bluuebunny/crossref_metadata_2025_split huggingface dataset:
Searching the NCBI Databases Using Entrez
Authors: Murphy Juliane , Baxevanis Andreas D. 
Published: June 2004

AbstractOne of the most widely‐used interfaces for the retrieval of information from biological databases is the NCBI Entrez system. Entrez capitalizes on the fact that there are pre‐existing, logical relationships between the individual entries found in numerous public databases. The existence of such natural connections, mostly biological in nature, argued for the development of a method through which all the information about a particular biological entity could be found without having to sequentially visit and query disparate databases. Two Basic Protocols describe simple, text‐based searches, illustrating the types of information that can be retrieved through the Entrez system. An Alternate Protocol builds upon the first Basic Protocol, using additional, built‐in features of the Entrez system, as well as providing alternative ways to issue the initial query. The Support Protocol reviews how to save frequently‐issued queries.
Amplification of Sequences from Affected Individuals
Authors: Watkins Hugh C. 
Published: June 2004

AbstractThis unit describes methods for obtaining DNA sequences from an individual affected by a genetic disorder. Target sequences that may be present at very low copy number in patient samples are amplified by the polymerase chain reaction (PCR). The first describes harvesting of mRNA from peripheral lymphocytes, which can even be utilized for genes with tissue‐specific patterns of expression. This technique has the advantage of ease of access to appropriate patient samples and also may provide a more efficient means of screening coding sequences than would analysis of individual exons. An alternate protocol describes modifications in PCR conditions that facilitate mutation analysis and sequencing. When the genomic sequence for a given candidate gene is known, the second may be used to obtain appropriate sequences for analysis of individual exons or noncoding regions of interest.
Detection of Mutations by RNase Cleavage
Authors: Watkins Hugh C. , Goldrick Marianna 
Published: June 2004

AbstractRibonucleases can specifically recognize and cleave RNA at the site of sequence mismatches in RNA‐DNA or RNA‐RNA hybrids. The cleavage products are then characterized by gel electrophoresis. In this unit, a procedure is presented for RNase cleavage of 32P‐labeled riboprobes (transcribed from a cloned copy of the normal sequence) that have been annealed to amplified sequences of a candidate gene or cDNA obtained from affected individuals. A explains how to prepare riboprobes from a genomic or cDNA template obtained from a nonmutant individual. An alternate protocol describes cleavage of RNARNA hybrids using a nonisotopic RNase cleavage mutation assay. Sequential PCR and in vitro transcription steps generate sufficient quantities of duplex RNA targets so that the cleavage products can be detected on a gel by ethidium bromide staining. The unit also discusses the use of alternative ribonucleases for cleaving singlebase mismatches.
Mismatch Detection Using Heteroduplex Analysis
Authors: Børresen Anne‐Lise 
Published: June 2004

AbstractThis protocol describes a technique that can be used to identify point mutations or single‐base polymorphisms in heterozygous individuals. This technique takes advantage of the fact that heteroduplex molecules containing single‐base mismatches can be separated under particular conditions of gel electrophoresis from nearly identical molecules containing no mismatches. RNA or DNA from a potentially heterozygous individual is amplified by PCR, and the products are denatured and allowed to renature, forming heteroduplexes. Renatured PCR products are run on nondenaturing mutation‐detection‐enhancement polyacrylamide gels (HydroLink MDE gels). On this gel, hybrid molecules containing a mismatch migrate more slowly than their corresponding homoduplexes. This protocol describes the analysis of unlabeled PCR products; however, radiolabeled PCR products can also be analyzed by this method.
Single‐Strand Conformation Polymorphism Analysis Using Capillary Electrophoresis
Authors: Larsen Lars Allan , Christiansen Michael , Vuust Jens , Andersen Paal Skytt 
Published: June 2004

AbstractSingle‐strand conformation polymorphism (SSCP) is one of the most frequently used mutation detection methods. This unit describes a method of SSCP with automated analysis by capillary electrophoresis in order to increase the capacity and throughput. A protocol is provided for sample preparation. For a medium throughput laboratory, a single capillary instrument, as described in this unit, may be quite sufficient. In many cases, however, screening for mutations in large population groups requires a high throughput, and this is best obtained through the use of a multi‐capillary instrument, as discussed.
Preparation and Culture of Products of Conception and Other Solid Tissues for Chromosome Analysis
Authors: Warburton Dorothy 
Published: June 2004

AbstractIn clinical settings, chromosome studies are usually performed on solid tissue other than solid tumors for one of two reasons: the tissue biopsy is the only tissue available from the patient, or tissues other than the standard peripheral blood lymphocytes must be examined because of suspected mosaicism. The,, anddescribe a method for culturing tissue samples to be used for preparation of metaphase chromosomes and subsequent examination or for biochemical or DNA analysis. Thedescribes mechanical disruption of the specimen to provide free access of the cells to the culture surface, whereasdescribes a more efficient enzymatic disruption procedure.anddescribe methods of collecting tissue samples., which can sometimes be applied to placental tissue, involves use of mitotically active chorionic villus tissue to prepare metaphase chromosome spreads directly without the need for cell culture.
Diagnosis of Fanconi Anemia by Diepoxybutane Analysis
Authors: Auerbach Arleen D. 
Published: June 2004

AbstractFanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive pancytopenia and a high risk of malignancies, particularly acute myelogenous leukemia (AML). Diepoxybutane (DEB) analysis is the preferred test for FA because other agents have higher rates of false‐positive and false‐negative results. in this unit applies the DEB test to rule out a diagnosis of FA using a peripheral blood sample from the patient. provides instructions for working with DEB. describes staining slides for chromosome‐breakage analysis that is performed on unbanded metaphase preparations. provides a detailed method for applying the DEB test to cultured fibroblasts that grow as a monolayer attached to the bottom of the flask. outlines methods for using the DEB test for prenatal diagnosis of FA, utilizing fetal cells obtained by chorionic villus sampling (CVS), amniocentesis, or fetal blood sampling.Fanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive pancytopenia and a high risk of malignanci.
Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH)
Authors: Schwartz Stuart , Micale Mark A. , Becker Laurie 
Published: June 2004

AbstractAlthough FISH has been used to clarify deletions or structural rearrangements, recent work has focused increasingly on its applications to interphase analysis. This unit describes preparation of uncultured amniotic fluid cells for FISH analysis. Cells are swollen, then slides are prepared using either a cytocentrifuge or standard methods. These are then fixed and permeabilized for subsequent FISH. An describes attachment of amniocytes to a glass or plastic surface followed by hypotonic swelling, fixation, and permeabilization for subsequent FISH. Interphase FISH analysis of amniotic fluid cells is also described.
Molecular Cytogenetic Analysis of Telomere Rearrangements
Authors: Lese Christa M. , Ledbetter David H. 
Published: June 2004

AbstractGenomic imbalances involving the telomeric regions of human chromosomes, which contain the highest gene concentration in the genome, are proposed to have severe phenotypic consequences. For this reason, it is important to identify telomere rearrangements and assess their contribution to human pathology. This unit describes the structure and function of human telomeres and outlines several FISH‐based methodologies that can be employed to study these unique regions of human chromosomes.
Molecular Analysis of Fragile X Syndrome
Authors: Nolin Sarah L. , Dobkin Carl , Brown W. Ted 
Published: June 2004

AbstractThe gene responsible for fragile X syndrome, fragile X mental retardation‐1 (FMR1), contains an unstable repeat sequence of (CGG)n. Additionally, an upstream promoter region of the gene—a CpG island—is abnormally methylated in most affected individuals. Amplification of CGG repeats and abnormal methylation show a correlation with affected status. The first basic and alternate protocols in this unit outline the use of the polymerase chain reaction (PCR) to rapidly detect the size of the repeat amplification. The second basic protocol describes the assessment of fragile X syndrome by direct Southern blot hybridization of genomic DNA digested with a pair of restriction enzymes, one of which is methylation‐sensitive. Extent of amplification and level of methylation can be simultaneously detected. A combination of the two techniques can be used to characterize the genotypes of individual members in identified fragile X families.
Analysis of Trinucleotide Repeats in Myotonic Dystrophy
Authors: Surh Linda C. , Mahadevan Mani , Korneluk Robert G. 
Published: June 2004

AbstractMyotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat in the 3‐untranslated region of a gene encoding a serine‐threonine protein kinase. There is no one method to detect the entire range of expansion sizes possible in affected patients, so current diagnostic approaches rely on analyzing samples by hybridization of both polymerase chain reaction (PCR)‐amplified CTG repeats (CTG‐PCR) and genomic DNA. In this unit, the describes the analysis of PCR‐amplified repeats transferred to a nylon membrane by Southern blotting and hybridized to an alkaline phosphatase‐labeled probe. The first support protocol describes a vacuum blotting technique for rapid transfer of the PCR product to the nylon membrane and the second support protocol describes the use of a radiolabeled oligonucleotide probe for hybridization. Analysis of genomic DNA by similar hybridization techniques is outlined in the second basic protocol.Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat
Detection of Nonrandom X Chromosome Inactivation
Authors: Thouin Melissa M. , Giron James M. , Hoffman Eric P. 
Published: June 2004

AbstractThis unit describes a PCR‐based assay for distinguishing between the two X chromosomes in female cells and assessing the percentage of cells having each parental X chromosome active. Methylation of CpG residues in gene promoters is a major mechanism of transcriptional silencing. In mammalian female cells, hypermethylation is the way in which one X chromosome is inactivated. The X‐inactivation assay described in the relies on methylation sensitivity. In this unit, the highly polymorphic and therefore typically heterozygous (CAG)n region of the 5 end of the coding region of the human androgen receptor gene (HUMARA), at Xq11.2, is used to distinguish and compare the methylation activity of the X chromosomes.
Amplification‐Refractory Mutation System (ARMS) Analysis of Point Mutations
Authors: Little Stephen 
Published: June 2004

AbstractThe amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base changes or small deletions. ARMS is based on the use of sequence‐specific PCR primers that allow amplification of test DNA only when the target allele is contained within the sample. Following an ARMS reaction the presence or absence of a PCR product is diagnostic for the presence or absence of the target allele. The protocols detailed here outline methods that can be used to analyze human genomic DNA for one or more mutations. The describes the development and application of an ARMS test for a single mutation; the extends this to multiplex ARMS for the analysis of two or more mutations. The describes a rapid DNA extraction method from blood or mouthwash samples that yields DNA compatible with the type of tests described.The amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change The amplification‐refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change
Molecular Analysis of Oxidative Phosphorylation Diseases for Detection of Mitochondrial DNA Mutations
Authors: Shoffner John M. 
Published: June 2004

AbstractOxidative phosphorylation (OXPHOS) diseases are caused by inherited or spontaneously occurring mutations in the mitochondrial DNA (mtDNA) or the nuclear DNA. Mutations in the mtDNA can be classified into two groups, rearrangements and point mutations. This unit describes a method for detecting rearrangements of the mtDNA, which involves Southern blot hybridization. Another protocol detects mtDNA point mutations using restriction analysis of polymerase chain reaction (PCR) products. The Southern blot method requires an mtDNA‐specific probe.
Protein Truncation Test
Authors: Vossen Rolf , den Dunnen Johan T. 
Published: June 2004

AbstractThe protein truncation test detects mutations at the protein level that lead to premature translation termination. The method has evolved considerably since is original publication in this manual. This thoroughly revised unit describes what is now the preferred method for performing the protein truncation test. Transcription and translation are performed in separate reactions; during translation, biotin‐labeled or N‐terminally tagged proteins are synthesized. The translation products are detected on immunoblots via chemiluminescence. An using coupled in vitro transcription/translation and radiolabeled proteins is also presented.
Internet Resources in Medical Genetics
Authors: Pagon Roberta A. , Tarczy‐Hornoch Peter , Hanson Nancy 
Published: June 2004

AbstractThis unit presents an overview of the most commonly used web‐based information resources for (1) clinicians seeking to apply molecular genetic testing to patient care or to understand the molecular basis of inherited diseases (e.g., GeneTests, or OMIM), (2) consumers seeking support group information or scientific and management information (e.g., Genetic Alliance, or The Family Village), and (3) professional societies. This unit presents an overview of the most commonly used web‐based information resources for (1) clinicians seeking to apply. This unit presents an overview of the most commonly used web‐based information resources for (1) clinicians seeking to apply.
Genotyping of Apolipoprotein E: Comparative Evaluation of Different Protocols
Authors: Ingelsson Martin , Shin Youngah , Irizarry Michael C. , Hyman Bradley T. , Lilius Lena , Forsell Charlotte , Graff Caroline 
Published: June 2004

AbstractDisease‐associated gene polymorphisms provide both scientific insight into pathophysiological mechanisms and clinical information regarding risk and progression. Of special interest is the ɛ4 allele of the apolipoprotein E gene, which has emerged as a substantial risk factor for late‐onset forms of Alzheimer disease and also influences the risk of cardiovascular disease. Genotyping of apolipoprotein E can be performed by several methods; presented here are a quality and cost‐benefit analysis of four different protocols on a cohort of 42 clinical samples is included in the unit. Each method resulted in genotyping with a high sensitivity and specificity. The newer microtiter‐plate‐based high‐throughput techniques, fluorescence polarization and SNaPshot analysis, were as reliable as the traditional techniques of restriction fragment length polymorphism analysis and reverse hybridization. The reverse hybridization method tends to be more cost‐ and time‐effective when the number of analyses is limited, although economy of scale favors fluorescence polarization or SNaPshot analysis in larger studies. The latter approaches also provide the flexibility to investigate other polymorphic disease markers.
Methylation‐Specific PCR
Authors: Herman James G. , Baylin Stephen B. 
Published: June 2004

AbstractMethylationspecific PCR is a rapid method used to determine the methylation status of DNA. Not only does methylation affect the expression of genes in normal cells, but it is now known that disease processes such as cancer can result in abnormal DNA methylation patterns. Methylationspecific PCR can be used to investigate imprinted genes, to assess human tumors for clonality by studying genes inactivated on the X chromosome, and to examine abnormally methylated CpG islands in neoplasia.
Constructing Tissue Microarrays for Research Use
Authors: Schweizer Martina Storz , Schumacher Lela , Rubin Mark A. 
Published: June 2004

AbstractTissue microarrays (TMAs) were recently developed to facilitate tissue‐based research. TMAs can be used for any type of study where standard tissue slides have previously been used. However, there are numerous advantages in using TMAs. TMAs allow for screening of a large number of tissue samples under similar experimental conditions. They are also useful in that they conserve tissue and resources and can greatly multiply the number of experiments that can be performed with a limited amount of tissue samples. The basic protocol in this unit discusses how to prepare and cut tissue microarray slides. The resulting arrays may then be constructed manually or automatically, as described in the support protocols.
Genetic Analyses on DNA Microarrays
Authors: Stein Lincoln D. 
Published: June 2004

AbstractA great stir has coursed through the genetics community with the advent in 1996 of “chip technologies,” a series of related techniques that allow DNA hybridization‐based studies to be performed with unprecedented speed and parallelism. All chip technologies have in common a microarray, a small glass chip approximately one square centimeter in area, to which nucleotide sequences are bound. Fluorescently labeled nucleic acids are hybridized to the microarray and imaged with a laser scanner or fluorescence microscope. This unit offers an overview of the two dominant technologies, cDNA microarrays and oligonucleotide chips. It concludes with a discussion regarding how well microarrays perform real‐world expression analysis.
Analysis of Expression Data: An Overview
Authors: Grewal Anoop , Lambert Peter , Stockton Jordan 
Published: June 2004

AbstractMicroarray‐based methods generate large amounts of data that must be stored, visualized, and analyzed appropriately. The unit discusses normalization of transcriptional profiling data, statistically acceptable ways of validating measurements, database issues, and analysis of normalized data. This unit concentrates on the software package GeneSpring.
Biosafety in Handling Gene Transfer Vectors
Authors: Braun Andrew 
Published: June 2004

AbstractThis unit is devoted to safety issues that must be considered when generating and working with vectors for gene therapy. A short description of the relevant regulatory bodies and mechanisms is followed by a general discussion of containment and safety procedures. This is followed by vector‐specific guidelines for nine commonly used vector systems, including adenovirus, vaccinia, HSV‐1, lentivirus, and non‐viral vectors. The unit also outlines the problems encountered in taking a promising vector to clinical trials.This unit is devoted to safety issues that must be considered when generating and working with vectors for gene therapy.
Preparation of Adenovirus‐Polylysine‐DNA Complexes
Authors: Cotten Matt , Baker Adam , Birnstiel Max L. , Zatloukal Kurt , Wagner Ernst 
Published: June 2004

AbstractThis unit describes preparation of adenovirus‐polylysine‐DNA complexes, which is useful for transfection of DNA into a variety of cell types. A DNA complex is prepared with biotinylated adenovirus and streptavidin‐polylysine, coupled to transferrin, and used to transfect cells. Several support protocols describe methods for adenovirus growth and purification, biotinylation, inactivation with psoralen, and quantitation of the adenovirus particles. Additional support protocols descibes preparation of streptavidin‐polylysine and transferrin‐polylysine, necessary for the basic procedure. The DNA used for transfection must be free of lipopolysaccharide (LPS), and two methods for removing LPS are described. A more direct polylysine‐virus linkage that is simple and requires no exotic reagents can be used for transfection. This protocol requires polylysine‐modified adenovirus, prepared as described. An alternate protocol describes transfecting cells with free virus and DNA condensed with a polycation.
Adenoviral Vectors
Authors: He Tong‐Chuan 
Published: June 2004

AbstractAdenoviral vectors have been widely used as efficient gene delivery vehicles for gene therapy and vaccine development, as well as gene function studies. The recently developed AdEasy system is a simple and efficient method for rapid generation of recombinant adenoviruses. Unlike traditional adenoviral vectors, the viral backbone described here is supplied as a supercoiled plasmid rather than as a linear viral DNA, facilitating backbone amplification. The recombination step is performed in E. coli rather than in mammalian cells, taking advantage of the high efficiency of homologous recombination in bacteria. This unit includes the complete set of protocols needed to generate recombinant adenoviruses using the AdEasy system.
Liposome Vectors for In Vivo Gene Delivery
Authors: Whitemore Mark , Li Song , Huang Leaf 
Published: June 2004

AbstractGene therapy with nonviral, cationic liposomebased vectors has demonstrated promising results in a variety of in vivo models and some clinical trials. In contrast to viral vectors, cationic liposomebased vectors usually induce just mild inflammatory reactions, making them suitable for repeated injections in animals. This unit describes the preparation and delivery of two liposomebased vectorsGene therapy with nonviral, cationic liposomebased vectors has demonstrated promising results in a variety of in vivo models.
Development of Molecular Genetic Interventions for HIV Infection
Authors: Woffendin Clive , Ranga Udaykumar , Nabel Gary J. 
Published: June 2004

AbstractThis unit includes a set of protocols for the ex vivo transfer of genes into CD4+ T cells, to be used in the initial evaluation of genes protecting against HIV infection in gene therapy protocols. The describes isolating and expanding CD4+ T cells from the patient. The cells are then transduced by either retroviral transduction or particle‐mediated gene transfer and reinfused into the patient. To monitor the effectiveness of gene transfer, genomic DNA is prepared from the patient's cells. Detection of vector DNA by PCR analysis of the patient's genomic DNA following gene transfer is also described in detail.
Gene Delivery to the Airway
Authors: Duan Dongsheng , Zhang Yulong , Engelhardt John F. 
Published: June 2004

AbstractThis unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of human bronchial xenografts as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols.