Azide and trans-cyclooctene dUTPs: incorporation into DNA probes and fluorescent click-labelling
Azide and trans-cyclooctene dUTPs: incorporation into DNA probes and fluorescent click-labelling
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DOI：
10.1039/c5an00158g
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发表时间：
2015-01-01
期刊：
影响因子：
4.2
通讯作者：
Brown, Tom
中科院分区：
文献类型：
--
作者：
Ren, Xiaomei;El-Sagheer, Afaf H.;Brown, Tom
5-叠氮甲基dUTP和两个5-反式-环辛烯dUTP与TCO和尿嘧啶碱基之间的不同接头已经通过引物延伸、逆转录和PCR扩增掺入DNA中。对于叠氮甲基dUTP，即使在修饰的dUTP未补充dTTP时，PCR反应也是成功的。在一种情况下，使用该方法将335个叠氮甲基dU残基掺入523个碱基对扩增子中。5-叠氮甲基dUTP被发现是一个更好的底物DNA聚合酶比反式环辛烯dUTP。然而，环辛烯DNA和四嗪Cy 3-染料之间的逆电子需求Diels-Alder反应比叠氮DNA和双环[6.1.0]壬-4-炔Cy 3染料之间的应变促进反应更有效。
5-Azidomethyl dUTP and two 5-trans-cyclooctene dUTPs with different linkers between the TCO and the uracil base have been incorporated into DNA by primer extension, reverse-transcription and PCR amplification. For azidomethyl dUTP the PCR reaction was successful even when the modified dUTP was not supplemented with dTTP. In one case 335 azidomethyl dU residues were incorporated into the 523 base pair amplicon using this methodology. 5-Azidomethyl dUTP was found to be a better substrate for DNA polymerases than the trans-cyclooctene dUTPs. However, the inverse electron demand Diels-Alder reaction between cyclooctene DNA and a tetrazine Cy3-dye was more efficient than the strain-promoted reaction between azide DNA and a bicyclo [6.1.0] non-4-yne Cy3 dye.