RIBOZYME-ASSISTED METHOD FOR NUCLEIC ACID DETECTION

随着可用DNA序列信息的快速增加， 基于核酸的诊断是一个非常感兴趣的课题。 样品制备效率的提高，核酸 扩增和检测将允许大大增加使用 这些方法用于常规诊断目的。 最广泛的 检测前扩增DNA的常用方法是 聚合酶链反应（PCR），其仅限于扩增 DNA，需要使用昂贵的热循环仪。 等温 这些方法包括一系列酶促步骤， 复杂性、蛋白酶的成本和污染的风险 在多个管开口期间。 在这里，我们提出了创新， 等温扩增基于使用RNA作为 可降解探针和RNA催化其自身的能力 裂解和再结合反应。 通过利用RNA 催化，我们可以消除对所有蛋白质因子的需求， 除了RNA聚合酶和减少管的数量 开幕式 混合捕获和洗涤的其他创新 程序进一步提高了自动化的速度和简易性。 该程序适用于封闭管，多重荧光 扩增过程中的检测，允许快速筛选许多 不同的目标，同时尽量减少接触病原体的风险， 被放大的目标造成的实验室污染 建议的商业应用 DNA序列信息的日益可用性， 微生物基因组和疾病相关基因，包括 癌基因，使得基于核酸的方法极具吸引力 用于诊断病毒和微生物感染，检测食品 污染和基因筛查 然而，目前的方法， 虽然非常敏感，但对于常规来说太慢和昂贵 用于临床实验室。 根据NIST的数据， 业界预测体外DNA诊断市场将增长 从1997年的5亿美元增加到2005年的60亿美元。 这个市场 将倾向于便宜、简单、快速和容易的技术 自动化，允许完全自动化的自我诊断， 用于临床实验室常规使用或作为 用于检测大量样品的多基因的方法 可以在中心用于高通量的目标， 诊断测试 我们提出了几个改进的国家 的艺术走向这一目标。

With the rapid increase in available DNA sequence information, nucleic acid-based diagnostics is a subject of intense interest. Improvements in the efficiency of sample preparation, nucleic acid amplification, and detection would permit greatly increased use of such methods for routine diagnostic purposes. The most widely used method for amplifying DNA prior to detection is the polymerase chain reaction (PCR), which is restricted to amplifying DNA and requires the use of costly thermal cyclers. Isothermal methods involve a sequence of enzymatic steps, with the attendant complexity, costs of the protein enzymes, and risk of contamination during multiple tube openings. Here we propose innovations in isothermal amplification based on the use of RNA as an amplifiable probe and the ability of RNA to catalyze its own cleavage and rejoining reactions. By taking advantage of RNA catalysis, we can eliminate requirements for all protein factors except an RNA polymerase and reduce the number of tube openings. Additional innovations in hybrid capture and wash procedures further increase the speed and ease of automation. The procedure lends itself to closed-tube, multiplex fluorescent detection during amplification, permitting rapid screening of many different targets while minimizing risks of exposure to pathogens or laboratory contamination by amplified targets. PROPOSED COMMERCIAL APPLICATION The increasing availability of DNA sequence information on microorganism genomes and genes of disease relevance, including oncogenes, makes nucleic acid-based methods extremely attractive for diagnosis of viral and microbial infections, detection of food contamination, and genetic screening. However, current method, while extremely sensitive, are too slow and expensive for routine use in clinical laboratories. According to NIST, the biotechnology industry projects the market for in vitro DNA diagnostics to grow from 500 million dollars in 1997 to 6 billion by 2005. This market will favor techniques that are inexpensive, simple, rapid, and easily automated, permitting completely automated diagnosis in a self- contained machine for routine use in clinical laboratories or as a method for testing large numbers of samples for multiple genetic targets that could be used in a center for high-throughput diagnostic testing. We propose several improvements in the state of the art toward that goal.