NUCLEIC ACID AMPLIFICATION BY TWO-ENZYME, SELF-SUSTAINED SEQUENCE REPLICATION.

摘要

Novel methods are provided for nucleic acid amplification by continuous, substantially isothermal, self-sustained sequence replication ("3SR") utilizing RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, RNAse H activity and DNA-dependent RNA polymerase activity. In one of the methods, before enzymatic activities can be provided by only two enzymes, a reverse transcriptase and a DNA-dependent RNA polymerase. The methods may employ two or three enzymes to provide the necessary enzymatic activities. Thus, in certain of the methods, an exogenous source of RNAse H, such as E. coli RNAse H, is employed in combination with a reverse transcriptase and a DNA-dependent RNA polymerase. In other of the methods of the present invention, reaction media are employed in which the inherent RNAse H activity of retroviral reverse transcriptases is effective to provide high levels of amplification so that only two enzymes, reverse transcriptase and DNA-dependent RNA polymerase, are required. Novel compositions for carrying out the methods of the present invention are also provided.