bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dialysis and Concentration of Protein Solutions
Authors: Andrew Sarah M. , Titus Julie A. , Zumstein Louis 
Published: June 2004

AbstractConventional dialysis separates small molecules from large molecules by allowing diffusion of only the small molecules through selectively permeable membranes. This appendix describes dialysis of large‐ and small‐volume samples using cellulose membranes with pore sizes designed to exclude molecules above a selected molecular weight. A describes preparation of membranes for dialysis and discusses issues related to the selection of membranes including commercial kits.
Column Chromatography of Antibodies and Fragments
Authors: Smith John A. 
Published: June 2004

AbstractSince both size‐exclusion (SE) chromatography (also known as gel filtration) and ion‐exchange (IEX) chromatography are the fundamental methods on which antibody and fragment purifications are based, they are dealt with in detail in this appendix. The procedures for preparing the column matrix, for pouring the chromatography column, and for estimating the appropriate size of the column for the amount of antibody and fragment that are to be purified are all described.
Monoclonal Antibodies to Rat Leukocyte Surface Antigens, MHC Antigens, and Immunoglobulins
Authors: Puklavec Michael J. , Barclay A. Neil 
Published: June 2004

AbstractThe CD nomenclature used for human‐leukocyte surface antigens is now being widely applied to naming their homologs in other species. This appendix catalogs those CD antigens that have been clearly defined in the rat. There are also many other antigens defined in the rat, but only those for which good biochemical data are available, such as amino acid sequences, are given here. The most commonly used antibodies are summarized.
Construction of Reference Genetic Maps
Authors: Buetow Kenneth H. 
Published: June 2004

AbstractThis unit details the specialized resources and procedures used for constructing reference genetic maps. Construction of such maps in humans represents a subset of the linkage analysis problem. Objectives include the addition of a new locus to an established map, development of a detailed map of loci in a localized area, and construction of de novo maps. Conceptually, the procedures for updating a reference map through the addition of a subset of new loci are similar to those used in establishing linkage for a disease locus. However, construction of new maps of multiple loci is most efficiently accomplished using different family resources that permit the use of accumulated typing resources and alternative, highly efficient statistical methods.
Use of LINKAGE Programs for Linkage Analysis
Authors: Speer Marcy C. 
Published: June 2004

AbstractThis unit provides an introduction to human genetic linkage analysis for cases in which the genetic model is known (parametric or lod score linkage analysis). The LINKAGE computer package is used for computer analysis because it remains a training standard. The concepts presented for the LINKAGE programs, however, translate directly to other commonly used programs including the FASTLINK and VITESSE programs. This unit includes seven examples selected to illustrate many of the key concepts involved in performing linkage analysis. In Examples 1 through 4, a large pedigree segregating an autosomal dominant disease with reduced penetrance is studied for linkage to various markers. In Example 5, the effects of misspecification of the marker allele frequency on linkage analysis are demonstrated.
Disease Associations and Family‐Based Tests
Authors: Ewens Warren J. , Spielman Richard S. , Kaplan Norman L. , Martin Eden R. 
Published: June 2004

AbstractThis unit describes the statistical techniques necessary for performing family‐based association studies (e.g., the TDT) for genetic polymorphisms. Such studies have become increasingly important in the identification of genes that confer an increased risk to disease, particularly for common diseases with a complex etiology. The family‐based approach avoids some of the problems often encountered when applying the traditional case‐control design to genetic studies. The unit includes the Sib TDT (S‐TDT) method, which allows application of the principle of the TDT to sibships without parental data. This extension of TDT is potentially valuable for studying late onset diseases.
Human Mapping Databases
Authors: Talbot Conover , Cuticchia A. Jamie 
Published: June 2004

AbstractThis unit concentrates on the data contained within two human genome databasesGDB (Genome Database) and OMIM (Online Mendelian Inheritance in Man)and includes discussion of different methods for submitting and accessing data. An understanding of electronic mail, FTP, and the use of a World Wide Web (WWW) navigational tool such as Netscape or Internet Explorer is a prerequisite for utilizing the information in this unit.
Construction of Small‐Insert Libraries Enriched for Short Tandem Repeat Sequences by Marker Selection
Authors: Pulido Jacqueline C. , Duyk Geoffrey M. 
Published: June 2004

AbstractThese protocols construct a representative small‐insert genomic DNA library in a phagemid vector. First, size‐selected DNA fragments are ligated into a phagemid vector. In the second protocol, the resulting small‐insert phagemid library is propagated in a bacterial strain combining mutations at the dut and ung loci, which permit incorporation of uracil in place of thymidine during DNA replication. Infection of the phagemid library with M13 helper phage permits recovery of this library as single‐stranded DNA (ssDNA). Finally, this uracil‐substituted ssDNA is used as a template for primer extension using an oligonucleotide whose sequence corresponds to the STR class of interest [e.g., (GATA)10] as primer. The products of this primer‐extension reaction are transformed into an E. coli strain maintaining wild‐type genes at the dut and ung loci. Under these conditions, uracil‐substituted ssDNA will be restricted from growing by the host‐encoded uracil‐N‐glycosylase, while the primer‐extended products are capable of replicating.
Colony Hybridization to Screen for Microsatellites
Authors: Hudson Thomas J. 
Published: June 2004

AbstractSimple sequence length polymorphisms (SSLPs) for use as genetic markers are derived from the microsatellite repeat sequences found abundantly throughout eukaryotic genomes. Microsatellite DNA is identified in libraries of cloned DNA by colony hybridization to oligonucleotide probes containing simple sequence repeat (SSR) DNA, a common procedure in molecular biology laboratories. Plasmid or phagemid colonies are transferred to a nylon filter, grown briefly, fixed, and hybridized with one or more 32P‐labeled oligonucleotide probes. This protocol is adapted for the rapid processing of many filters at once and optimized for identification of long stretches of uninterrupted repetitive DNA sequences.
Characterization of (CA)n Microsatellite Repeats from Large‐Insert Clones
Authors: Litt Mike , Browne David 
Published: June 2004

AbstractThe most laborious part of developing (CA)n microsatellite repeats as genetic markers is constructing DNA clones to permit determination of sequences flanking the microsatellites. When cosmids or large‐insert phage clones are used as primary sources of (CA)n repeat markers, they have traditionally been subcloned into plasmid vectors such as pUC18 or M13 mp18/19 cloning vectors to obtain fragments of suitable size for DNA sequencing. This unit presents an alternative approach whereby a set of degenerate sequencing primers that anneal directly to (CA)n microsatellites can be used to determine sequences that are inaccessible with vector‐derived primers. Because the primers anneal to the repeat and not to the vector, they can be used with subclones containing inserts of several kilobases and should, in theory, always give sequence in the regions directly flanking the repeat. Degeneracy at the 3 end of each of these primers prevents elongation of primers that have annealed out‐of‐register.The most laborious part of developing (CA)n microsatellite repeats as genetic markers is constructing DNA clones to permit.
Genotyping by Ligation Assays
Authors: Nickerson Deborah A. , Ankener Wendy , Delahunty Claire , Kwok Pui‐Yan 
Published: June 2004

AbstractThis unit describes two methods of genotyping DNA sequences containing known nucleotide variations. The first protocol describes a colorimetric method for genotyping DNA samples amplified by the polymerase chain reaction (PCR) using an oligonucleotide ligation assay (OLA). The second protocol describes the ligase chain reaction (LCR), a method for simultaneously amplifying and genotyping genomic DNA samples. A describes the preparation of modified biotin‐ and digoxigenin‐labeled oligonucleotide primers.
High‐Throughput Genotyping Using the TaqMan Assay
Authors: Ranade Koustubh 
Published: June 2004

AbstractThis unit describes the use of the 5‐nuclease allelic discrimination assay, or TaqMan, for genotyping with single nucleotide polymorphisms (SNPs).In this method, the region flanking the polymorphism, typically 100 to 150 bp, is amplified in the presence of two probes, each specific for one or the other allele. Probes have a fluor called “reporter” at the 5 end but do not fluoresce when free in solution because they have a “quencher“ at the 3 end that absorbs fluorescence from the reporter. The presence of two probes allows one to detect both alleles in a single tube. Moreover, because probes are included in the PCR, genotypes are determined without any post‐PCR processing, a feature that is unavailable with most other genotyping methods. The design of probes and primers is described first, followed by PCR conditions, and finally methods for assigning genotypes in an automated fashion are discussed.
High‐Throughput Genotyping with Primer Extension Fluorescent Polarization Detection
Authors: Kwok Pui‐Yan 
Published: June 2004

AbstractThe primer extension assay with fluorescence polarization (FP) detection is a versatile assay for single nucleotide polymorphism (SNP) genotyping. With proper design, this homogeneous assay works under universal conditions. Fluorescence polarization occurs when a fluorescent dye is excited by plane‐polarized light and is detected if the fluorescent dye is part of a large molecule. In assays where small fluorescent molecules are turned into large fluorescent products, FP provides a simple way to determine if the reaction has occurred without the need for separation or purification of the reaction mixture. In the primer extension assay, DNA polymerase incorporates the complementary, allelic nucleotide onto the SNP primer designed to anneal to the target DNA one base upstream of the polymorphic site. When a fluorescently labeled nucleotide is incorporated, high FP is observed for that dye and the genotype of the DNA sample is determined.
Construction of Somatic Cell Hybrids
Authors: Jackson Cynthia L. 
Published: June 2004

AbstractSomatic cell hybridization is the method of choice to separate a chromosome of interest from the full chromosome complement and obtain a permanent source of the chromosome. This unit begins with the choice of fusion techniques and selectable markers for hybrids containing a chromosome of interest. The first set of protocols outline the production of whole‐cell hybrids by fusion of two cell lines: a monolayer (adherent) recipient and a donor that may be adherent or grown in suspension. The second set of protocols outline the production of micronuclei containing a limited number of chromosomes, and enucleation of the micronuclei to form microcells for fusion with recipient cells. Support protocols describe the preparation and use of cloning cylinders to isolate colonies in tissue culture, subcloning of whole‐cell hybrid populations to isolate lines that have segregated additional chromosomes, purification of microcell preparations, and molecular and cytogenetic methods for characterizing.
Chromosome Preparation from Cultured Peripheral Blood Cells
Authors: Bangs Charles D. , Donlon Timothy A. 
Published: June 2004

AbstractChromosome preparations currently provide the only direct view of the genome as a whole. Although molecular methods allow a more detailed analysis of specific regions of the genome, the study of genetics is not complete without an appreciation of the metaphase cell. The stimulated T cell system described in this unit is the most widely used means of obtaining large numbers of mitotic cells for genetic analyses. Synchronization of the cell cycle in culture is described, combined with direct inhibition of chromosome condensation, to yield longer high‐resolution prophase or prometaphase preparations. Such preparations are used for detailed analysis of microdeletions or subtle rearrangements, fine breakpoint analysis, and refined mapping. Microscope slide preparation of mitotic chromosomes from harvested cell culture suspensions is also explained in the support protocol.
Microscopy and Image Analysis
Authors: Difilippantonio Michael J. , Ried Thomas , McNamara George , Bieber Frederick R. 
Published: June 2004

AbstractThis unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). Computerized image analysis systems currently used in clinical cytogenetics are also discussed. Scanning and transmission electron microscopy as well as confocal microscopy are not covered in this unit despite their usefulness as invaluable tools for contemporary studies of biological systems.
High‐Resolution FISH Analysis
Authors: Heng Henry H.Q. , Tsui Lap‐Chee , Windle Bradford , Parra Irma 
Published: June 2004

AbstractMap order, orientation, and gap or overlap distance of closely linked DNA probes may be determined using fluorescent hybridization to decondensed DNA. The linear arrangement of released chromatin fibers not only simplifies the task of gene ordering, but also provides higher resolution with probes separated by greater distances than can be achieved in FISH with intact interphase nuclei. The first basic protocol of this unit describes an alkaline lysis procedure for generating free chromatin from cultured cells for FISH analysis. A support protocol describes an empirical approach to optimize conditions for preparation of free chromatin. An provides a method for producing free chromatin from cultured lymphocytes with drug treatment. The second basic protocol, high‐resolution FISH mapping with free chromatin, is a modification of the method used for FISH mapping of interphase nuclei.
Comparative Genomic Hybridization
Authors: DeVries Sandy , Gray Joe W. , Pinkel Daniel , Waldman Frederic M. , Sudar Damir 
Published: June 2004

AbstractComparative Genomic Hybridization (CGH) is a powerful molecular cytogenetic technique that permits assessment of DNA copy number on a genome‐wide scale. Of note, this methodology uses tumor DNA as a probe for fluorescence in situ hybridization (FISH) to normal metaphase chromosomes and does not require dividing cells from the tumor specimen. This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.
Construction of YAC Libraries with Large Inserts
Authors: Foote Simon , Denny Christopher 
Published: June 2004

AbstractThe yeast artificial chromosome (YAC) cloning system makes it possible to clone large pieces of genomic DNA into yeast. Libraries have been made containing clones with inserts in the megabase‐pair range. The basic protocol in this unit describes preparation of YAC vectors and transformation of ligated DNA into yeast spheroplasts. A support protocol describes titration of Lyticase to make spheroplasts. Additional support protocols detail two methods for partial digestion of genomic DNA: EcoRI restriction endonuclease‐EcoRI methylase competition and the partial digestion of genomic DNA by use of limiting amounts of Mg2+, respectively.
Construction of Chromosome Jumping and Linking Libraries in E. coli
Authors: Drumm Mitchell L. 
Published: June 2004

AbstractChromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant point on the same chromosome without cloning the intervening sequences as in chromosome walking. A linking library consists of genomic fragments that each contain a particular restriction site with a marker inserted into it. Because the sequences flanking the marker represent the termini of two contiguous genomic restriction fragments, the linking clones can be used, in conjunction with a specific jumping library made with the same restriction enzyme, to expedite jumping between consecutive restriction fragments. Separate protocols present strategies for constructing general and specific jumping libraries from total genomic DNA. An additional protocol details construction of a linking library from flow‐sorted chromosomes. Support protocols provide instructions for preparing genomic insert DNA, supF gene fragments, and Chromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant.
Screening Large‐Insert Libraries by PCR
Authors: Chinault A.Craig , Sternberg Nat L. 
Published: June 2004

AbstractThis unit describes procedures for screening large‐insert libraries by multistep polymerase chain reaction (PCR) analysis of DNA samples from clone pools. In the basic protocol, PCR amplification and agarose gel electrophoresis are used to identify successively smaller pools of DNA or colonies that contain clones with the appropriate‐sized amplification product. In the last screening step, individual clones are identified. The first and second support protocols describe the preparation of DNA and yeast‐cell pools for screening, and the first alternate protocol describes the preparation of crude lysates suitable for PCR from individual clones or small‐scale pools.
Rescuing YAC‐Insert Ends as E. coli Plasmids
Authors: Hermanson Gary G. , Evans Glen A. 
Published: June 2004

AbstractEnd clones from YACs (terminal fragments of the YAC‐insert DNA cloned into a plasmid vector) are essential ingredients for contig building using chromosome walking strategies, for fluorescent in situ hybridization experiments, and for generating repeat‐free probes for pulsed‐field gel electrophoresis or genetic linkage analysis. The describes a method for rescuing the CEN (centromere) ends of YACs constructed in the vector pYAC4. However, because this method relies on XhoI and SalI sites, which are relatively rare in the mammalian genome, it is not always possible to obtain a subclone in this manner. An alternate protocol presents a method utilizing integrative plasmid‐rescue vectors to facilitate the isolation of both ends of any YAC clone even in the absence of convenient restriction enzyme sites.
Deriving Probes From Large‐Insert Clones by PCR Methods
Authors: Albertsen Hans , Thliveris Andrew , Riley John H. , Munroe David J. , Watkins Paul , Basson Craig T. 
Published: June 2004

AbstractThis unit describes several polymerase chain reaction (PCR)‐based methods to obtain DNA fragments from clones with large inserts without prior knowledge of the insert DNA sequence. The protocols can be categorized into three groups: (1) methods to generate DNA fragments at random representing the entire length of the cloned insert, (2) methods to generate DNA fragments representing the extremities of an insert, and (3) methods to generate complex probes suitable for fluorescence in situ hybridization. Support protocols describe direct cloning of these PCR products and the isolation of total yeast DNA from yeast artificial chromosome (YAC) clones.
Introduction of Large Insert DNA into Mammalian Cells and Embryos
Authors: Reeves Roger H. , Cabin Deborah E. , Lamb Bruce 
Published: June 2004

AbstractThis unit provides a set of protocols for introducing large insert DNA into cultured mammalian cells and embryos. Two different methods, spheroplast fusion and lipofection, are described for effecting transfer of YACs or gel‐purified YAC DNA into cells. Additional protocols discuss preparing and transferring BACs into cells by lipofection and into embryos by microinjection.
Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
Authors: Osoegawa Kazutoyo , de Jong Pieter J. , Frengen Eirik , Ioannou Panayiotis A. 
Published: June 2004

AbstractThis unit describes the construction of BAC and PAC libraries. Two vectors, pCYPAC2 and pPAC4 have been used for preparing PAC libraries, and a new BAC vector pBACe3.6 has been developed for construction of BAC libraries. A support protocol describes preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, simultaneous dephosphorylation with alkaline phosphatase, and subsequent purification through pulsed‐field gel electrophoresis (PFGE). For the preparation of high‐molecular weight DNA for cloning, support protocols provide procedures for embedding total genomic DNA from lymphocytes or animal tissue cells, respectively, in InCert agarose. Another support protocol details the next steps for the genomic DNA: partial digestion with MboI or with a combination of EcoRI endonuclease and EcoRI methylase, and subsequent size fractionation by preparative PFGE. The final support protocol covers the isolation of BAC and PAC plasmid DNA for analyzing clones.
Selective Isolation of Mammalian Genes by TAR Cloning
Authors: Kouprina Natalay , Larionov Vladimir 
Published: June 2004

AbstractThis unit describes procedures for selective isolation of specific chromosomal regions and entire mammalian genes as circular yeast artificial chromosomes (YACs). The isolation is based on transformation‐associated recombination (TAR) in yeast between genomic DNA and a linearized vector containing targeting sequences homologous to a region of interest. The details preparation of highly competent yeast spheroplasts and transformation of the spheroplasts by genomic DNA along with a TAR vector. Support protocols describe preparation of chromosome‐sized genomic DNA in solid agarose plugs, identification of positive clone pools among primary yeast transformants using PCR, and a quick method for preparing PCR templates from lysed spheroplasts. PCR analysis of each clone in the positive pool is performed to identify individual clones containing the gene of interest. A final support protocol describes retrofitting TAR‐isolated YACs into bacterial artificial chromosomes (BACs).
Isolation of Exons from Cloned DNA by Exon Trapping
Authors: Nisson Paul E. , Watkins Paul C. , Krizman David B. 
Published: June 2004

AbstractExon trapping is an RNA polymerase chain reaction (PCR) method to clone expressed sequences or exons directly from mammalian genomic DNA. The basic protocol in this unit describes the method for trapping internal exons from cosmid clones and the second basic protocol describes trapping of 3 terminal exons. An describes 3 terminal exon trapping, which avoids subcloning of target DNA by ligating it to the vector for direct transfection. A describes a rapid cloning procedure using uracil DNA glycosylase.
Identifying Transcribed Sequences in Arrayed Bacteriophage or Cosmid Libraries
Authors: Hochgeschwender Ute , Brennan Miles B. 
Published: June 2004

AbstractThis unit describes methods for identifying bacteriophage or cosmid clones that contain sequences present in the mRNA of a cell line or tissue. In the , a radiolabeled cDNA probe is synthesized by reverse transcription of poly(A)+ RNA isolated from the cell line or tissue of interest. The probe is incubated with DNA‐cellulose to remove highly repetitive sequences before it is used for hybridization analysis of filters from a phage or cosmid genomic library. After identification of positive clones from the library screen, the same probe can be used to screen Southern blots of restriction enzyme‐digested DNA from the positive clones. Support protocols describe preparation and testing of DNA‐cellulose.