Amplification of assay reporters by nucleic acid replication

• US 5,985,548 A
• Filed: 02/13/1995
• Issued: 11/16/1999
• Est. Priority Date: 02/04/1993
• Status: Expired due to Term

• Current Assignee: PerkinElmer Life Sciences, Inc.
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Hatfield, Tina Marie, Collier, David Nash, Moran, John Richard, Ebersole, Richard Calvin, Hendrickson, Edwin R.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Rees, Diane
• Application Number:
  US08/256,627
• Time in Patent Office: 1,737 Days
• Field of Search: 435/6, 435/5, 435/91.2, 435/7.1, 435/7.9, 536/24.3-24.33, 536/26.6
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals

Abstract

A method for the amplified detection of an analyte, wherein amplification is achieved by replication of a target nucleic acid sequence which has been immobilized in response to analyte.

Claims

1. A method for simultaneous multiple measurements of a multiplicity of different immobilized non-nucleic acid analytes in a single test sample comprising the steps of:

i) providing multiple immobilized capture reagents, each comprising at least one member of a binding pair which binds to a nonnucleic acid analyte wherein each capture reagent binds, through said at least one member of the binding pair, to a different non-nucleic acid analyte;

ii) providing said nonnucleic acid analytes and contacting them with said capture agents to form immobilized non-nucleic acid analytes;

iii) providing at least one reporter conjugate in the test sample, each conjugate comprising a member of a binding pair conjugated to a unique target nucleic acid sequence wherein each reporter conjugate binds through the member of its binding pair to a unique non-nucleic acid analyte thereby forming analyte-dependent reporter complexes;

iv) contacting the analyte-dependent reporter complexes with a nucleic acid replication composition capable of replicating said target nucleic acid sequences;

v) replicating the target nucleic acid sequences; and

vi) simultaneously detecting the replicated target nucleic acid sequences so as to obtain simultaneous multiple measurements of said unique nonnucleic acid analytes.

2. The method of claim 1, wherein each reporter conjugate is comprised of a binding pair chemically attached to a target nucleic acid sequence.

3. The method of claim 2 wherein each target nucleic acid sequence may be differentiated on the basis of sequence length or sequence identity.

4. The method of claim 3 wherein said target sequences can be replicated by sets of primers that are specific for each target sequence.

7. The method of claim 1, further comprising additionally adding to the sample one or more reference nucleic acid sequences and the means for replicating said reference sequences, whereby said reference sequences are replicated in addition to said target sequences and thereby serving as internal controls for more accurate detection and quantitation of more than one non-nucleic acid analyte and wherein the reference nucleic acid sequences are also attached to binding pairs to form reporter conjugates.

8. The method of claim 1, wherein the nucleic acid replication composition provided at step (iv) additionally comprises at least one replication control comprising reference sequences, replicating said reference sequences at step iv) concurrently with at least one of said target sequences and at step v) detecting and separately quantitating the replicated reference sequences and replicated target sequences, to determine a ratio of the concentration of replicated reference sequences to the concentration of replicated target sequences, thereby determining from said ratio the concentration of one or more nonnucleic acid analytes.

9. The method of claim 1 further comprising at step (iii) providing at least one immobilized reference nucleic acid sequence and at step (iv) contacting the reporter complexes with said nucleic acid replication composition wherein said composition additionally comprises the means to replicate the reference sequence of said immobilized reference nucleic acid sequences, at step (v) replicating said reference sequences concurrently with said target sequences and at step (vi) detecting and separately quantitating said replicated reference sequences to determine a ratio of the concentration of the replicated reference sequences to the concentration of replicated target sequences thereby determining from said ratio the concentration of one or more nonnucleic acid analytes.

10. The method of claim 1, 2, 4 or 7, wherein the nucleic acid sequence replication is accomplished using a thermal-stable nucleic acid polymerase.

11. The method of claim 1, 2, 4 or 7, wherein the target or reference nucleic acid sequence contains at one end a first primer binding sequence, and contains at the other end a sequence which is complementary to the first primer binding sequence, thereby enabling replication with a single primer.

12. The method of claim 1, 2, 4 or 7, wherein nucleic acid sequence replication is accomplished using primers which contain sequences at their 5'"'"' ends which are not complementary to the target or reference sequences.

13. The method of claim 1, 2, 4 or 7, wherein nucleic acid sequence replication is accomplished using a thermal-stable ligase.

14. The method of claim 1, 2, 4 or 7, wherein at least one signal generating moiety is incorporated within the replicated nucleic acid sequences.

15. The method of claim 14, wherein said signal-generating moieties are radioactive.

16. The method of claim 14, wherein said signal-generating moieties are luminescent.

17. The method of claim 14, wherein said signal-generating moieties are chemiluminescent.

18. The method of claim 14, wherein said signal generating moieties are enzymes.

19. The method of claim 14, wherein said signal generating moieties are fluorescent.

20. The method of claim 19, wherein said fluorescent moieties are positioned within the replicated sequences to enable energy transfer between said fluorescent moieties.

21. The method of claim 19, wherein said fluorescent moieties are positioned no more than about 12 bases apart within the replicated nucleic acid sequences.

22. The method of claim 1, 2, 4 or 7 wherein primers or ligand labeled bases are labeled with a first member of a binding pair are incorporated within the amplified nucleic acid sequences.

23. The method of claim 22, wherein detection and quantitation of the replicated nucleic acid sequences is accomplished by immobilizing the replicated nucleic acid sequences by deposition of said sequences onto an immobilized capture reagent comprising a second member of the binding pair, and further detecting said immobilized replicated nucleic sequences.

24. The method of claim 22, wherein said first member of a binding pair is biotin.

25. The method of claim 1, further comprising after step (iii) separating the replicated nucleic acid sequences from the non-incorporated signal generating moieties using size separation techniques;

and (iv) detecting the replicated nucleic acid sequences.

5. A method of detecting non-nucleic acid analytes comprising the steps of:

a) forming an analyte-dependent reporter complex by contacting an immobilized capture reagent which binds to a nonnucleic acid analyte, with a nonnucleic acid analyte, and a reporter conjugate wherein said reporter conjugate is comprised of a member of a binding pair which also binds to the nonnucleic acid analyte and is conjugated to a reporter enzyme;

b) contacting the analyte-dependent reporter complex with a nucleic acid replication substrate comprising a target nucleic acid sequence conjugated to a moiety capable of detectable activation by said reporter enzyme to produce a detectably activated nucleic acid replication intermediate which deposits onto an immobilized receptor capable of binding to said complex thereby producing a deposited nucleic acid replication product;

c) contacting the deposited nucleic acid replication product with a nucleic acid replication composition capable of replicating said target nucleic acid sequence;

d) replicating a target nucleic acid sequence from the deposited nucleic acid sequences

e) detecting said replicated target nucleic acid sequence.

6. A method of detecting non-nucleic acid analytes comprising the steps of:

a) forming an analyte-dependent reporter complex by contacting an immobilized capture reagent which binds to a nonnucleic acid analyte, with a nonnucleic acid analyte, and a reporter conjugate wherein said reporter conjugate is comprised of a member of a binding pair which also binds to the nonnucleic acid analyte and is conjugated to a reporter enzyme;

b) contacting the analyte-dependent reporter complex with a nucleic acid replication substrate comprising a target nucleic acid sequence conjugated to a moiety capable of detectable activation by said reporter enzyme to produce a detectably activated nucleic acid replication intermediate which deposits onto an immobilized receptor capable of binding to said complex thereby producing a deposited nucleic acid replication product;

c) contacting the deposited nucleic acid replication product with a nucleic acid replication composition capable of replicating said target nucleic acid sequence to produce a deposited nucleic acid replication binding pair complex,

d) replicating a target nucleic acid sequence of the deposited nucleic acid sequences;

e) detecting said replicated target nucleic acid sequence.

26. A method for the detection and quantitation of a nonnucleic acid analyte or analytes comprising:

a) adding to an analyte sample an immobilized capture reagent comprising a least one member of a binding pair;

b) adding simultaneously or sequentially with step (a) at least one ligand reporter conjugate comprising a target nucleic acid sequence conjugated to a ligand wherein the ligand of the reporter conjugate will compete with the analyte for binding to the immobilized capture reagent to form immobilized analyte complexes and/or immobilized ligand complexes;

c) washing unbound analyte and unbound ligand reporter conjugate away from any immobilized analyte complex and/or immobilized ligand complexes;

d) contacting washed and immobilized analyte complexes and/or immobilized ligand complexes with a nucleic acid replication composition capable of replicating the target nucleic acid in any of the immobilized ligand complexes;

e) detecting the presence of replicated target nucleic acid sequences as a means of determining competitive binding of the ligand reporter conjugate and the analyte to the immobilized capture reagent thereby detecting and quantitating the presence of the nonnucleic acid analyte or analytes in the sample.

27. A method for the amplified detection of at least one nonnucleic acid analyte comprising the steps of:

a) contacting a nonnucleic acid analyte sample with immobilized capture reagent capable of binding to the analyte whereby at least one nonnucleic acid analyte is immobilized;

b) adding simultaneously or sequentially with step (a) at least one reporter conjugate to the sample wherein said reporter conjugate comprises a target nucleic acid sequence conjugated to a member of a binding pair which is capable of binding, to said nonnucleic acid analyte;

c) separating any excess reporter conjugates which has remained free in solution from the sample;

d) contacting free reporter conjugates of step (c) with a nucleic acid replication composition capable of replicating said target nucleic acid;

e) replicating the target nucleic acid sequence of the free reporter conjugates of step (d);

f) detecting and quantitating the replicated target nucleic acids of step (e) whereby the presence of at least one analyte in the sample is determined.