Method for the amplification and detection of a nucleic acid fragment of interest

• US 20030087271A1
• Filed: 06/20/2002
• Published: 05/08/2003
• Est. Priority Date: 03/01/1996
• Status: Abandoned Application

• Current Assignee: Edwin R. Hendrickson, Michael P. Perry, Richard C. Ebersole, Sandra Fitzpatrick-Mcelligott
• Original Assignee: Edwin R. Hendrickson, Michael P. Perry, Richard C. Ebersole, Sandra Fitzpatrick-Mcelligott
• Inventors: Fitzpatrick-McElligott, Sandra, Ebersole, Richard C., Hendrickson, Edwin R., Perry, Michael P.
• Application Number:
  US10/176,422
• Publication Number:
  US 20030087271A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

A method is provided for the replication and detection of a specific nucleic acid target using a detection probe. The probe is present throughout the amplification reaction but does not participate in the reaction in that it is not extended. The probe contains sequence complementary to the replicated nucleic acid analyte for capture of the analyte by hybridization. Additionally the probe or analyte contains at least one reactive ligand to permit immobilization or reporting of the probe/analyte hybrid.

Claims

1. A method for the detection of a target nucleic acid analyte sequence in a nucleic acid replication reaction, comprising the steps of:

(i) contacting at least one target nucleic acid sequence with a nucleic acid replication composition, said composition further comprising;

(a) a first label capable of incorporating into a replicating nucleic acid;

(b) a detection probe, wherein said probe contains

(1) a second label;

(2) a target domain; and

(3) a replication inhibitor moiety that renders the detection probe non-participatory in the replication reaction;

(ii) replicating the target nucleic acid sequence in the replication composition of step (i) to produce a target nucleic acid analyte and under reaction conditions that permit the formation of an analyte/probe hybrid wherein said hybrid consists of the detection probe and at least one replicated-strand of said target analyte nucleic acid;

(iii) immobilizing said analyte/probe hybrid through either said first or second label; and

(iv) detecting the presence said immobilized analyte/probe hybrid.

2. A method of claim 1 wherein said first or second label is a reactive ligand.

3. The method of claim 1 wherein the replication inhibiting moiety is selected from the group consisting of dideoxynuleotides, a sequence of mismatched nucleotides, 3′

phosphate and 3′

deoxynucleotides.

4. The method of claim 3 where in the 3′

deoxynucleotides is cordycepin.

5. The method of claim 2 wherein the reactive ligand is a member of a binding pair wherein binding pairs are selected from group consisting of pairs of antigens and antibodies, haptens and anti-haptens biotin and avidin, biotin and streptavidin, folic acid and folate binding protein complementary nucleic acid segments;

protein A or G/immunoglobulins; and

binding pairs which form covalent bonds.

6. A method for the detection of a target nucleic acid analyte sequence in a nucleic acid replication reaction, comprising the steps of:

(i) contacting at least one target nucleic acid sequence with a nucleic acid replication composition containing a homogeneous detection probe system conprising at least one pair of probes said pair consisting of;

(a) a first, signal generating probe comprising a first member of a reporter pair, a target domain, a first probe binding domain and replication inhibitor moiety, and;

(b) a second, signal modifying probe comprising a second member of a reporter pair and a second probe binding domain complementary to said first probe binding domain wherein the first and second members of the reporter pair are capable of reacting with each other to produce a detectable signal;

(ii) replicating the target nucleic acid sequence in the replication composition of step (i) to produce a nucleic acid analyte and under reaction conditions that permit the formation of an analyte/probe hybrid wherein said hybrid consists of said target analyte nucleic acid and the signal generating detection probe; and

(iii) detecting the presence said analyte/probe hybrid.

7. A method of claim 6 wherein the signal generating detection probe lacks a replication inhibitor moiety and the signal modifying detection probe are linked by a nucleic acid tether.

8. A method of claim 6 wherein the signal generating detection probe lacks a replication inhibitor moiety and the signal modifying detection probe are linked by a molecular spacer.

9. A method of claim 6 wherein the replication inhibiting moiety is selected from the group consisting of dideoxynuleotides, a sequence of mismatched nucleotides, 3′

phosphate, a molecular spacer, and 3′

deoxynucleotides.

10. The method of claim 9 where in the 3′

deoxynucleotides is cordycepin.

11. The method of claim 6 wherein said reporter pair is selected from the group consisting of fluorophores and enzymes.

12. A homogenous detection probe system comprising at least one pair of probes consisting of a first signal generating probe comprising a first member of a reporter pair, a target domain, a first probe binding domain and replication inhibitor moiety and a second signal modifying probe comprising a second member of a reporter pair and a second probe binding domain complementary to said first probe binding domain wherein the first and second members of the reporter pair are capable of reacting with each other to produce a detectable signal.

13. A detection probe comprising a reporter, a target domain and a replication inhibitor moiety consisting of cordycepin.