Real-time DNA sequencing using detection of pyrophosphate release
Real-time DNA sequencing using detection of pyrophosphate release
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DOI：
10.1006/abio.1996.0432
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发表时间：
1996-11-01
影响因子：
2.9
通讯作者：
Nyren, P
中科院分区：
文献类型：
--
作者：
Ronaghi, M;Karamohamed, S;Nyren, P
一种不需要电泳法的实时DNA测序方法已经被开发出来。该方法依赖于通过酶发光无机焦磷酸(PPI)检测分析(Elida)(Nyren，P.(1987)anal)检测DNA聚合酶活性。生物化学。167,235-238)，DNA聚合反应中形成的PPI被三磷酸腺苷硫酰酶转化为三磷酸腺苷，三磷酸腺苷的产生由萤火虫荧光素酶持续监测。在测序过程中，将固定化单链模板用于重复的脱氧核苷酸延伸循环。当互补碱基被掺入时，ELIDA中的实时信号被观察到，与掺入的核苷酸的量成比例。用脱氧腺苷α-硫代三磷酸(dATPα-硫代三磷酸)代替天然的脱氧腺苷三磷酸(dATPαS)可提高信噪比，dATPαS可被DNA聚合酶有效利用，但不被荧光素酶识别。作为模型，对15个碱基的单链聚合酶链式反应产物进行了测序。讨论了以自动方式并行处理多个样品的可能性。(C)1996年学术出版社。
An approach for real-time DNA sequencing without the need for electrophoresis has been developed. The approach relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (Nyren, P. (1987) Anal. Biochem. 167, 235-238), The PPI formed in the DNA polymerase reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the sequencing procedure, immobilized single-stranded template was used in a repeated cycle of deoxynucleotide extension. Real-time signals in the ELIDA, proportional to the amount of incorporated nucleotide, were observed when complementary bases were incorporated. An increased signal-to-noise ratio was obtained by substitution of deoxyadenosine alpha-thiotriphosphate (dATP alpha S) for the natural deoxyadenosine triphosphate, dATP alpha S is efficiently used by the DNA polymerase, but is not recognized by the luciferase. As a model, 15 bases of a single-stranded PCR product were sequenced. The possibility for parallel processing of many samples in an automated manner is discussed. (C) 1996 Academic Press, Inc.