Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P
Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.
Anal Biochem. 1996 Nov 1;242(1):84-9. doi: 10.1006/abio.1996.0432.
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) (Nyrén, 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.
一种无需电泳的实时DNA测序方法已被开发出来。该方法依赖于通过酶促发光无机焦磷酸（PPi）检测法（ELIDA）检测DNA聚合酶活性（尼伦，P.（1987年）《分析生物化学》167卷，235 - 238页）。DNA聚合酶反应中形成的PPi通过ATP硫酸化酶转化为ATP，萤火虫荧光素酶持续监测ATP的产生。在测序过程中，固定化的单链模板用于脱氧核苷酸延伸的重复循环。当互补碱基掺入时，在ELIDA中观察到与掺入核苷酸量成比例的实时信号。通过用脱氧腺苷α - 硫代三磷酸（dATPαS）替代天然脱氧腺苷三磷酸，获得了更高的信噪比，dATPαS能被DNA聚合酶有效利用，但不被荧光素酶识别。作为一个模型，对单链PCR产物的15个碱基进行了测序。讨论了以自动化方式并行处理多个样品的可能性。
