Termination of DNA synthesis by N6-alkylated, not 3-O-alkylated, photocleavable 2-deoxyadenosine triphosphates
Termination of DNA synthesis by N6-alkylated, not 3-O-alkylated, photocleavable 2-deoxyadenosine triphosphates
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DOI：
10.1093/nar/gkm689
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发表时间：
2007-10-01
影响因子：
14.9
通讯作者：
Metzker, Michael L.
中科院分区：
文献类型：
--
作者：
Wu, Weidong;Stupi, Brian P.;Metzker, Michael L.
人类基因组计划促进了许多物种的测序，但目前的桑格方法过于昂贵、劳动力密集且耗时，无法完成人类基因组的医学重测序。在“下一代”技术中，循环可逆终止（CRT）是一种很有前途的方法，其目标是以很少的成本和精力产生准确的序列信息。这种方法的基础是可逆终止子（RT），其化学和生物学特性直接影响测序技术的性能。在这里，我们发现了 RT 化学中的一种新范例，将可光裂解的 2-硝基苄基连接到 2'-脱氧腺苷三磷酸 (dATP) 的 N-6 位，一旦掺入，就会终止 DNA 合成。 N-6-(2-硝基苯甲基)-dATP 的 3'-OH 基团保持未封闭状态，为几种市售 DNA 聚合酶提供有利的掺入和终止特性，同时保持对错配掺入的良好辨别力。用紫外线去除 2-硝基苄基后，天然核苷酸就会恢复，不会产生分子疤痕。一项五碱基实验说明了通过均聚物重复进行的精妙逐步添加，证明了 N-6-(2-硝基苄基)-dATP 作为 CRT 测序理想 RT 的适用性。
The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the 'next-generation' technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N-6-position of 2'-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3'-OH group of the N-6-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N-6-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.