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2019/10/14 13:36:54
该试剂盒的设计目的是研究是否有一种化合物插入到DNA双螺旋结构中,或结合在沟槽中,导致DNA解开。这是许多dna修饰酶抑制剂的特征,如拓扑异构酶抑制剂m-amsacrine。插入器包含平面的,通常是多环芳香族结构,可以插入到双螺旋DNA分子的碱基之间。能够插入DNA或在凹槽中结合的化合物可以导致DNA的局部解绕,从而导致DNA扭曲的减少。如果一个DNA分子在这种化合物的存在下被切割并重新连接(例如通过拓扑异构酶),那么结果就是松弛的、未缠绕的DNA;去除酶和化合物后,它会转化成超螺旋DNA。在这些条件下形成的超螺旋DNA表明是插入物。这是DNA解开实验的基础。超螺旋(或放松)质粒DNA与测试化合物进行短暂孵育,然后由小麦胚芽topo i进行放松。然后提取酶和药物,凝胶电泳分析质粒。超螺旋状构造表明该化合物是一种插入剂或沟槽粘合剂。如果化合物不是插入物,那么产物将是松弛质粒。应使用放松质粒进行对照反应,以证明该化合物不是简单地作为topo I的抑制剂,从而检查检测结果。该试剂盒(产品代码DUKSR001)包含所有您需要做的分析,包括:-超螺旋pBR322(50μg),放松pBR322(25μg),小麦胚芽威尼斯平底渔船我(250 U),分析缓冲(1毫升)和稀释缓冲(1毫升)。我们还提供工具包,其中包含topo I和缓冲区,但只有(i) pBR322(产品代码DUKR002)的宽松形式。这将显示一个化合物是否为插入物,但如果它也抑制topo I(即可能出现假阴性),则不会显示(ii) pBR322的超卷形式(产品代码DUKS003)。这将显示化合物是否为插入剂,但如果它是topo I的抑制剂,则会得到相同的结果(即可能出现假阳性)。如下图所示:
This kit is designed to investigate whether a compound intercalates into the DNA double-helix, or binds in the groove, leading to unwinding of the DNA. This is a characteristic of a number of inhibitors of DNA-modifying enzymes such as the topoisomerase inhibitor m-amsacrine.
Intercalators contain planar, normally polycyclic, aromatic structures which can insert between the bases of the double-helical DNA molecule.  Compounds which are able to intercalate into DNA or bind in the groove can lead to local unwinding of the DNA leading to a decrease in the twist of the DNA. If a DNA molecule is nicked and rejoined (for example by a topoisomerase) in the presence of such a compound, then the result is relaxed, underwound DNA; on removal of the enzyme and compound this converts to supercoiled DNA. The supercoiled DNA formed in these conditions is indicative of an intercalator.
This is the basis of the DNA unwinding assay. Supercoiled (or relaxed) plasmid DNA is incubated briefly with the test compound prior to relaxation by the wheat germ topo I. The enzyme and drug are then removed by extraction and the plasmid analysed by gel electrophoresis. Supercoiled topoiosmers indicate the compound is an intercalator or groove binder. If the compound is not an intercalator then the product will be relaxed plasmid.
The assay should be checked by performing a control reaction using relaxed plasmid to show that the compound is not simply acting as an inhibitor of the topo I.
The kit (product code DUKSR001) contains all you need to do the assay including:-
Supercoiled pBR322 (50 μg), relaxed pBR322 (25 μg), wheat germ topo I (250 U), Assay Buffer (1 ml) and Dilution Buffer (1 ml).
We also supply kits which contain topo I and buffers but only:-
(i) the relaxed form of pBR322 (product code DUKR002). This will show if a compound is an intercalator but not if it also inhibits the topo I (i.e. false-negatives are possible)
(ii) the supercoiled form of pBR322 (product code DUKS003). This will show if a compound is an intercalator but the same result will be given if it is an inhibitor of topo I (i.e. false-positives are possible)
This is illustrated in the scheme below:

优势供应产品:
Cat No. Product
DUKSR001 DNA Unwinding Assay Kit
100 assays
DUKR002 DNA Unwinding Assay Kit - Relaxed only
100 assays
DUKS003 DNA Unwinding Assay Kit - Supercoiled only
100 assays

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