Method for amplification and detection of RNA and DNA sequences

• US 5,783,391 A
• Filed: 07/12/1995
• Issued: 07/21/1998
• Est. Priority Date: 03/10/1988
• Status: Expired due to Fees

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: Rossi, John J.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/501,647
• Time in Patent Office: 1,105 Days
• Field of Search: 435/6, 435/91.2, 935/77.78
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

A method of detecting and identifying specific human nucleic acid sequences contained in human nucleic acids in a human blood or tissue sample, comprising: (1) amplifying at least one portion of the specific nucleic acid sequence present in the sample; (2) transcribing the amplification product with an RNA polymerase to produce multiple RNA copies of each copy of specific nucleic acid sequence comprising the amplification product; and (3) identifying the transcription product.

Claims

1. The method which comprises:

(i) providing an RNA sample which may have a target nucleotide sequence;

(ii) providing a first primer and a second primer for reverse transcriptase-polymerase chain reaction amplification of said RNA sample

at least one of said first and second primers having a 5'"'"' terminal moiety which is a recognition sequence for an RNA polymerase,

(iii) subjecting said RNA sample to reverse transcriptase polymerase chain reaction amplification including thermal denaturation between cycles denaturation between cycles, to produce a first amplification product;

said amplification product having said recognition sequence for an RNA polymerase of said at least one of said first and second primers,

(iv) providing an RNA polymerase which recognizes the said recognition sequence of said at least one of said primers;

(v) subjecting said first amplification product of step (iii) to transcription by said RNA polymerase of step (iv) to produce a second amplification product;

(vi) subjecting said second amplification product of step (v) to reverse transcriptase polymerase chain reaction amplification to produce a third amplification product;

(vii) determining whether said target nucleic acid sequence is present in said third amplification product.

2. The claim 1 method in which said at least one of said first and second primers has a 5'"'"' terminal label which is a radioisotope or a fluorophore.

3. The claim 1 method in which said at least one of said first and second primers has a 5'"'"' ³² P radioisotope label.

4. The claim 1 method in which said at least one of said first and second primers has a 5'"'"' recognition sequence for T7 RNA polymerase.

5. The claim 1 method in which said RNA sample is obtained from human blood or tissue sample.

6. The claim 1 method in which said RNA sample is an HIV-1 RNA sample.