Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-06
2001-07-03
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S025320, C536S025400, C536S025600, C536S026600
Reexamination Certificate
active
06255051
ABSTRACT:
BACKGROUND OF THE INVENTION
The principle of hybridization serves as the basis upon which methods of detecting nucleic acids is founded. Conventional methods for detecting the presence of a particular nucleic acid sequence involves employing a complementary sequence, the probe sequence which is usually labeled, and incubating this labeled sequence with the sample putatively containing the sample of interest. If the polynucleotide sequence of the target nucleic acid is complementary to the polynucleotide sequence of the probe, then the two (under suitable conditions) will hybridize. If there is hybridization, then the hybridization complex can be detected. Variations of this general protocol have been developed over time. Sensitivity of the detection assay is critical especially when detecting the presence of nucleic acids that are in low concentrations.
To improve the sensitivity in nucleic acid assays, different methods of amplification have developed. One approach is to amplify the number of target molecules. Polymerase Chain Reaction, or PCR, is such a method that is employed to increase the copy number of a target polynucleotide sequence which results in the amplification of the original target sequence. Ausubel, F. M., et al. (eds.),
Current Protocols in Molecular Biology,
Green Publishing Associates and Wiley-Interscience, 5
th
ed., (1991), vol. 2, pp. 15.1.1-15.3.8.; 15.4.1-15.4.6. Strand Displacement Amplification (SDA) and Transcription Mediated Amplification (TMA) are two other examples of methods that are used to increase or amplify the target polynucleotide sequence. All of these methods require proteins, for example, the enzymes that are used in these procedures to catalyze the necessary reactions, which constrain the conditions under which the assay can be performed. Due to the necessary presence of these enzymes in these particular assays, critical and stringent reactions must be established and maintained to insure the efficacy of these stated methods.
It would be desirable to have a nucleic acid hybridization assay that is sensitive enough to amplify a signal that is generated during a hybridization-displacement assay rather than the target sequence itself, yet avoids the above-mentioned complications.
SUMMARY OF THE INVENTION
The invention pertains to novel and commercially useful methods for analyzing nucleic acids. The present invention provides for a highly specific hybridization-based identification system of nucleic acids using multiple sequential polynucleotide displacement reactions which result in gain or amplification of a detectable signal.
The core of the present invention provides multiple rounds of polynucleotide displacement wherein the displaced polynucleotide of the preceeding displacement reaction is transferred to contact the next probe complex where it becomes the target for a new cycle of displacement.
In one embodiment of the invention, a method of detecting a target polynucleotide sequence within a biological sample is disclosed. This detection method involves a series of sequential hybridization and displacement reactions, that utilize probe complexes formed by hybridization between complementary polynucleotide sequences which are hybridized to one another to form a probe complex. This hybridization complex is subsequently contacted with a polynucleotide sequence which will compete with one of the constituent polynucleotide sequences of the complex. The target nucleotide sequence is one such sequence that will compete off one the constituents found in the probe complex, generally the first probe complex formed. This competition results in the generation of a displaced polynucleotide sequence. Based on the physicochemical principle of affinity, the competing polynucleotide sequence will displace one of the two polynucleotide sequences comprising the probe complex. The displaced polynucleotide sequence(s) then serve, as a signal which can be detected in any number of ways, or be used to compete off another constituent polynucleotide sequence in a different probe complex. Multiple displacement can be least one or more displacement events other performed with, or without gain, of signal at each displacement step.
In another embodiment, the present invention pertains to a method of detecting a target polynucleotide sequence within a biological test sample using a recursive cycle. Several cycles of probe and displacement complex formation generating multiple displacement polynucleotide sequences in which one of these displaced polynucleotide sequences is identical to the original target polynucleotide sequence, thereby facilitating cycling back to the first displacement complex and proceeding through the sequential hybridization and displacement cycles again. This process of recycling through probe and displacement complex formation provides for the amplification of the assay signal(s) prior to detection.
In another embodiment of the invention, a method for detecting a target polynucleotide sequence in a nucleic acid molecule within a sample using a set of heterogenous signals is described. In this embodiment, cycles of hybridization and displacement take place in which a bifurcation event occurs resulting in the production of multiple and distinct hybridization/displacement cycles, thereby generating multiple heterogenous signals for one particular target polynucleotide sequence.
In still another embodiment, the invention pertains to a method of detecting different target polynucleotide sequences in one sample by generating a homogenous signal. Probe complexes are formed by hybridizing a first polynucleotide sequence, that is partially or completely complementary to the target polynucleotide sequence, with a second polynucleotide sequence (which is separate and distinct from the target sequence). This probe complex formation occurs for all of the target polynucleotide sequences to be analyzed in the sample. Hybridization and displacement cycles take place independently using different target polynucleotide sequences designated for analysis. However, the assay is constructed in such a way as to generate a homogenous polynucleotide sequence signal that accounts for all of the target polynucleotide sequences analyzed.
In another embodiment, the invention pertains to a method for detecting a target polynucleotide sequence in a nucleic acid molecule using an immobilizing surface. Cycles of hybridization and displacement occur throughout this method. However, the hybridization complexes are immobilized to a surface and the displaced polynucleotide sequence is free in solution. The displaced polynucleotide sequence liberated from a hybridization complex, in particular, a displacement complex by employing a competing polynucleotide sequence to displace a polynucleotide sequence constituent of the complex, can be transferred to another surface in order to continue the reaction cycles, for example, another displacement complex. The surfaces involved can be coextensive or can be separated from each other by, for example, size-exclusion membranes. Alternatively, the surfaces can be spatially separated as exemplified by using different conical tubes as representing different surfaces. This embodiment also embraces the use of solid support matrixes in which reaction stations can be integral to the matrix itself. The reactants for a particular event, such as probe complex or displacement complex formation, can be placed into these reaction stations, thereby isolating these individual reaction events. Movement of reaction substrates or products from one station to the next can be accomplished via assisted or unassisted migration through the matrix, or alternatively, mechanical transfer using, for example, a pipette.
A diagnostic kit for determining the presence of a target polynucleotide sequence within a biological sample is also disclosed in the present invention. The kit comprises a first probe complex and at least one second probe complexes. The first probe complex comprises a first polynucleotide sequence comprising a sequence complementary to a ta
Abrams Ezra S.
Boles T. Christian
Hammond Philip W.
Muir Andrew R.
Campbell Eggerton A.
Hamilton Brook Smith & Reynolds P.C.
Mosaic Technologies Inc.
Tung Joyce
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