Full Description
Laboratory studies in hemostasis have traditionally focused on abn- malities of platelet function or the quantitative and qualitative disorders that affect the proteins involved in blood coagulation. However, over the last 10 years there has been an explosion in our understanding of the molecular bases that underlie many of the inherited and acquired disorders of hemostasis. Many of these disorders are now routinely diagnosed and assessed by methods that involve genotypic analysis. Indeed in the late 1990s the distinction between molecular methods for research and for routine diagnosis is becoming incre- ingly blurred. The techniques and approaches that are used in hemostasis are manifold and published in isolation in a variety of publications. The aim, therefore, of this volume Hemostasis and Thrombosis Protocols is to pull together, into a single volume, the variety of techniques that are frequently used in the field of hemostasis. We have targeted this volume at laboratories who wish to move into the field of molecular hemostasis or who may already have some expe- ence in this area but wish to develop new areas of research and diagnosis. The chapters are wide-ranging and hopefully provide a broad overview of the d- fering applications in which these standard techniques can be used. Though the articles may appear relatively specific, the techniques contained within them are applicable to the study of many different disorders and we hope that they provide a series of ideas and concepts well-suited to problem solving.
Contents
Hemostasis.- Basic Techniques.- Isolation of DNA and RNA.- Amplification of DNA and RNA by PCR.- Direct Sequencing of PCR Products.- Solid-Phase Sequencing of Biotinylated PCR Products with Streptavidin-Coated Magnetic Beads.- Automated DNA Sequencing.- Detection of DNA by Silver Staining.- Promoter Studies in Hemostasis.- Methods of Mutational Analysis.- Detection of Mutations and Polymorphisms in Clotting Factors by Denaturing Gradient Gel Electrophoresis.- Screening for Mutations in DNA by Single-Stranded Conformation Polymorphism (SSCP) Analysis.- Screening for DNA Heteroduplexes in the Factor VII Gene Using Ethylene Glycol Gel Electrophoresis of Solvent-Treated 32P-Labeled PCR Products.- Detection of Mutations Causing Hemophilia A Using an In Vitro Coupled Transcription and Translation System.- Screening for Mutations in the Human Antithrombin Gene by Hydrolink D-5000™ and MDE™ Gel Electrophoresis.- Methods for Analyzing Inherited/Acquired Disorders of Hemostasis.- Detection of Mutations in Hemophilia A Patients by Chemical Cleavage of Mismatch Method.- Inversion Mutation Analysis in Hemophilia A by Restriction Enzyme Analysis and Southern Blotting.- Hemophilia B Mutational Analysis.- Screening for Candidate Mutations Causing von Willebrand's Disease (vWD).- Use of Intron 40 VNTR I in vWD Gene Tracking.- Multimeric Analysis of von Willebrand Factor.- Identification of Mutations in the Human Factor VII Gene.- Molecular Analysis in Factor XI Deficiency.- Mutational Analysis in Antithrombin Deficiency.- Ectopic Transcript Analysis in Human Antithrombin Deficiency.- Mutational Analysis of the Human Protein C Gene.- Analysis of the Protein S Gene in Protein S Deficiency.- Screening for the G to A Transition at Position 20210 in the 3?-Untranslated Region (UTR)of the Prothrombin Gene.- Screening for the Factor V Leiden Mutation.- Multiplex PCR for Detection of the Prothrombin 3?-UTR (G20210A) Polymorphism and the Factor V Leiden Mutation.- Isoelectric Focusing and Immunodetection of Plasma Antithrombin.- Characterization of Heparin Binding Variants of Antithrombin by Crossed Immunoelectrophoresis in the Presence of Heparin.- The Determination of Amino Acid Sequence Abnormalities in Proteins by HPLC Peptide Analysis.- Platelet and Megakaryocyte Analysis.- Molecular Biological Identification and Characterization of Inherited Platelet Receptor Disorders.- In Vitro Expansion of Megakaryocytes from Peripheral Blood Hematopoietic Progenitors.- Molecular Biology Studies with Primary Megakaryocytes.
