DNA Unwinding Assay Kit
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:
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多谢……
各位大侠：小弟最近做个长片段的CDNA与T载体的连接，片段大小6.4K，载体选用的是pUC19和pGEMT-easy,反转录酶用的是RevertAidTirstStrandcDNASynthesisKit(#K1621)，CDNA第二链的扩增用的是LATaq。我是用纯病毒的RNA做模板，操作步骤严格按照说明做的，也曾经有一次获得过电泳图清晰显示的是全长6.4KB左右的片段，与载体连接，结果筛选到的几个克隆，插入的片段仅有2KB左右，奈何？可最近几次再用此反转录试剂盒却不能得到所需片段。在此，小弟想问问大家，你们是否遇到同样的问题，是如何解决的？所用的试剂（载体和酶的选择）和方法是什么，可以告诉后来者，共同进步吧。
