert genomic DNA and to generate extensive contiguous overlapping sets of clones (contigs). The three approaches are sequence‐tagged site (STS) content mapping, repetitive‐element hybridization fingerprinting, and Alu‐PCR fingerprinting. Methods for isolating the necessary BAC DNA suitable for automated fluorescent sequencing and generating new STS markers are discussed in support protocols. An alternate protocol presents repetitive‐element hybridization fingerprinting to detect overlaps and build contigs with full‐genomic YAC libraries.
Building Larger YACs by Recombination
Authors: Silverman Gary A.
Published: June 2004

Despite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact, but are represented as a collection of overlapping clones or contigs. Fortunately, the relatively high frequency and fidelity of homologous recombination in Saccharomyces cerevisiae can be used to reconstruct intact genes within a single clone by splicing together overlapping DNA segments. This unit describes two protocols for carrying out such homologous recombination; one relies on the meiotic phase of the yeast cycle, while the other utilizes the mitotic phase of the yeast life cycle.Despite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact.
Transfer of YAC Clones to New Hosts by Karyogamy‐Deficient Mating
Authors: Spencer Forrest , Simchen Giora
Published: June 2004

This unit provides a protocol for moving yeast artificial chromosome (YAC) clones to new yeast hosts using basic microbial techniques and pulsed‐field gel analysis. In contrast to other methods that can be used to transfer YAC clones, this requires neither optimization to achieve high‐efficiency DNA‐mediated transformation of chromosome‐sized DNA nor specialized equipment for tetrad dissection and analysis. Instead, chromosome (YAC) transfer is selected in rare segregants (“YACductants”) from a yeast mating that is rendered incomplete in most cell pairings by the presence of a kar1 (karyogamy‐deficient) mutation in either parental strain. The in this unit details the transfer of a YAC clone from yeast strain AB1380 (host to nearly all existing YAC libraries) to YPH925, a strain with nonreverting genetic markers compatible with existing plasmid constructs useful in YAC modification.
Navigating Public Physical Mapping Databases
Authors: Stein Lincoln D.
Published: June 2004

This unit provides concise overviews of the many physical mapping resources available and relates them to the genetic and transcript maps. Useful information on resolution of the maps, how to access them, and how to interpret them is compiled and presented in a clear fashion. Especially useful is a set of detailed protocols describing how to construct an STS marker and how to map it by means of available yeast artificial chromosomes (YACs). An additional protocol describes accessing EST marker maps.
Direct Selection of cDNAs Using Genomic Contigs
Authors: Lovett Michael
Published: June 2004

In the , uncloned cDNA or cDNA from a library with highly repetitive sequences suppressed are hybridized to biotinylated genomic DNAs. The selected sequences are amplified by PCR and rehybridized to create a secondary‐selected enriched cDNA library. Biotinylation of cloned genomic DNA is presented in the first support protocol. Linker addition and suppression of repeats in uncloned DNA is presented in the second support protocol and insert amplification and suppression of repeats from libraries is detailed in the third support protocol.In the , uncloned cDNA or cDNA from a library with highly repetitive sequences suppressed are hybridized to biot.
Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR
Authors: Albertsen Hans , Thliveris Andrew
Published: June 2004

This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template.This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA.