Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing
Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing
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DOI：
10.1021/bc015591c
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发表时间：
2002-05-01
影响因子：
4.7
通讯作者：
Kumar, S
中科院分区：
文献类型：
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作者：
Nampalli, S;McDougall, MG;Kumar, S
二新戊酰基-5-羧基荧光素 N-羟基琥珀酰亚胺酯 1 和 5-炔丙基氨基-2',3'-二脱氧尿苷三磷酸 5 用马来酰亚胺、卤代乙酰胺和巯基反应性官能团进行修饰，通过硫键参与交叉共轭反应，生成荧光标记的硫醚交叉共轭终止子 10 和 11。将它们的 DNA 测序潜力与酰胺交叉共轭终止子 13，通过直接偶联 5-羧基荧光素 NHS 酯与 18-ddUTP 9 合成。这些终止子（10、11 和 13）与 Thermo Sequenase II DNA 聚合酶结合，在热循环测序实验中表明，硫醚交叉共轭终止子 10 和酰胺交叉共轭终止子 13 是良好的终止底物，产生令人满意的终止底物单色凝胶图像和电泳图，而其他硫醚交叉共轭和马来酰亚胺衍生的 11 经历了意想不到的 pH 和温度诱导分解，没有显示出掺入的荧光特征。
Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.