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    • 8. 发明申请
    • BIOLUMINESCENCE REGENERATIVE CYCLE (BRC) FOR NUCLEIC ACID QUANTIFICATION
    • 用于核酸定量的生物发光再生循环(BRC)
    • WO03087388A9
    • 2004-05-21
    • PCT/US0220690
    • 2002-06-28
    • UNIV LELAND STANFORD JUNIORHASSIBI ARJANGPOURMAND NADER
    • HASSIBI ARJANGPOURMAND NADER
    • C12Q1/68C07H21/04C12P19/34
    • C12Q1/6825C12Q1/689C12Q2565/301C12Q2545/114C12Q2531/113
    • The present invention concerns methods of quantifying nucleic acids using a bioluminescence regenerative cycle (BRC). In BRC, steady state levels of bioluminescence result from processes that produce pyrophosphate. Pyrophosphate reacts with APS in the presence of ATP sulfurylase to produce ATP. The ATP reacts with luciferin in a luciferase-catalyzed reaction, producing light and regenerating pyrophosphate. The pyrophosphate is recycled to produce ATP and the regenerative cycle continues. Because the kinetic properties of ATP sulfurylase are much faster than luciferase, a steady state results wherein concentrations of ATP and pyrophosphate and the rate of light production remain relatively constant. Photons are counted over a time interval to determine the number of target molecules present in the initial sample. The BRC process has a controllable dynamic range up to seven orders of magnitude and is sensitive enough to detect a few thousand molecules of target nucleic acid.
    • 本发明涉及使用生物发光再生循环(BRC)定量核酸的方法。 在BRC中,稳定状态的生物发光水平来自产生焦磷酸的过程。 焦磷酸在ATP硫酸化酶存在下与APS反应产生ATP。 ATP在萤光素酶催化的反应中与萤光素反应,产生光并再生焦磷酸。 焦磷酸盐被再循环生产ATP,再生循环继续进行。 由于ATP硫酸化酶的动力学性质比萤光素酶快得多,所以其中ATP和焦磷酸盐的浓度以及光产生速率保持相对恒定的稳态结果。 在一段时间内对光子进行计数以确定初始样品中存在的靶分子的数量。 BRC过程的可控动态范围可达7个数量级,并且足够灵敏以检测几千个目标核酸分子。