Method for amplification and detection of RNA and DNA sequences

• US 5,869,249 A
• Filed: 11/12/1996
• Issued: 02/09/1999
• 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:
  US08/747,489
• Time in Patent Office: 819 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78, 536/24.33
• 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 first and second primers for 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) providing a cellular marker sequence;

(iv) providing third and fourth primers for polymerase chain reaction amplification of said cellular marker sequence;

(v) simultaneously subjecting said RNA sample and said cellular marker sequence to reverse transcriptase-polymerase chain reaction amplification with said first, second, third and fourth primers to produce a first amplification product;

(vi) providing an RNA polymerase which recognizes the recognition sequence for an RNA polymerase which is a 5'"'"' terminal moiety of at least one of said first and second primers of step (ii);

(vii) subjecting said first amplification product to transcription with said RNA polymerase of step (vi) to produce a second amplification product;

(viii) producing a first oligonucleotide probe having a sequence complementary to at least a portion of said target sequence in said RNA sample of step (i);

(ix) subjecting said second amplification product and said first probe to hybridization conditions; and

(x) determining whether step (ix) yields a hybridization product of said first probe and said second amplification product.

2. The method of claim 1, in which the 5'"'"' terminal moiety which is a recognition sequence for RNA polymerase is a T7 promoter sequence.

3. The method of claim 1, wherein the cellular marker sequence comprises a T-cell receptor sequence, a CD4 receptor beta chain constant region sequence or a beta-actin sequence.

4. In a process for amplifying a target nucleotide sequence present in an RNA sample by a reverse transcriptase polymerase chain reaction, the improvement which comprises:

(i) utilizing as at least one primer in said reverse transcriptase polymerase chain reaction a polynucleotide terminating 5'"'"' in a recognition sequence for an RNA polymerase to produce a reverse transcriptase polymerase chain reaction first amplification product having said 5'"'"' terminal polymerase recognition sequence;

(ii) amplifying said reverse transcriptase polymerase chain reaction product by transcription with an RNA polymerase corresponding to said 5'"'"' terminal recognition sequence of said reverse transcriptase polymerase chain reaction first amplification product to produce a second amplification product;

(iii) subjecting the second amplification product of step (ii) to hybridization conditions with a probe known to be complementary to at least a portion of said target nucleotide sequence; and

(iv) determining whether step (iii) results in hybridization of said probe to said amplification product.

5. The method which comprises

(i) simultaneously co-amplifying

(a) an HIV-1 mRNA sequence from clinical samples obtained from an AIDS patient;

and

(b) a T4 receptor mRNA sequence or T-cell receptor mRNA sequence to provide an amplification product;

(ii) quantifying the amount of each of said T4 receptor and said T-cell receptor in said amplification product to establish the severity of T-cell depletion in said clinical sample.

6. The method of claim 5, in which said co-amplifying is accomplished by the polymerase chain reaction.

7. The method of claim 5 or 6, in which the beta chain of the constant region of the T-cell receptor is amplified.