Analyte detection process using dual labeled probes

Details Analyte detection process using dual labeled probes Analyte detection process using dual labeled probes

1997-11-07

2001-09-25

Campbell, Eggerton A. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C435S183000, C436S094000, C536S023100, C536S024300, C536S024330, C536S025300, C536S025310

Reexamination Certificate

active

06294326

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The field of this invention is analyte detection. More particularly, the present invention relates to the detection of target nucleic acids in a test sample using a dual labeled oligonucleotide probe and heterogeneous detection methods.
BACKGROUND OF THE INVENTION
There are a number of methods for detecting the presence of a particular nucleic acid molecule in a sample. By way of example, detecting amplification products from the polymerase chain reaction (PCR), ligase chain reaction (LCR), gap ligase chain reaction (GLCR), Q&bgr; replicase, self-sustained sequence replication and strand displacement amplification (SDA), as a means for detecting a particular nucleic acid sequence, have all been described (See, e.g., Roche Molecular Systems, Inc.,
Current Opinion in Biotechnology
4:41-47, 1993). These amplification processes are becoming useful clinical diagnostic tools to, for example, construct assays which detect infectious organisms in a test sample. Additionally, amplification assays have been used in research and development fields as well as in forensic fields to, for example, detect genetic defects.
LCR described in European Patent Application EP-A-320-308; PCR and a variation of PCR known as reverse transcription PCR (RT-PCR) described in U.S. Pat. Nos. 4,683,202, 4,683,195, and 5,322,770 (the disclosures of which are incorporated herein by reference); and GLCR described in U.S. Pat. No. 5,427,930 (the disclosure of which is also incorporated herein by reference) are widely used in nucleic acid amplification based assays designed to detect a target sequence and therefore the organism which is associated with the target sequence. These amplification techniques typically employ primers or probes to repeatedly generate copies of a target nucleic acid sequence, which is usually a small region of a much larger nucleic acid sequence. Primers and probes are themselves nucleic acid sequences that are complementary to regions of a target sequence and under suitable conditions, hybridize or bind to the complementary portions of the target sequence. Copies of the target sequence are typically generated by the process of primer or probe extension that utilizes enzymes such as polymerase or ligase, separately or in combination, to add nucleotides (or probes) to the hybridized primers or probes. The nucleotides (or probes) that are added to the primers or probes are also complementary to the target sequence. Once the primers or probes have been sufficiently extended and/or ligated they are separated from the target sequence and a sequence complementary to the target sequence is formed. A new round of extension can then take place to amplify the number of complementary target sequences. Additionally, the sequences that are complementary to the target sequence can serve as templates for primer or probe extension to thereby amplify the number of target sequences. Hence multiple copies of the target sequence and its complementary sequence are produced.
The presence or amount of amplified target sequences and/or sequences complementary to the target sequence in a solution can be detected in a number of ways. Typically, the amplified sequences are detected using labeled primers that are incorporated into the amplified sequences, labeled probes that hybridize to the amplification products or a combination of both. For example, a sandwich assay format (variously referred to as a heterogeneous assay format) can be employed to detect the presence or amount of amplified nucleic acid sequences in a solution. Specifically, amplified sequences can be immobilized on a solid phase such as a suspension of microparticles coated with a capturing moiety. That capturing moiety binds to, for example, a capture hapten incorporated into the amplified sequences. Accordingly, when the sequences are contacted with the capture reagent, the sequences become immobilized on the solid support. A second hapten, also incorporated into the amplified sequences, can be used to detect the sequences immobilized to the solid support. In particular, the support bound sequences can be contacted with a conjugate comprising, for example, a fluorescent moiety conjugated to a member that specifically binds the second hapten. Thus, the fluorescent moiety also is immobilized to the solid phase by virtue of the second hapten and the fluorescent moiety can then be detected as an indication of the amplified sequences on the solid support.
More recently, homogeneous methods for detecting amplified sequences have been described. The so-called “Taq-Man” method, which is described in U.S. Pat. Nos. 5,210,015 and 5,538,848, the disclosures of which are incorporated herein by reference, is one such method. Briefly, the method uses a labeled oligonucleotide probe that specifically anneals to the target nucleic acid at a position downstream from a primer sequence (i.e., in the direction of primer extension). The probe contains both a reporter fluorescent molecule and a quencher molecule positioned on the probe such that the quencher molecule inhibits the detection of the fluorescent signal when the probe is intact. As amplification occurs (only in the presence of the target), the primer is extended by the action of a DNA polymerase and the probe is digested by the exonuclease activity of the polymerase. Upon such digestion of the probe, the quencher molecule is physically separated from the reporter molecule to enable detection of the fluorescent signal. In this manner, fluorescence increases with amplification and indicates the presence of the target sequence.
Unfortunately, however, there is no method for detecting amplified nucleic acid sequences using heterogeneous techniques such that the signal detected is imminently coupled to the amplification of the target sequence.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method for detecting the presence of a target sequence in a test sample. The process includes the steps of: (a) forming a reaction mixture comprising the test sample and any target sequence that may be contained therein, amplification reagents, and a primer and probe that hybridize to the same strand of the target sequence such that the probe is downstream from the primer; (b) degrading the probe in the reaction mixture to separate a first and a second label present on the intact probe where the first label is a specific binding member; (c) contacting the reaction mixture with a capture reagent to form a first specific-binding-member/capture reagent complex; and (d) detecting the second label associated with the first specific-binding-member/capture reagent complex, wherein a loss of signal indicates the presence of the target sequence. The reaction mixture can further include probe and/or primer sequences that hybridize to target sequence's complementary sequence. Additionally, the reaction may include additional probe and/or primer sequences that hybridize to another one or more target sequences to enable detection of multiple target sequences.
The second label on the probe can be a directly detectable label or it also may be a second specific binding member. In cases where the second label is a specific binding member, the method can further include the step of contacting the first specific-binding-member/capture reagent complex with a conjugate to detect the second label. The method can further include single or multiple wash steps that can be performed, for example, (i) after forming the first specific-binding-member/capture reagent complex and prior to contacting the so-formed complexes with a conjugate or detecting the second label, and/or (ii) after contacting the first specific-binding-member/capture reagent complex with the conjugate and prior to detecting the second label.
In one embodiment, the probe is degraded by including an enzyme having polymerase and 5′→3′ exonuclease activity in the reaction mixture. According to this embodiment, the target sequence is concomitantly amplified as the probe is degraded. In another embodiment, the pro

Affiliated with

Also associated with

Rating

Analyte detection process using dual labeled probes does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Analyte detection process using dual labeled probes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Analyte detection process using dual labeled probes will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2515884