bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quality Assurance and Quality Control in Clinical Cytogenetics
Authors: Watson Michael S. 
Published: June 2004

AbstractThe goal of any clinical laboratory should be to provide patients with the most accurate test results possible. This is accomplished through various overlapping programs that continuously monitor and optimize all aspects of a test, including the decision to request the test, the test itself, and the reporting of results to the referral source and patient. The levels at which test performance and accuracy can be optimized are encompassed under quality control (QC) and quality assurance (QA). This unit reviews quality control and assurance guidelines, in addition to discussing how to establish a quality assurance program.
Preparation of Metaphase Spreads from Chorionic Villus Samples
Authors: Jackson Laird , Gibas Longina M. , Barr Marie A. 
Published: June 2004

AbstractChorionic villi are composed of an outer layer of trophoblastic cells and an inner mesenchymal cell core, both of fetal origin. Cytogenetic analysis of chorionic villi can be accomplished using material prepared in either of two ways. In the culture method described in this unit, villi are disaggregated by mechanical and enzymatic methods, and the resulting cell suspension is used to establish primary cultures. Mesenchymal cells of the villus core are released by this procedure and the fibroblasts are actively proliferative in tissue culture. Cultures can be used for cytogenetic analysis after ˜1 week. In the “direct” technique, presented here in an , Langhans cells of the cytotrophoblast, actively dividing cells in first‐trimester villi, are synchronized and arrested in mitosis after a short incubation period, and metaphase spreads are prepared.Chorionic villi are composed of an outer layer of trophoblastic cells and an inner mesenchymal cell core, both of fetal origin
Analysis of Sister‐Chromatid Exchanges
Authors: German James , Alhadeff Becky 
Published: June 2004

AbstractTwo requirements for the cytogenetic analysis of sister‐chromatid exchanges (SCEs) in somatic cells are (1) a population of actively proliferating cells that will provide an adequate number of metaphases and (2) sister chromatids that in some way are differentially labeled or stained in the metaphases. SCEs can be recognized as abrupt discontinuities in the staining patterns of the two chromatids of a metaphase chromosome at what appear to be identical sites, with reciprocal switching from one chromatid to its sister. This protocol uses phytohemagglutinin (PHA)‐stimulated cultures of blood lymphocytes as a source of proliferating cells. The cells are incubated with the thymidine analog BrdU. Slides prepared from fixed cells with BrdU‐substituted chromosomes are treated with Hoechst 33258, exposed to light and heat, and then Giemsa‐stained to produce differentially stained chromosomes. The chromatids with bifilar substitution exhibit a lighter purple stain than their unifilarly substituted sister chromatids.
Determination of Chromosomal Aneuploidy Using Paraffin‐Embedded Tissue
Authors: Schofield Deborah E. 
Published: June 2004

AbstractTissue processed in pathology departments is generally fixed in formalin and embedded in paraffin blocks. Depending upon the nature of the question asked, two different approaches are taken to analyze this material and are presented in this unit. If information about the spatial relationship of one cell to another is not necessary, a thick section of tissue can be disaggregated and the resulting intact nuclei analyzed. The number of hybridization signals per nucleus is tabulated; variance from two hybridization signals per nucleus indicates aneuploidy. If the spatial relationship of one cell to another needs to be preserved, intact tissue sections on glass slides can be analyzed. Tissue processed in pathology departments is generally fixed in formalin and embedded in paraffin blocks.
Overview of Molecular Genetic Diagnosis
Authors: Korf Bruce R. , Pagon Roberta A. 
Published: June 2004

AbstractThis unit describes the two approaches for molecular genetic diagnosis, direct mutation analysis and linkage studies. Direct mutation analysis is possible if the gene responsible for a disorder has been cloned and techniques exist to detect mutations. Genetic linkage analysis is used when polymorphic markers are available that are closely linked to a disease gene but the gene itself has not been cloned, or when it is difficult or impossible to detect mutations in the gene. For direct mutation analysis, the unit offers a review of the types of mutations that can be detected and the strategies that can be employed for their detection. The unit then describes the types of disorders that are best suited for linkage‐based analysis and offers guidance for interpreting the data.
Quality Assurance and Quality Control in Diagnostic Molecular Biology
Authors: Murphy Patricia D. 
Published: June 2004

AbstractThe newest forms of clinical diagnostic testing are based upon tools gleaned from molecular biology. Manipulations of RNA and DNA from various tissue sources (e.g., skin, blood, urine, excised tumor) can provide valuable information about an individual's genetic status or the basis or potential for metastasis of a tumor. As of 2002, over 200 laboratories in the United States provide clinical services. Particularly when such information is to be used to provide genetic counseling or determine treatment, care must be taken to ensure accuracy in the generation and reporting of results. This unit outlines the aspects of quality assurance that are important in nucleic acid‐based diagnostic testing. More specifically, it describes the three elements of a quality assurance program: the directors qualifications, on‐site laboratory inspection, and interlaboratory comparison and proficiency testing. The unit also discusses how to evaluate if a particular genetic test is suitable for clinical testing.
Single‐Cell DNA and FISH Analysis for Application to Preimplantation Genetic Diagnosis
Authors: Chong Samuel S. , Gore‐Langton Robert E. , Hughes Mark R. , Miron Patricia Minehart 
Published: June 2004

AbstractThe goal of preimplantation genetic diagnosis (PGD) is to avoid transfer of embryos affected with a specific genetic disease or condition. This unit describes the steps involved in amplifying DNA from a single blastomere and specific assays for detecting a variety of DNA mutations. For some assays, whole‐genome amplification by primer‐extention preamplification (PEP) is performed prior to analysis. Support protocols describe the biopsy of one or two blastomeres from the developing preimplantation embryo, isolation for further investigation of all blastomeres from embryos shown to have the mutant allele, and isolation of single lymphocytes or lymphoblastoid cells as models for single‐cell DNA analysis. A procedure for FISH analysis on single interphase blastomeres is provided along with a support protocol for probe validation that is recommended as a preliminary step before performing any PGD FISH analysis.
Searching Online Mendelian Inheritance in Man (OMIM) for Information for Genetic Loci Involved in Human Disease
Authors: Baxevanis Andreas D. 
Published: June 2004

AbstractOnline Mendelian Inheritance in Man (OMIM) is a non‐sequence‐based information resource that can be of tremendous use to genomics researchers, physicians, and patients. There are two main ways in which a user can search the OMIM database. One may choose to search the OMIM database directly from the NCBI home page, as described in this unit. Alternatively, OMIM can be downloaded and run on any internal site where the user may want to keep from submitting data across the Web or where a local installation would be otherwise advantageous. describes how to navigate through an OMIM record. Online Mendelian Inheritance in Man (OMIM) is a non‐sequence‐based information resource that can be of tremendous use to genom Mendelian Inheritance in Man (OMIM) is a non‐sequence‐based information resource that can be of tremendous use to genomics researchers, physicians, and patients.
Overview of Genetic Diagnosis in Cancer
Authors: Korf Bruce R. 
Published: June 2004

AbstractBoth cytogenetic and molecular genetic studies can contribute to the management of patients with cancer. In some cases genetic markers are specific to particular tumor types and are useful in diagnosis. This can be helpful in distinguishing histologically similar tumors that may respond differently to treatment and can sometimes be of prognostic value. Genetic markers can also be tools for following the response of a tumor to therapy, providing a sensitive means of detection of relapse. This introductory unit considers some of the types of genetic changes that occur in association with malignancies and major approaches to their detection.
Metaphase Harvest and Cytogenetic Analysis of Solid Tumor Cultures
Authors: Fletcher Jonathan 
Published: June 2004

AbstractCytogenetic preparations of solid tumor specimens often demonstrate clonal chromosome aberrations. These clonal aberrations are useful in confirming the neoplastic nature of a given tumor and might also suggest a specific diagnosis or prognosis. Most solid tumors must be cultured for several days before metaphase cells can be obtained for cytogenetic analysis. Thedescribes harvesting of metaphase cells from cultures prepared from solid tumor specimens. The cells are fixed and then stained using a trypsin‐Giemsa (GTG) method to produce characteristic banding patterns for cytogenetic analysis. The firstdescribes disaggregation and short‐term tissue culture of solid tumors. The seconddescribes analysis of cells from lymph nodes infiltrated by lymphoma cells and the thirddescribes the direct analysis of cells from malignant effusion (fluid) samples.
Molecular Analysis of DNA Rearrangements in Leukemias and Non‐Hodgkin's Lymphomas
Authors: Longtine Janina , Fox Edward , Reynolds Carol , Sklar Jeffrey 
Published: June 2004

AbstractGenetic markers for leukemias and lymphomas include chromosomal translocations and antigen‐receptor gene rearrangements. Clonal rearrangements of immunoglobulin or T cell receptor (TCR) genes reflect clonal proliferations of lymphocytes, a characteristic feature of lymphoid neoplasia. These rearrangements can be detected as described in this unit by Southern blot hybridization or, in many instances, the polymerase chain reaction (PCR). Specific chromosomal translocations can also serve as markers for clonality, for malignant transformation, and for various defined subtypes of hematopoietic cancers. PCR protocols are described for detection of the two most commonly assayed translocations, t(9;22) of chronic myelogenous leukemia or acute lymphoblastic leukemia, and t(14;18) of follicular lymphomas.
Molecular Analysis of Gene Amplification in Tumors
Authors: Wasson Jonathon C. , Brodeur Garrett M. 
Published: June 2004

AbstractDetection of gene amplification in human cancer cells has both clinical and biological importance. Amplified genes can be classified in one of two categoriesas oncogenes or as genes conferring drug resistance. Both types of gene amplification may alter clinical management of the patient. The basic protocol describes preparation and quantitation of DNA from tumor tissue and the use of conventional Southern blot hybridization analysis to detect and quantify gene amplification. The first alternate protocol provides an approach to quickly screen tumor samples for gene amplification using slot blot hybridization analysis. The second alternate protocol describes the use of the polymerase chain reaction (PCR) for analyzing tumors that may be difficult to analyze because of degradation or limited amounts of DNA. A explains the proper methods for obtaining, processing, storing, and shipping tumor tissue for DNA analysis.
Differential Display of mRNA by PCR
Authors: Livesey F.J. , Hunt S.P. 
Published: June 2004

AbstractThis unit outlines the polymerase chain reaction (PCR)‐based technique of mRNA differential display, which identifies genes that are differentially expressed between cells or tissues. The basic protocol describes the actual differential display PCR reaction along with details of the identification, reamplification, and cloning of candidate differentially expressed genes. A support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.
One‐Step Enzymatic Purification of PCR Products for Direct Sequencing
Authors: Bum Kim Jae , Blackshaw Seth 
Published: June 2004

AbstractThe polymerase chain reaction (PCR) has become a powerful and widespread method for analyzing DNA. This protocol offers convenient enzymatic purification of PCR products for direct sequencing without the need for further subcloning, precipitation, or column purification. All reactions may be performed in 96‐well PCR plates and are compatible with large‐scale sequencing. This method is designed to be convenient and rapid. It is suitable for a variety of PCR templates, including plasmid, lambda, and genomic DNA.
Production of Pseudotype‐Retroviral Vectors
Authors: Yee Jiing‐Kuan 
Published: June 2004

AbstractRetrovirus pseudotype is defined as the genome of one retrovirus encapsidated by the envelope protein of a second virus. The host range of the pseudotype is that of the virus donating the envelope protein. Two procedures that use 293GP cells, which are derived from human kidney 293 cells, are described here. The first is based on the high transient transfection efficiency of 293 cells. The retroviral construct and an expression plasmid for VSV‐G are co‐transfected into 293GP cells that stably express MLV gag and pol proteins. Transiently generated virus is then harvested during consecutive days following DNA transfection. The second procedure involves stable 293GP cell lines containing the VSV‐G gene under the control of a promoter whose activity is regulated by tetracycline. Cell lines containing the retroviral vector of interest are established under noninduced conditions. Infectious virus can be harvested following the induction of VSV‐G expression in these cell lines.Retrovirus pseudotype is defined as the genome of one retrovirus encapsidated by the envelope protein of a second virus.
Production of Recombinant Adeno‐Associated Viral Vectors
Authors: Haberman Rebecca A. , Kroner‐Lux Gabriele , Samulski Richard Jude 
Published: June 2004

AbstractRecombinant adeno‐associated viral (rAAV) vectors are capable of delivering genes to both dividing and nondividing cells. This property, along with the physical stability of the virions, permits transduction of cells both in tissue culture and in the whole animal. In this unit, protocols for the adenovirus‐free production of rAAV vectors, their purification, and their quantitation are provided. The production and purification of the rAAV vectors are described, using two different methods. Two assays for determining rAAV titers are also presented: dot blot and in vitro infection with a transgene expression assay.
Construction of Replication‐Defective Herpes Simplex Virus Vectors
Authors: Goins William F. , Marconi Peggy , Krisky David , Wolfe Darren , Glorioso Joseph C. , Ramakrishnan Ramesh , Fink David J. 
Published: June 2004

AbstractAdvances in identification and characterization of gene products responsible for specific diseases of the nervous system have opened opportunities for novel therapies using gene transfer vectors for gene replacement. Herpes simplex virus (HSV)‐based vectors are particularly well suited for gene delivery to neurons of the central and peripheral nervous systems. The authors have developed methods to delete HSV‐1 IE gene functions and to subsequently introduce foreign genes into the HSV‐1 genome using homologous recombination. This unit describes methods for generating cell lines that complement multiple essential gene deletion mutants as well for generating such replication‐defective virus recombinants and inserting foreign DNA sequences into replication‐defective viral genomes, the last step in preparing a vector. Three support protocols describe methods for preparing virus stocks, titering virus, and preparing viral DNA.
Ex Vivo and In Vivo Gene Delivery to the Brain
Authors: Suhonen Jaana , Ray Jasodhara , Blömer Ulrike , Gage Fred H. 
Published: June 2004

AbstractThis unit describes methods for grafting genetically modified cells for ex vivo delivery of specific genes into the rat brain and direct delivery of transgenes to brain cells in vivo using recombinant viral vectors. These methods assess the function of a gene in the brain. The ex vivo approach of gene transfer to the nervous system depends on genetic manipulation of cells in vitro prior to grafting of the cells into the brain to enable production of a transgene at physiologically significant levels for a long period of time. This unit also includes procedures for transcardial perfusion to fix the tissue prior to analysis of expression and for sectioning of brains by use of a freezing sledge microtome. Thionine staining of tissue sections is also described.
Gene Delivery to Muscle
Authors: Springer Matthew L. , Rando Thomas A. , Blau Helen M. 
Published: June 2004

AbstractThe delivery of genes to skeletal muscle by myoblast implantation, DNA injection, or viral transduction has therapeutic applications for human neuromuscular and systemic disorders, many of which are now represented by transgenic or “knockout” mouse models. This unit describes the isolation and retroviral transduction of mouse myoblasts, the injection of myoblasts and plasmid DNA into mouse muscle, and histological methods for analyzing the recipient muscle. A procedure describing the injection of plasmid DNA into muscle with or without electric charge is also included.
Cancer Vaccines
Authors: Greten Tim F. , Jaffee Elizabeth M. 
Published: June 2004

AbstractThis unit describes the use of retroviral vectors that can be successfully employed for gene transfer into both primary tumor cultures and established cell lines. The unit includes procedures for assaying the stability of the vaccine following gene transfer. Techniques for maintaining the retroviral producer lines and titering the retroviral vectors are also described. A protocol for frozen storage of the vaccine (transduced tumor cells) is provided. In addition, methods are described for characterizing the vaccine cells following gene transfer. Directions for testing the expression of the transferred gene in the transfected tumor line are also given. The final protocol provides suggestions for designing in vivo animal experiments and discusses what has to be observed in a clinical setting.
Isolation of DNA from Forensic Evidence
Authors: Bing David H. , Bieber Frederick R. , Holland Mitchell M. , Huffine Edwin F. 
Published: June 2004

AbstractThis unit covers the many and varied methods for extracting DNA from such diverse specimens as blood, tissue, stamps and envelopes, and cigarette butts, among others. Modifications to the methods that allow the DNA to be used for either PCR or Southern blotbased analyses are also included.
Use of Mouse Models for the Analysis of Human Disease
Authors: Semsarian Christopher 
Published: June 2004

AbstractThe first half of this unit discusses the two main approaches to developing genetically engineered mouse models of human disease (i.e., transgenesis and gene targeting). The experimental steps for each method are discussed. Potential pitfalls, advantages, and limitations are also addressed. The second half of the unit describes two mouse models of hypertrophic cardiomyopathy, which illustrate the value of studying such models of human disease.The first half of this unit discusses the two main approaches to developing genetically engineered mouse models of human disease.
Use of Zebrafish Models for the Analysis of Human Disease
Authors: Zhu Hao , Zon Leonard I. 
Published: June 2004

AbstractThe zebrafish has emerged as a powerful animal model for human diseases. While it has long informed us about the biology of early development, it has recently come into favor for the investigation of clinically relevant problems. Genes conserved from fish to humans can be rapidly analyzed using the zebrafish embryo in what is essentially a transparent in vivo assay. This unit describes methodologies including genetic screening, targeted knockdowns, ectopic overexpression, and transgenesis.
Enzymatic Labeling of DNA
Authors: Tabor Stanley , Struhl Kevin 
Published: June 2004

AbstractThis appendix describes the preparation of uniformly labeled radioactive probes by nick translation and random oligonucleotide‐primed synthesis, as well as the end‐labeling of oligonucleotides by T4 polynucleotide kinase. Accompanying these protocols are methods for removing unincorporated dNTP precursors with spin columns and for measuring the specific activity of a probe by acid precipitation.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C. 
Published: June 2004

AbstractThe first section of this appendix discusses basic principles of sterile technique, while the second section discusses medium preparation. The basic protocol describes subculturing of a monolayer culture grown in petri plates or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An alternate protocol describes freezing suspension cells.
Preparation of DNA from Fixed, Paraffin‐Embedded Tissue
Authors: Fox Edward A. 
Published: June 2004

AbstractPurification of DNA from fixed, paraffin‐embedded tissue (PET) requires the removal of paraffin, proteins, and, in some cases, heavy metal ions. The resulting DNA is not generally suitable for Southern blot analysis. However, the polymerase chain reaction (PCR) can be used to amplify short regions of this DNA. The Basic Protocol is rapid and involves only a small number of manipulations, thus minimizing the possibility of contamination. The Alternate Protocol involves additional steps including extractions with xylenes and phenol/chloroform. This procedure is recommended as the primary approach when tissue has not been fixed in an optimal manner or as a secondary approach when the Basic Protocol has failed to produce satisfactory DNA.
Analysis of RNA by Northern Blot Hybridization
Authors: Brown Terry , Mackey Karol 
Published: June 2004

AbstractSpecific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Because they are single‐stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Thedescribes blotting and hybridization of RNA fractionated in an agarose‐formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot‐blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a.