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    • 4. 发明申请
    • SEQUENCE ANALYSIS OF SACCHARIDE MATERIAL
    • 食品材料的序列分析
    • WO1996013606A1
    • 1996-05-09
    • PCT/GB1995002541
    • 1995-10-30
    • CANCER RESEARCH CAMPAIGN TECHNOLOGY LIMITEDGALLAGHER, John, ThomasTURNBULL, Jeremy, EwanHOPWOOD, John, Joseph
    • CANCER RESEARCH CAMPAIGN TECHNOLOGY LIMITED
    • C12Q01/34
    • C12Q1/34G01N2333/924G01N2400/40
    • In a method of analysing saccharide material such as glycosaminoglycans (GAG's) to determine the sequence of monosaccharide units in oligosaccharide chains thereof, the saccharide chains are end referenced, e.g. by labelling or tagging at their reducing ends, and the saccharide material is subjected to a controlled partial depolymerisation using a selective scission reagent, for example low pH nitrous acid, which cleaves internal glycosidic linkages in accordance with a known linkage specificity so as to produce a mixed set of saccharide chain fragments having different lengths ranging throughout the full spectrum of possible lengths for the particular glycosidic linkage specificity of the selective scission reagent employed. Samples of the mixed set of saccharide chain fragments are then treated with selected exoenzymes including exoglycosidases that cleave only particular glycosidic linkages at the non-reducing end of saccharide chains and exosulphatases that selectively remove sulphate groups from monosaccharide residues at the non-reducing end of saccharide chains. These exoenzymes are applied to the samples either singly or in combination in accordance with a predetermined strategy. The treated samples are then analysed, conveniently by polyacrylamide gel electrophoresis (PAGE) to detect the chain fragments present which have a reducing end derived from the reducing end of the corresponding chain in the original saccharide material. Using PAGE, preferably gradient PAGE, the different samples are run in parallel tracks of the gel and a migration banding pattern is obtained that provides information which enables the monosaccharide sequence in the original saccharide material to be deduced.
    • 在分析诸如糖胺聚糖(GAG)等糖类物质以确定其糖链中的单糖单元序列的方法中,糖链为末端参考,例如, 通过在其还原末端标记或标记,并且使用选择性切断试剂(例如低pH亚硝酸)对糖类物质进行受控的部分解聚,所述选择性裂解试剂根据已知的连锁特异性切割内部糖苷键,从而产生 具有不同长度的混合组的糖链片段可以在所用选择性分裂试剂的特定糖苷键特异性的整个可能长度的全谱范围内。 混合的糖链片段的样品然后用选择的外切酶处理,所述外切酶包括外切酶,其仅在糖链的非还原末端处切割特定的糖苷键,并且在糖的非还原末端选择性地从单糖残基中除去硫酸根基团 链。 这些外酶根据预定的策略单独或组合应用于样品。 然后通过聚丙烯酰胺凝胶电泳(PAGE)方便地分析经处理的样品,以检测存在的链段,其具有衍生自原始糖类材料中相应链的还原末端的还原末端。 使用PAGE,优选梯度PAGE,不同的样品在凝胶的平行轨道中运行,并且获得提供能够推导原始糖类材料中的单糖序列的信息的迁移条带图案。