Reverse displacement assay for detection of nucleic acid...

Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o

Reexamination Certificate

Rate now

  [ 0.00 ] – not rated yet Voters 0   Comments 0

Details

C435S006120, C435S091100, C536S024300, C204S456000

Reexamination Certificate

active

06238927

ABSTRACT:

BACKGROUND OF THE INVENTION
The principle of hybridization is the property upon which most practical methods of detecting nucleic acid sequences are based. In general, methods for detecting the presence of particular target nucleic acid sequences involve employing a complementary sequence termed a probe, generally detectably labeled, and incubating this labeled probe sequence with the test sample thought to contain the target nucleic acid sequence. These complementary nucleic acid sequences hybridize to one another under suitable conditions to form probe-target hybridization complexes which can be identified through the presence of the detectable label. Various methods in which particular aspects of this basic process have been optimized for the purpose of addressing specific assay requirements have developed over time. (see Wetmur, 1991
, Critical Reviews in Biochemistry and Molecular Biology
26, 227-259).
While some homogeneous phase hybridization assays permit detection of the hybridized probe-target complex without removal of excess unhybridized probe sequences present in the assay solution (see Tyagi and Kramer, 1996,
Nature Biotechnology
14,303-308; Arnold, Waldrop, and Hanmmond, 1990, U.S. Pat. No. 4,950,613), very sensitive assays require separation of the hybridized complex from the unhybridized probe prior to the detection step. Many different methods have been employed to accomplish this separation, including several that rely on differences in physical characteristics between the two products. Other methods employ the use of a second nucleic acid sequence, described as a “capture” probe, for the purpose of separating the probe-target complex from the unhybridized probe. Capture probes are generally immobilized on a solid support and are selected to hybridize to a different portion of the target nucleic acid sequence than does the labeled probe. Thus, a tripartite capture probe-target-labeled probe complex forms which is bound to the solid support, while the unhybridized labeled probe remains unbound in solution, allowing the two products to be readily separated. Such assays are referred to as “sandwich” hybridizations (see, for example, Engelhardt and Rabbani, 1994, U.S. Pat. No. 5,288,609). Although widely used, these assays require a number of steps to perform and are quite time-consuming.
Alternative assay methods, termed displacement assays, were developed in an attempt to simplify the method of identifying nucleic acids. A schematic illustration of a standard displacement assay is depicted in
FIG. 1
(see Diamond, S. E., et al., 1988
, U.S. Pat. No.
4,766,062; Williams, J. I., et al. 1988, U.S. Pat. No. 4,766,064; Vary, 1987,
Nucleic Acids Res.
15, 6883-6897; Vary et al., 1986,
Clinical Chemistry
32, 1696-1701). In a standard displacement assay, a tether nucleic acid sequence, complementary to the target nucleic acid sequence, is hybridized to a shorter, detectably-labeled signal nucleic acid sequence, complementary to a specific subsequence of the tether sequence. The signal nucleic acid is fully base-paired with the tether nucleic acid in this signal-tether complex, but the longer tether nucleic acid retains a single-stranded region. Upon the introduction of a test sample containing the target nucleic acid, the target hybridizes to the single-stranded portion of the tether component. Since the target is homologous to the entire length of the tether, a homologous strand exchange reaction with the signal nucleic acid is initiated, and the target displaces the signal from the tether. This strand exchange reaction proceeds rapidly in the direction of signal nucleic acid displacement because the target is longer and forms a more stable duplex with the tether (see Green, C. and Tibbetts, C., 1981
, Nucleic Acids Res.
9, 1905-1918). The amount of displaced labeled signal nucleic acid is measured to determine the amount of target nucleic acid in the sample. The tether component of the probe complex can also be linked to a solid support, so that separation of the solid and solution phases results in isolation of the signal nucleic acid.
Unfortunately, despite their advantages, standard displacement assays do have certain drawbacks. For example, when target nucleic acid hybridizes to a tether not hybridized to a signal nucleic acid, or when a displaced signal nucleic acid hybridizes to a tether nucleic acid which was not previously hybridized to a signal nucleic acid, a decrease in the detection signal produced per unit of target hybridized results. Moreover, if the hybridized complex is not stable, an undesirable background signal can be introduced, which complicates interpretation of the assay results and reduces the sensitivity of the assay.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a probe-tether complex useful in a novel reverse displacement assay. This assay eliminates many of the difficulties inherent in the standard displacement assay, while retaining the key advantages over other solution phase hybridization reactions. The probe-tether complex of the invention is formed by contacting a first probe nucleic acid sequence complementary to the target nucleic acid sequence with a second tether nucleic acid sequence complementary to at least one subsequence of the first nucleic acid sequence under conditions suitable for hybridization between the two sequences. The probe-tether complex of the invention contains at least one double stranded segment and at least one single stranded segment.
In one aspect, the present invention pertains to a method for detecting the presence of a target nucleic acid in a test sample in which the sample to be tested for the presence of the target nucleic acid is introduced into a solution containing the probe-tether complex, under conditions suitable for hybridization to occur between the first probe nucleic acid sequence and the target nucleic acid sequence, so that the second tether nucleic acid sequence is displaced from the probe-tether complex, and a second complex is formed by the hybridization of the first probe nucleic acid sequence and the target nucleic acid sequence. The presence of the target nucleic acid can then be detected in the test sample.
In a preferred embodiment, one of the nucleic acid sequences contains a detectable label. In a particularly preferred embodiment, the probe nucleic acid contains a detectable label.
In a preferred embodiment, one of the nucleic acids is immobilized on a solid support. In a particularly preferred embodiment, the tether nucleic acid sequence is immobilized on a solid support.
In a preferred embodiment, one of the nucleic acids is immobilized on an electrophoretic medium. In a particularly preferred embodiment, the tether nucleic acid sequence is immobilized on an electrophoretic medium.
In another aspect, the present invention pertains to a method of detecting a target nucleic acid sequence in a test sample in which the method utilizes multiple sequential nucleic acid displacement reactions. In this method, the sample to be tested for the presence of the target nucleic acid is introduced into a solution containing the probe-tether complex, under conditions suitable for hybridization to occur between the first probe nucleic acid sequence and the target nucleic acid sequence, so that the tether nucleic acid sequence is displaced from the probe-tether complex, and a second hybridization complex, termed a displacement complex, is formed by the hybridization of the probe nucleic acid sequence and the target nucleic acid sequence. The tether nucleic acid sequence displaced by the target nucleic acid sequence is then available to form a probe-tether complex and the remainder of the steps of the method can be repeated. The sequence of steps can be repeated as many times as desired. The presence of the target nucleic acid can then be detected in the test sample.
In a preferred embodiment, one of the nucleic acid sequences contains a detectable label. In a particularly preferred embodiment, the probe nucleic acid contains a detecta

LandOfFree

Say what you really think

Search LandOfFree.com for the USA inventors and patents. Rate them and share your experience with other people.

Rating

Reverse displacement assay for detection of nucleic acid... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Reverse displacement assay for detection of nucleic acid..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Reverse displacement assay for detection of nucleic acid... will most certainly appreciate the feedback.

Rate now

     

Profile ID: LFUS-PAI-O-2441885

  Search
All data on this website is collected from public sources. Our data reflects the most accurate information available at the time of publication.