Patent ID: 7074558
Filing Date: 2006-07-11
Classification: C12Q

Abstract:
1. A method of amplifying a target nucleic acid in an aqueous solution with a first and a second primer, said method comprising: i.) transcribing an intermediate duplex with a phage-encoded RNA polymerase to form a sense transcription product having a 5′ end and a 3′ end, wherein said intermediate duplex comprises a double-stranded nucleic acid, wherein said double-stranded DNA molecule comprises a first and a second strand, wherein said first strand comprises in the following order from 5′ to 3′: wherein said second strand comprises in the following order from 5′ to 3′: wherein said transcribing takes place in the presence of Mn++, with all four dNTPs, and with those rNTPs represented in said first spacer sequence; ii.) hybridizing said second primer to said sense transcription product to form a second primer-sense transcription product complex, wherein said second primer comprises in the following order from 5′ to 3′: iii.) extending said second primer-sense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a first amplification duplex; iv.) transcribing said first amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn++, with all four dNTPs, and with those rNTPs represented in said second spacer sequence, to form an antisense transcription product; v.) hybridizing said first primer to said antisense transcription product to form a first primer-antisense transcription product complex, wherein said first primer comprises in the following order from 5′ to 3′: vi.) extending said first primer-antisense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a second amplification duplex; and vii.) transcribing said second amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn++, with all four dNTPs, and with those rNTPs represented in said first spacer sequence to form said sense transcription product.