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Sequencing exon by exon to identify mutations is both laborious and time consuming. Screening techniques have been sought to identify mutations without the need for this. Rapid detection of single base changes have focused on identification of the heteroduplex formed when wild-type and ...

The polymerase chain reaction (PCR) provides a rapid method for generating a large amount of a defined region of DNA without recourse to cloning. However, direct sequencing of this amplified material is tedious, time-consuming, and frequently generates large amounts of normal sequence d ...

Hemophilia A is an X-linked disorder that leads to a defect in blood coagulation. This is caused by mutations in the factor VIII gene, which results in its activity being reduced or abolished in the blood-clotting cascade. The factor VIII gene is 186 kb long with 26 exons, varying from 69 bp (exon 5) to 3106 bp (exon ...

In the last few years a number of methods have been developed that, in combination with the PCR method (1), have vastly improved the detection of mutations and polymorphisms. The most frequently used screening methods for mutation hunting in bleeding disorders are denaturing gradient gel ele ...

Intron 22 of the factor VIII gene contains a 9.5kb region of DNA that is repeated on at least two other locations telomeric to, and at least 500 kb from, the gene. These regions are termed intron 22 homologous regions (int22h) (1) and contain the Factor VIII associated gene (F8A).

Since the cloning of the factor IX gene in 1982 (1), there have been several strategies employed for the identification of mutations in the mutationally heterogeneous hemophilia B population. Initially, such strategies inevitably employed Southern blotting to screen for gross delet ...

von Willebrand factor (vWF) is a large, complex glycoprotein that exists in plasma and platelets, and is synthesized by megakaryocytes and endothelial cells. vWF plays an essential role in hemostasis in at least two ways. It is involved in platelet adhesion to the damaged vascular endothelium ...

Von Willebrand Factor (vWF) in normal plasma is composed of a series of high molecular multimers, ranging in size from 8�105 to over 15�106 Daltons (1). The multimeric structure of vWF was first investigated by two-dimensional crossed immunoelectrophoresis (2D-IEP). In 1974, Kernoff et al. (2) u ...

Common alleles or polymorphisms form the basis of human diversity, and some of these polymorphisms closely linked on the same chromosome with a defective gene have been used for gene tracking in many genetic disorders. The success of any linkage analysis also relies on the nature of the polymorph ...

It has been recognized from the early 1800s that activation of coagulation can be initiated by the exposure of subendothelial layers (tissue factor), but it was the 1940s before factor VII (FVII) was included in this event (1,2). The discovery of hemophilia A and B supported the hypothesis that the int ...

Factor XI (FXI) is the zymogen precursor of an active serine protease that participates in the contact phase of coagulation. Synthesized in the liver, it circulates in the plasma in a noncovalent complex with high molecular weight kininogen (1) at a normal concentration of 5 μg/mL. (For clinical pu ...

Human antithrombin is a single-chain glycoprotein of MW 58 kDa and the most important plasma inhibitor of the coagulation serine proteases. It is a member of the serine protease inhibitor (SERPIN) family of proteins and in common with several other members of this family, its inhibitory activi ...

A number of reports have demonstrated that it is possible to identify correctly spliced low-level transcripts for tissue-specific genes in a number of non-specific tissues (1–3). The number of transcripts is low (approx 1 copy every 500–1000 cells) (2), but as they are initiated at the normal mRNA st ...

The single gene for protein C is located at position q13–q14 on chromosome 2 (1). Two groups have described human genomic clones of protein C isolated from phage l charon libraries using cDNA for human protein C as hybridization probes (2,3). The gene is approximately 11 kb long and is composed of 9 exons and 8 in ...

Protein S (PS) is a 71-kDa vitamin K-dependent glycoprotein first identified in human plasma by DiScipio and colleagues in 1977 (1), a year after the discovery of the anticoagulant protein C (PC) (2,3). A few years later, Walker demonstrated that PS acts as a cofactor for activated protein C (APC) in the pro ...

The prothrombin gene has recently been investigated as a candidate gene for venous thrombosis risk in selected individuals with a history of venous thrombosis (1). A genetic variation in the 3′-untranslated region (UTR) of the prothrombin gene, a G to A transition at nucleotide position 20210, ...

The first studies of plasma protein variation relied predominantly on conventional electrophoretic methods in media such as starch, agar, or cellulose acetate, which separated molecules on the basis of their relative differences in net charge and size. The introduction of isoelectr ...

The first update of the antithrombin mutation database published in 1993 used a revised classification for antithrombin variants (1). Currently, two deficiency states are recognized: type 1 deficiency is characterized by a parallel reduction in immunological and functional plas ...

Current techniques for DNA amplification and sequencing have greatly simplified the identification of genetic mutations underlying disorders of abnormal protein production. The need, however, for the direct characterization of an amino acid abnormality in a defective prote ...

Recently, there have been several reports describing the in vitro proliferation and differentiation of megakaryocytic progenitor cells, isolated from either bone marrow (BM) or peripheral blood (PB), into relatively pure human mega-karyocytes (1,2). These culture systems orig ...