Long-read pyrosequencing using pure 2′-deoxyadenosine-5′-O′-(1-thiotriphosphate) Sp-isomer
Long-read pyrosequencing using pure 2′-deoxyadenosine-5′-O′-(1-thiotriphosphate) Sp-isomer
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DOI：
10.1006/abio.2001.5494
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发表时间：
2002-02-01
影响因子：
2.9
通讯作者：
Nyrén, P
中科院分区：
文献类型：
--
作者：
Gharizadeh, B;Nordström, T;Nyrén, P
焦磷酸测序是一种非电泳的DNA测序方法，它使用基于内切酶的酶系统来真实的实时监测DNA合成，迄今为止仅限于短片段DNA的测序。为了增加焦磷酸测序中的信噪比，天然dATP被dATPalphaS（M. Ronaghi等人，1996年，《肛门》242，84-89）。所应用的dATPaS是两种异构体（Sp和Rp）的混合物。我们在此表明，通过在焦磷酸测序中引入纯的2 ′-脱氧腺苷-5 ′-O ′-（1-硫代三磷酸）Sp-异构体，可以获得实质上更长的读数。纯的Sp-异构体允许使用较低的核苷酸浓度，并提高了通过多聚（T）区读取的可能性。通常，通过使用纯Sp-异构体可以获得加倍的读取长度。可以在不同类型的模板上产生50至100个碱基的焦磷酸测序数据。更长的读段将使许多新的应用成为可能，例如医学上重要微生物的鉴定和分型，以及用于突变筛查和克隆检查的DNA片段的重测序。(C)2002年，Elsevier Science。
Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking. (C) 2002 Elsevier Science.