TERMINAL REPEAT AMPLIFICATION METHOD

摘要

This invention relates to a process for amplifying a specific nucleic acid sequence or its complement at a relatively constant temperature and without serial addition of reagents. The process provides in a single reaction medium an RNA polymerase, DNA polymerase, a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single or double-stranded RNA or DNA, and ribonucleoside and deoxyribonucleoside triphosphates. The process then provides an RNA first template in the reaction medium. The RNA first template comprises a sequence complementary to a specific nucleic acid sequence, minus-sense sequences for a promoter and initiation site that are recognized by the RNA polymerase, and a 5'-terminal sequence that is complementary to at least the minus-sense sequence of the initiation site. Thus, the RNA first template has an inverted repeat sequence which could fold into a 5'-terminal stem-loop structure. The DNA polymerase uses the RNA first template to synthesize a DNA second template that together comprise an RNA-DNA hybrid. The DNA second template has plus-sense sequences of the promoter and the initiation site, and a 3'-terminal priming sequence that is complementary to the plus-sense sequence of the initiation site. The ribonuclease then hydrolyses an RNA which comprises the RNA-DNA hybrid, allowing the 3'-terminal priming sequence to hybridize to the plus-sense sequence of the initiation site in the DNA second template. The DNA polymerase then uses the DNA second template to synthesize the promoter by extending the 3'-terminal priming sequence of the DNA second template. The resulting partially double-stranded DNA has a promoter oriented toward the apex of a stem-loop structure. The RNA polymerase then recognizes the promoter and transcribes the DNA second template, thereby providing copies of the RNA first template. The process thereafter maintains the reaction conditions for a time sufficient to achieve a desired amplification of the specific nucleic acid sequence or its complement. This invention includes a kit containing the reagents of this invention.