Patent ID: 7074558

Claim:
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 5â€² end and a 3â€² 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 5â€² to 3â€²: a phage-encoded RNA polymerase recognition sequence, a first spacer sequence comprising a sequence of from 12 to 20 nucleotides that consists of one nucleotide type or two different nucleotide types, and a first target complementary sequence which can bind to a segment of said target nucleic acid, wherein said second strand comprises in the following order from 5â€² to 3â€²: a second target complementary sequence which can bind to a segment of said target nucleic acid, a second spacer sequence comprising a sequence of from 12 to 20 nucleotides that consists of one nucleotide type or two different nucleotide types, and a phage-encoded RNA polymerase recognition sequence, 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 5â€² to 3â€²: a phage-encoded RNA polymerase recognition sequence, said second spacer sequence, and said second target complementary sequence which can bind to a 3â€² segment of said target nucleic acid; 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 5â€² to 3â€²: a phage-encoded RNA polymerase recognition sequence, said first spacer sequence, and said first target complementary sequence which can bind to a 5â€² segment of said target nucleic acid; 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.