Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-30
2001-08-07
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300
Reexamination Certificate
active
06270972
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for identifying nucleic acid sequences using two or mere hybridization probes which hybridize to the same nucleic acid sequence.
2. Description of Related Art
A variety of assays have been developed to detect the presence of a particular nucleic acid sequence, hereinafter referred to as a target sequence, through selective hybridization of a hybridization probe to the target sequence. In order for a hybridization probe to hybridize to a target sequence, the hybridization probe must contain a nucleic acid sequence that is at least partially complementary to the target sequence. The complementary sequence of the probe must also be sufficiently long so that the hybridization probe selectively hybridizes to the target sequence over non-target sequences.
In addition to hybridizing a hybridization probe to a target sequence, the hybridization probe must be detectable. A variety of sandwich hybridization assays have been developed which identify a target nucleic acid sequence through the hybridization of a target nucleic acid sequence to two different hybridization probes. The first step of the assay generally involves the hybridization of a target nucleic acid sequence to a first hybridization probe. The hybridized pair is then generally immobilized to a solid support. A second hybridization probe containing a detectable marker is then hybridized to the target sequence, thereby enabling the target sequence hybridized to the first hybridization probe to be detected.
Sandwich hybridization assays require the use of two different hybridization probes where each hybridization probe hybridizes to a separate, non-overlapping portion of the target nucleic acid sequence. However, it is not always possible to design two hybridization probes to a target sequence where each hybridization probe hybridizes to different nonoverapping portions of the target sequence. As a result, some prior art sandwich hybridization assays employ hybridization probes that are not specific for the target nucleic acid. This significantly limits the quantitative accuracy of the assay for detecting target nucleic acid sequences in a sample.
It is an object of the present invention to provide an efficient hybridization assay for the identification of target nucleic acid sequences using two or more hybridization probes which hybridize to the same sequence of a target nucleic acid.
SUMMARY OF THE INVENTION
A method for detecting a target nucleic acid sequence in a sample is provided using two or more hybridization probes which hybridize to the same sequence of a target nucleic acid.
According to an embodiment of the method, a sample containing a target nucleic acid is contacted with a first hybridization probe and a second hybridization probe under conditions favorable for hybridization. The first and second hybridization probes are able to simultaneously hybridize to the target nucleic acid sequence, the first and second hybridization probes including a first fraction of hybridization probes which include a first complexing agent capable of forming a binding pair with a second complexing agent and a second fraction of hybridization probes which include a detectable marker, the second fraction of hybridization probes having the same ratio of first hybridization probes to second hybridization probes as the first fraction.
After the sample containing the target nudeic acid is contacted with the first and second hybridization probes, the first complexing agent attached to the first fraction of hybridization probes is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, the target nucleic acids hybridized to the first fraction of hybridization probes become immobilized on to the solid support Once immobilized, the immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second fraction of hybridization probes.
In order to facilitate the detection and quantification of the detectable marker, as well as the isolation of the immobilized target sequences, the first fraction of hybridization probes is preferably detachably linked to the solid support. This may be accomplished by incorporating a detachable linker between the first fraction of hybridization probes and the first complexing agent, between the solid support and the second complexing agent or between the first and second complexing agents. The presence of the released target sequences may be determined by detecting the presence of the detectable marker or by detecting the presence of the released target sequence itself.
When a detachable linker is employed, purification of the target nucleic acid sequence hybridized to the hybridization probes can be accomplished by releasing the target nucleic acid sequences from the solid support, dehybridizing the target sequence from the hybridization probes and isolating the released dehybridized target nucleic acid sequence, for example by gel electrophoresis. Thus, using a detachable linker, the target sequence may readily be isolated and made available for further analysis, such as sequencing.
The first complexing agent is preferably an antigen, antibody, biotin, a biotin derivative or analogue, avidin or an avidin derivative and analogue. The detectable marker is preferably a radioisotope, an isotope measurable by AMS, a fluorescent molecule, a chemiluminescent molecule, an antibody, the nucleic acid itself or an enzymatically modifiable substrate, the modified enzymatic substrate being analytically detectable.
A kit is also provided for detecting a target nudeic acid sequence in a sample. The kit may include a first fraction of hybridization probes having a first hybridization probe and a second hybridization probe, the first and second hybridization probes being able to simultaneously hybridize to the target nucleic acid sequence and a first complexing agent attached to the first and second hybridization probes, the first complexing agent forming a binding pair with a second complexing agent. The kit also includes a second fraction of hybridization probes having the first and second hybridization probes used in the first fraction wherein the ratio of the first to second hybridization probes in the second fraction is approximately equal to the ratio of first to second hybridization probes in the first fraction. The second fraction of hybridization probes also include,s a detectable marker for detecting the target sequence. Optionally, the kit may further include a second complexing agent attached to a solid support as well as instructions for using the kit.
The assay of the present invention may also be readily adapted for the diagnosis of disease, the occurrence of which is associated with and/or identifiable by the presence or absence of a target nucleic acid sequence. According to this embodiment of the invention, the hybridization probe or probes are designed to selectively hybridize to a nudeic acid sequence associated with and/or characteristic of a disease. The present invention also relates to a kit for diagnosing disease using the competitive hybridization assay of t he present invention.
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pa
Bogen Kenneth T.
Lucas Joe N.
Straume Tore
Arthur Lisa B.
The Regents of the University of California
Weitz David J.
Wilson Sonsini Goodrich & Rosati
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