【摘要】 分别以5-碘-2’-脱氧尿苷、5-碘-2’-脱氧胞苷、7-去氮-7-碘-2’-脱氧腺苷及7-去氮-7-碘-2’-脱氧鸟苷为原料,以二硫键为可裂解连接单元,通过多步反应合成了四色荧光标记不同碱基的脱氧核糖核苷酸;研究了该类荧光标记核苷酸作为可逆终端在DNA合成测序中的应用.所得产物结构经1H NMR,31P NMR及HRMS表征,并对其进行了DNA高通量测序的测试.结果表明,该类荧光标记核苷酸作为DNA合成测序的可逆终端能够满足高通量测序的生化反应要求,具有较好的应用前景.更多还原

【Abstract】 DNA sequencing is one of the important means of biological research. The development of accurate and high-throughput DNA-sequencing methods has a strong impact on biomedical research. Sequencing by synthesis( SBS) approaches is one of the most important techniques of DNA sequencing. A series of SBS methods have been investigated and implemented. In SBS methods,the principal thing is to synthesize four fluorescently labelled cyclic reversible terminators as the sequencing reagents. These cyclic reversible terminators need to connect the fluorescein through a cleavable linker. In chemical biology,disulfide bridges,which are known to be efficiently and rapidly cleaved by mild reducing agents,have been used in a wide range of applications including drug delivery,functional and structural proteomics,DNA and tumor imaging. The disulfide-based cleavable linker is commonly used due to its rapid cleavage and straightforward synthesis. In this work,starting from 5-iodo-2’-deoxyuridine,5-iodo-2’-deoxycyridine,7-deazo-7-iodo-2’-deoxyadenosine and7-deazo-7-iodo-2’-deoxyguanosine,four kinds of fluorescence labelled necleotides with four different bases through disulfide were synthesized and characterized. The synthesized fluorescence labelled nucleotides can be recognized and accepted by an identified DNA polymerase,incorporated faithfully into DNA strand,followed by cleaved completely by DTT within 1 min. These results demonstrate that the fluorescence labelled nucleotides have potential value as reversible terminators in DNA sequencing by synthesis.更多还原