Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1995-04-24
2001-05-22
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091200, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06235465
ABSTRACT:
TECHNICAL FIELD
This invention is in the field of nucleic acid hybridization assays. More specifically, it relates to novel nucleic acid probes for detecting HTLV-1.
BACKGROUND ART
HTLV-1 is a human lymphotrophic retrovirus which causes adult T-cell leukemia/lymphoma and tropic spastic paraparesis/HTLV-1-associated myelopathy. These HTLV-1 associated diseases are endemic in Japan and the Caribbean, with sporadic occurrences in the U.S. Detection of HTLV-1 is typically done by immunological or polymerase chain reaction assays (see, e.g., Meytes, et al.,
Lancet
336(8730):1533-1535, 1990).
Commonly owned U.S. Pat. No. 4,868,105, issued Sep. 19, 1989describes a solution phase nucleic acid sandwich hybridization assay in which analyte nucleic acid is first hybridized in solution to a labeling probe set and to a capturing probe set in a first vessel. The probe-analyte complex is then transferred to a second vessel that contains a solid-phase-immobilized probe that is substantially complementary to a segment of the capturing probes. The segments hybridize to the immobilized probe, thus removing the complex from solution. Having the analyte in the form of an immobilized complex facilitates subsequent separation steps in the assay. Ultimately, single stranded segments of the labeling probe set are hybridized to labeled probes, thus permitting the analyte-containing complex to be detected via a signal generated directly or indirectly from the label.
Commonly owned European Patent Application (EPA) 883096976 discloses a variation in the assay described in U.S. Pat. No. 4,868,105, issued Sep. 19, 1998, in which the signal generated by the labeled probes is amplified. The amplification involves the use of nucleic acid multimers. These multimers are branched polynucleotides that are constructed to have a segment that hybridizes specifically to the analyte nucleic acid or to a nucleic acid (branched or linear) that is bound to the analyte and iterations of a second segment that hybridize specifically to the labeled probe. In the assay employing the multimer, the initial steps of hybridizing the analyte to label or amplifier probe sets and capturing probe sets in a first vessel and transferring the complex to another vessel containing immobilized nucleic acid that will hybridize to a segment of the capturing probes are followed. The multimer is then hybridized to the immobilized complex and the labeled probes in turn hybridized to the second segment iterations on the multimer. Since the multimers provide a large number of sites for label probe attachment, the signal is amplified. Amplifier and capture probe sequences are disclosed for Hepatitis B virus,
Neisseria gonorrhoeae
, penicillin and tetracycline resistance in
N. gonorrhoeae
, and
Chlamydia trachomatis.
Commonly owned copending application Ser. No. 558,897, filed Jul. 27, 1990, describes the preparation of large comb-type branched polynucleotide multimers for use in the above-described solution phase assay. The combs provide greater signal enhancement in the assays than the smaller multimers.
DISCLOSURE OF THE INVENTION
One aspect of the invention is a synthetic oligonucleotide useful as an amplifier probe in a sandwich hybridization assay for HTLV-1 nucleic acid comprising a first segment having a nucleotide sequence substantially complementary to a segment of HTLV-1 nucleic acid, and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide acid multimer.
Another aspect of the invention is a synthetic oligonucleotide useful as a capture probe in a sandwich hybridization assay for HTLV-1 nucleic acid comprising a first segment having a nucleotide sequence substantially complementary to a segment of HTLV-1 nucleic acid; and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide bound to a solid phase.
Another aspect of the invention is a solution sandwich hybridization assay for detecting the presence of HTLV-1 nucleic acid in a sample, comprising
(a) contacting the sample under hybridizing conditions with an excess of (i) an amplifier probe oligonucleotide comprising a first segment having a nucleotide sequence substantially complementary to a segment of HTLV-1 nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer and (ii) a capture probe oligonucleotide comprising a first segment having a nucleotide sequence that is substantially complementary to a segment of HTLV-1 nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
(b) contacting the product of step (a) under hybridizing conditions with said oligonucleotide bound to the solid phase;
(c) thereafter separating materials not bound to the solid phase;
(d) contacting the product of step (c) under hybridization conditions with the nucleic acid multimer, said. multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide;
(e) removing unbound multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with the labeled oligonucleotide;
(g) removing unbound labeled oligonucleotide; and
(h) detecting the presence of label in the solid phase complex product of step (g).
Another aspect of the invention is a kit for the detection of HTLV-1 nucleic acid in a sample comprising in combination
(i) a set of amplifier probe oligonucleotides wherein the amplifier probe oligonucleotide comprises a first segment having a nucleotide sequence substantially complementary to a segment of HTLV-1 nucleic acid and a second segment having a nucleotide sequence substantially complementary to an oligonucleotide unit of a nucleic acid multimer;
(ii) a set of capture probe oligonucleotides wherein the capture probe oligonucleotide comprises a first segment having a nucleotide sequence that is substantially complementary to a segment of HTLV-1 nucleic acid and a second segment that is substantially complementary to an oligonucleotide bound to a solid phase;
(iii) a nucleic acid multimer, said multimer comprising at least one oligonucleotide unit that is substantially complementary to the second segment of the amplifier probe polynucleotide and a multiplicity of second oligonucleotide units that are substantially complementary to a labeled oligonucleotide; and
(iv) a labeled oligonucleotide.
MODES FOR CARRYING OUT THE INVENTION
Definitions
“Solution phase nucleic acid hybridization assay” intends the assay techniques described and claimed in commonly owned U.S. Pat. No. 4,868,105 and EPA 883096976.
A “modified nucleotide” intends a nucleotide monomer that may be stably incorporated into a polynucleotide and which has an additional functional group. Preferably, the modified nucleotide is a 5′-cytidine in which the N
4
-position is modified to provide a functional hydroxy group.
An “amplifier multimer” intends a branched polynucleotide that is capable of hybridizing simultaneously directly or indirectly to analyte nucleic acid and to a multiplicity of polynucleotide iterations (i.e., either iterations of another multimer or iterations of a labeled probe). The branching in the multimers is effected through covalent bonds and the multimers are composed of two types of oligonucleotide units that are capable of hybridizing, respectively, to analyte nucleic acid or nucleic acid hybridized to analyte nucleic acid and to a multiplicity of labeled probes. The composition and preparation of such multimers are described in EPA 883096976 and U.S. Ser. No. 558,897 filed Jul. 27, 1990, the disclosures of which are incorporated herein by reference.
The term “amplifier probe” is intended as a branched or linear polynucleotide that is constructed to have a segment that hybridizes specifically to the analyte nucleic acid and iterations of a second segmen
Kolberg Janice A.
Urdea Michael S.
Bayer Corporation
Fredman Jeffrey
Morrison & Foerester LLP
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