Incorporation of reporter molecule-labeled nucleotides by DNA polymerases.: II.: High-density labeling of natural DNA
Incorporation of reporter molecule-labeled nucleotides by DNA polymerases.: II.: High-density labeling of natural DNA
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DOI：
10.1093/nar/gkg371
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发表时间：
2003-05-15
影响因子：
14.9
通讯作者：
Amacker, M
中科院分区：
文献类型：
--
作者：
Tasara, T;Angerer, B;Amacker, M
利用连接到可检测报告或官能团的核苷酸对核酸进行修饰是现代分子生物学的重要实验工具。这增强了DNA或RNA的检测，并扩大了核酸的催化谱系。在这里，我们提出了广泛的修饰脱氧核糖核苷5‘-三磷酸(DNTPs)的评价，涵盖了所有四个自然存在的核苷酸碱基，用于DNA修饰的潜在用途。使用不同的模型和天然DNA模板对30个带有荧光或非荧光报告基团的修饰dNTP进行了系统的单独和组合高密度掺入的评估。此外，我们使用不同修改的dNTP底物并排比较了A族(Taq)和B族(Vent(R)exo(-))型DNA聚合酶的掺入效率。我们的结果显示，家族B型DNA聚合酶Vent(R)exo(-)具有优异的性能，当所有天然dNTP完全被其生物素标记的dNTP类似物取代时，它能够完全合成300bP的DNA产物。此外，我们对荧光染料修饰的dNTPs的各种组合进行了系统的测试，从而能够同时标记最多四种不同修饰的dNTPs。
The modification of nucleic acids using nucleotides linked to detectable reporter or functional groups is an important experimental tool in modern molecular biology. This enhances DNA or RNA detection as well as expanding the catalytic repertoire of nucleic acids. Here we present the evaluation of a broad range of modified deoxyribonucleoside 5'-triphosphates (dNTPs) covering all four naturally occurring nucleobases for potential use in DNA modification. A total of 30 modified dNTPs with either fluorescent or non-fluorescent reporter group attachments were systematically evaluated individually and in combinations for high-density incorporation using different model and natural DNA templates. Furthermore, we show a side-by-side comparison of the incorporation efficiencies of a family A (Taq) and B (Vent(R) exo(-)) type DNA polymerase using the differently modified dNTP substrates. Our results show superior performance by a family B-type DNA polymerase, Vent(R) exo(-), which is able to fully synthesize a 300 bp DNA product when all natural dNTPs are completely replaced by their biotin-labeled dNTP analogs. Moreover, we present systematic testing of various combinations of fluorescent dye-modified dNTPs enabling the simultaneous labeling of DNA with up to four differently modified dNTPs.