ISOTHERMAL, INVITRO AMPLIFICATION OF NUCLEIC-ACIDS BY A MULTIENZYME REACTION MODELED AFTER RETROVIRAL REPLICATION
ISOTHERMAL, INVITRO AMPLIFICATION OF NUCLEIC-ACIDS BY A MULTIENZYME REACTION MODELED AFTER RETROVIRAL REPLICATION
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DOI：
10.1073/pnas.87.5.1874
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发表时间：
1990-03-01
影响因子：
11.1
通讯作者：
GINGERAS, TR
中科院分区：
文献类型：
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作者：
GUATELLI, JC;WHITFIELD, KM;GINGERAS, TR
通过利用逆转录病毒复制所必需的三种酶活性：逆转录酶、核糖核酸酶H和一种依赖DNA的RNA聚合酶，靶核酸序列可在等温条件下于体外呈指数式复制（扩增）。通过模拟借助于cDNA中间体进行RNA复制的逆转录病毒策略，该反应积累原始靶标的cDNA和RNA拷贝。产物积累相对于时间呈指数式，这表明新合成的cDNA和RNA可作为一系列连续转录和逆转录反应的模板。在1 - 2小时的温育后会发生一千万倍的扩增，其初始扩增速率为每2.5分钟10倍。这种自我维持的序列复制系统可用于稀有RNA和DNA的检测以及核苷酸序列分析。文中还讨论了其与逆转录病毒复制各方面的相似性。
A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.