US 2020/0048682 A1Published application

Methods for performing multiplexed real-time pcr

AssigneeRoche Molecular Systems, Inc.

InventorsIgor Kozlov, Amar Gupta, Randall Saiki, Alison Tsan

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Abstract

The present invention describes methods for performing higher multiplexed real-time PCR for detection and quantitation of target nucleic acids using tagged hydrolysis probes.

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Claims22

Dependent claims

A method for amplification and detection of a target nucleic acid in a sample comprising the steps of:

(a) contacting said sample containing said target nucleic acid in a single reaction vessel with
(i) one pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to opposite strands of a subsequence of said target nucleic acid;
(ii) an oligonucleotide probe that comprises an annealing portion and a tag portion, wherein the tag portion is attached to an internal position on the annealing portion located between 1 nucleotide and 10 nucleotides downstream from the 5′ terminus of the annealing portion and comprises a nucleotide sequence non-complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence at least partially complementary to the target nucleic acid sequence and hybridizes to a region of said subsequence of said target nucleic acid that is bounded by said pair of oligonucleotide primers, wherein said probe further comprises an interactive dual label comprising a reporter moiety located on the 5′ terminus of said tag portion and a first quencher moiety located on the 5′ terminus of said annealing portion and wherein said reporter moiety is separated from said first quencher moiety by a nuclease susceptible cleavage site;
and wherein said tag portion is reversibly bound in a temperature-dependent manner to a quenching molecule that comprises at least a second quencher moiety and optionally additional quencher moieties capable of quenching said reporter moiety when said quenching molecule is bound to said tag portion;
(b) amplifying said target nucleic acid by PCR using a nucleic acid polymerase having 5′ to 3′ nuclease activity such that during an extension step of each PCR cycle, the nuclease activity of the polymerase allows cleavage and separation of the tag portion from the first quencher moiety on the annealing portion of the probe;
(c) measuring a suppressed signal from the reporter moiety at a first temperature at which the quenching molecule is bound to the tag portion;
(d) increasing temperature to a second temperature at which the quenching molecule is not bound to the tag portion;
(e) measuring a temperature corrected signal from the reporter moiety at the second temperature;
(f) obtaining a calculated signal value by subtracting the suppressed signal detected at the first temperature from the temperature corrected signal detected at the second temperature;
(g) repeating steps (b) through (f) through multiple PCR cycles;
(h) measuring the calculated signal values from the multiple PCR cycles to detect the presence of the target nucleic acid.

8 more elements

A method for detecting two or more target nucleic acid sequences in a sample comprising the steps of:

(a) contacting said sample suspected of containing said two or more target nucleic acid sequences in a single reaction vessel with
(i) a first pair of oligonucleotide primers with nucleotide sequences that are complementary to each strand of a first target nucleic acid sequence, and a second pair of oligonucleotide primers with nucleotide sequences that are complementary to each strand of a second target nucleic acid sequence;
(ii) a first oligonucleotide probe comprising a nucleotide sequence at least partially complementary to the first target nucleic acid sequence and anneals within the first target nucleic acid sequence bounded by the first pair of oligonucleotide primers, wherein said first oligonucleotide probe comprises a fluorescent moiety capable of generating a detectable signal and a first quencher moiety capable of quenching the detectable signal generated by the fluorescent moiety, wherein the fluorescent moiety is separated the first quencher moiety by a nuclease susceptible cleavage site;
(iii) a second oligonucleotide probe comprising two distinct portions, an annealing portion comprising a nucleotide sequence at least partially complementary to the second target nucleic acid sequence and anneals within the second target nucleic acid sequence bounded by the second pair of oligonucleotide primers, wherein the annealing portion comprises a second quencher moiety located on the 5′ terminus of the annealing portion;
and a tag portion attached to an internal position on the annealing portion located between 1 nucleotide and 10 nucleotides downstream from the 5′ terminus of the annealing portion and comprising a nucleotide sequence that is non-complementary to the two or more target nucleic acid sequences, wherein the tag portion comprises a fluorescent moiety that is identical to the fluorescent moiety on the first oligonucleotide probe and whose detectable signal is capable of being quenched by the second quencher moiety on the annealing portion, wherein said fluorescent moiety is separated from the second quenching moiety by a nuclease susceptible cleavage site;
(iv) a quenching oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the second oligonucleotide probe and hybridizes to the tag portion to form a duplex, wherein said quenching oligonucleotide comprises a third quencher moiety which quenches the detectable signal generated by the fluorescent moiety on the tag portion when the quenching oligonucleotide is hybridized to the tag portion;
(b) amplifying the first and second target nucleic acid sequences by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5′ to 3′ nuclease activity such that during an extension step of each PCR cycle, the 5′ to 3′ nuclease activity of the nucleic acid polymerase allows cleavage and separation of the fluorescent moiety from the first quenching moiety on the first oligonucleotide probe, and cleavage and separation of the fluorescent moiety on the tag portion from the second quenching moiety on the annealing portion of the second oligonucleotide probe, wherein at the extension step the quenching oligonucleotide remains hybridized to the tag portion;
(c) measuring a fluorescent signal at a first temperature at which the quenching oligonucleotide is hybridized to the tag portion from the second oligonucleotide probe;
(d) increasing temperature to a second temperature, which is higher than the first temperature, at which the quenching oligonucleotide is not hybridized to the tag portion from the second oligonucleotide probe;
(e) measuring a fluorescent signal at the second temperature;
(f) obtaining a calculated signal value by subtracting the fluorescent signal detected at the first temperature from the fluorescent signal detected at the second temperature
(g) repeating steps (b) through (f) in multiple PCR cycles to produce desired quantity of amplification products from the first and second target nucleic acid sequences;
(h) determining the presence of the first target nucleic acid sequence from the fluorescent signals detected at the first temperature from the multiple PCR cycles and the presence of the second target nucleic acid sequence from the calculated signal values from the multiple PCR cycles.

10 more elements

A method for detecting two or more target nucleic acid sequences in a sample comprising the steps of:

(a) contacting said sample suspected of containing said two or more target nucleic acid sequences in a single reaction vessel with
(i) a first pair of oligonucleotide primers with sequences that are complementary to each strand of a first target nucleic acid sequence, and a second pair of oligonucleotide primers with sequences that are complementary to each strand of a second target nucleic acid sequence;
(ii) a first oligonucleotide probe comprising two distinct portions, a first annealing portion comprising a sequence at least partially complementary to the first target nucleic acid sequence and anneals within the first target nucleic acid sequence bounded by the first pair of oligonucleotide primers, wherein the first annealing portion comprises a first quencher moiety located on the 5′ terminus of the first annealing portion and is blocked at the 3′ terminus to prohibit extension by a nucleic acid polymerase;
and a first tag portion attached to an internal position on the first annealing portion located between 1 nucleotide and 10 nucleotides downstream from the 5′ terminus of the first annealing portion and comprising a nucleotide sequence that is non-complementary to the two or more target nucleic acid sequences, wherein the first tag portion comprises a fluorescent moiety whose detectable signal is capable of being quenched by the first quencher moiety on the first annealing portion, wherein said fluorescent moiety is separated from the first quenching moiety by a nuclease susceptible cleavage site;
(iii) a first quenching oligonucleotide comprising a sequence at least partially complementary to the first tag portion of the first oligonucleotide probe and hybridizes to the first tag portion to form a duplex, wherein said first quenching oligonucleotide comprises a second quenching moiety which quenches the detectable signal generated by the fluorescent moiety on the first tag portion when the first quenching oligonucleotide is hybridized to the first tag portion;
(iv) a second oligonucleotide probe comprising two distinct portions, a second annealing portion comprising a sequence at least partially complementary to the second target nucleic acid sequence and anneals within the second target nucleic acid sequence bounded by the second pair of oligonucleotide primers, wherein the second annealing portion comprises a third quencher moiety located on the 5′ terminus of the second annealing portion and is blocked at the 3′ terminus to prohibit extension by a nucleic acid polymerase;
and a second tag portion attached to an internal position on the second annealing portion located between 1 nucleotide and 10 nucleotides downstream from the 5′ terminus of the second annealing portion and comprising a nucleotide sequence that is non-complementary to the two or more target nucleic acid sequences, and has a different nucleic acid sequence or different nucleotide modifications compared to the nucleotide sequence of the first tag portion of the first oligonucleotide probe, wherein the second tag portion comprises a fluorescent moiety that is identical to the fluorescent moiety on the first oligonucleotide probe and whose detectable signal is capable of being quenched by the third quencher moiety on the second annealing portion, wherein said fluorescent moiety is separated from the third quenching moiety by a nuclease susceptible cleavage site;
(v) a second quenching oligonucleotide comprising a sequence at least partially complementary to the second tag portion of the second oligonucleotide probe and hybridizes to the second tag portion to form a duplex, wherein said second quenching oligonucleotide comprises a fourth quenching moiety which quenches the detectable signal generated by the fluorescent moiety on the second tag portion when the second quenching oligonucleotide is hybridized to the second tag portion;
wherein the duplex between the second quenching oligonucleotide and the second tag portion of the second oligonucleotide probe has a higher melting temperature (Tm) value than the duplex between the first quenching oligonucleotide and the first tag portion of the first oligonucleotide probe;
(b) amplifying the first and second target nucleic acid sequences by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5′ to 3′ nuclease activity such that during an extension step of each PCR cycle, the 5′ to 3′ nuclease activity of the nucleic acid polymerase allows
(i) cleavage and separation of the fluorescent moiety on the first tag portion from the first quenching moiety on the first annealing portion of the first oligonucleotide probe, wherein at the extension step the first quenching oligonucleotide remains hybridized to the first tag portion, and
(ii) cleavage and separation of the fluorescent moiety on the second tag portion from the third quenching moiety on the second annealing portion of the second oligonucleotide probe, wherein at the extension step the second quenching oligonucleotide remains hybridized to the second tag portion;
(c) increasing temperature to a first temperature at which the first quenching oligonucleotide is not hybridized to the first tag portion from the first oligonucleotide probe and the second quenching oligonucleotide remains hybridized to the second tag portion from the second oligonucleotide probe;
(d) measuring a fluorescent signal at the first temperature;
(e) increasing temperature to a second temperature which is higher than the first temperature, at which the second quenching oligonucleotide is not hybridized to the second tag portion from the second oligonucleotide probe;
(f) measuring a fluorescent signal at the second temperature;
(g) obtaining a calculated signal value by subtracting the fluorescent signal detected at the first temperature from the fluorescent signal detected at the second temperature;
(h) repeating steps (b) through (g) in multiple PCR cycles to produce desired quantity of amplification products from the first and second target nucleic acid sequences;
(I) determining the presence of the first target nucleic acid sequence from the fluorescent signals detected at the first temperature from the multiple PCR cycles and the presence of the second target nucleic acid sequence from the calculated signal values from the multiple PCR cycles.

16 more elements

Dependencies are inferred from the claim text.

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Abstract

The present invention describes methods for performing higher multiplexed real-time PCR for detection and quantitation of target nucleic acids using tagged hydrolysis probes.

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Claims22

Dependent claims

A method for amplification and detection of a target nucleic acid in a sample comprising the steps of:

(a) contacting said sample containing said target nucleic acid in a single reaction vessel with
(i) one pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to opposite strands of a subsequence of said target nucleic acid;
(ii) an oligonucleotide probe that comprises an annealing portion and a tag portion, wherein the tag portion is attached to an internal position on the annealing portion located between 1 nucleotide and 10 nucleotides downstream from the 5′ terminus of the annealing portion and comprises a nucleotide sequence non-complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence at least partially complementary to the target nucleic acid sequence and hybridizes to a region of said subsequence of said target nucleic acid that is bounded by said pair of oligonucleotide primers, wherein said probe further comprises an interactive dual label comprising a reporter moiety located on the 5′ terminus of said tag portion and a first quencher moiety located on the 5′ terminus of said annealing portion and wherein said reporter moiety is separated from said first quencher moiety by a nuclease susceptible cleavage site;
and wherein said tag portion is reversibly bound in a temperature-dependent manner to a quenching molecule that comprises at least a second quencher moiety and optionally additional quencher moieties capable of quenching said reporter moiety when said quenching molecule is bound to said tag portion;
(b) amplifying said target nucleic acid by PCR using a nucleic acid polymerase having 5′ to 3′ nuclease activity such that during an extension step of each PCR cycle, the nuclease activity of the polymerase allows cleavage and separation of the tag portion from the first quencher moiety on the annealing portion of the probe;
(c) measuring a suppressed signal from the reporter moiety at a first temperature at which the quenching molecule is bound to the tag portion;
(d) increasing temperature to a second temperature at which the quenching molecule is not bound to the tag portion;
(e) measuring a temperature corrected signal from the reporter moiety at the second temperature;
(f) obtaining a calculated signal value by subtracting the suppressed signal detected at the first temperature from the temperature corrected signal detected at the second temperature;
(g) repeating steps (b) through (f) through multiple PCR cycles;
(h) measuring the calculated signal values from the multiple PCR cycles to detect the presence of the target nucleic acid.

8 more elements

A method for detecting two or more target nucleic acid sequences in a sample comprising the steps of:

(a) contacting said sample suspected of containing said two or more target nucleic acid sequences in a single reaction vessel with
(i) a first pair of oligonucleotide primers with nucleotide sequences that are complementary to each strand of a first target nucleic acid sequence, and a second pair of oligonucleotide primers with nucleotide sequences that are complementary to each strand of a second target nucleic acid sequence;
(ii) a first oligonucleotide probe comprising a nucleotide sequence at least partially complementary to the first target nucleic acid sequence and anneals within the first target nucleic acid sequence bounded by the first pair of oligonucleotide primers, wherein said first oligonucleotide probe comprises a fluorescent moiety capable of generating a detectable signal and a first quencher moiety capable of quenching the detectable signal generated by the fluorescent moiety, wherein the fluorescent moiety is separated the first quencher moiety by a nuclease susceptible cleavage site;
(iii) a second oligonucleotide probe comprising two distinct portions, an annealing portion comprising a nucleotide sequence at least partially complementary to the second target nucleic acid sequence and anneals within the second target nucleic acid sequence bounded by the second pair of oligonucleotide primers, wherein the annealing portion comprises a second quencher moiety located on the 5′ terminus of the annealing portion;
and a tag portion attached to an internal position on the annealing portion located between 1 nucleotide and 10 nucleotides downstream from the 5′ terminus of the annealing portion and comprising a nucleotide sequence that is non-complementary to the two or more target nucleic acid sequences, wherein the tag portion comprises a fluorescent moiety that is identical to the fluorescent moiety on the first oligonucleotide probe and whose detectable signal is capable of being quenched by the second quencher moiety on the annealing portion, wherein said fluorescent moiety is separated from the second quenching moiety by a nuclease susceptible cleavage site;
(iv) a quenching oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the second oligonucleotide probe and hybridizes to the tag portion to form a duplex, wherein said quenching oligonucleotide comprises a third quencher moiety which quenches the detectable signal generated by the fluorescent moiety on the tag portion when the quenching oligonucleotide is hybridized to the tag portion;
(b) amplifying the first and second target nucleic acid sequences by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5′ to 3′ nuclease activity such that during an extension step of each PCR cycle, the 5′ to 3′ nuclease activity of the nucleic acid polymerase allows cleavage and separation of the fluorescent moiety from the first quenching moiety on the first oligonucleotide probe, and cleavage and separation of the fluorescent moiety on the tag portion from the second quenching moiety on the annealing portion of the second oligonucleotide probe, wherein at the extension step the quenching oligonucleotide remains hybridized to the tag portion;
(c) measuring a fluorescent signal at a first temperature at which the quenching oligonucleotide is hybridized to the tag portion from the second oligonucleotide probe;
(d) increasing temperature to a second temperature, which is higher than the first temperature, at which the quenching oligonucleotide is not hybridized to the tag portion from the second oligonucleotide probe;
(e) measuring a fluorescent signal at the second temperature;
(f) obtaining a calculated signal value by subtracting the fluorescent signal detected at the first temperature from the fluorescent signal detected at the second temperature
(g) repeating steps (b) through (f) in multiple PCR cycles to produce desired quantity of amplification products from the first and second target nucleic acid sequences;
(h) determining the presence of the first target nucleic acid sequence from the fluorescent signals detected at the first temperature from the multiple PCR cycles and the presence of the second target nucleic acid sequence from the calculated signal values from the multiple PCR cycles.

10 more elements

A method for detecting two or more target nucleic acid sequences in a sample comprising the steps of:

(a) contacting said sample suspected of containing said two or more target nucleic acid sequences in a single reaction vessel with
(i) a first pair of oligonucleotide primers with sequences that are complementary to each strand of a first target nucleic acid sequence, and a second pair of oligonucleotide primers with sequences that are complementary to each strand of a second target nucleic acid sequence;
(ii) a first oligonucleotide probe comprising two distinct portions, a first annealing portion comprising a sequence at least partially complementary to the first target nucleic acid sequence and anneals within the first target nucleic acid sequence bounded by the first pair of oligonucleotide primers, wherein the first annealing portion comprises a first quencher moiety located on the 5′ terminus of the first annealing portion and is blocked at the 3′ terminus to prohibit extension by a nucleic acid polymerase;
and a first tag portion attached to an internal position on the first annealing portion located between 1 nucleotide and 10 nucleotides downstream from the 5′ terminus of the first annealing portion and comprising a nucleotide sequence that is non-complementary to the two or more target nucleic acid sequences, wherein the first tag portion comprises a fluorescent moiety whose detectable signal is capable of being quenched by the first quencher moiety on the first annealing portion, wherein said fluorescent moiety is separated from the first quenching moiety by a nuclease susceptible cleavage site;
(iii) a first quenching oligonucleotide comprising a sequence at least partially complementary to the first tag portion of the first oligonucleotide probe and hybridizes to the first tag portion to form a duplex, wherein said first quenching oligonucleotide comprises a second quenching moiety which quenches the detectable signal generated by the fluorescent moiety on the first tag portion when the first quenching oligonucleotide is hybridized to the first tag portion;
(iv) a second oligonucleotide probe comprising two distinct portions, a second annealing portion comprising a sequence at least partially complementary to the second target nucleic acid sequence and anneals within the second target nucleic acid sequence bounded by the second pair of oligonucleotide primers, wherein the second annealing portion comprises a third quencher moiety located on the 5′ terminus of the second annealing portion and is blocked at the 3′ terminus to prohibit extension by a nucleic acid polymerase;
and a second tag portion attached to an internal position on the second annealing portion located between 1 nucleotide and 10 nucleotides downstream from the 5′ terminus of the second annealing portion and comprising a nucleotide sequence that is non-complementary to the two or more target nucleic acid sequences, and has a different nucleic acid sequence or different nucleotide modifications compared to the nucleotide sequence of the first tag portion of the first oligonucleotide probe, wherein the second tag portion comprises a fluorescent moiety that is identical to the fluorescent moiety on the first oligonucleotide probe and whose detectable signal is capable of being quenched by the third quencher moiety on the second annealing portion, wherein said fluorescent moiety is separated from the third quenching moiety by a nuclease susceptible cleavage site;
(v) a second quenching oligonucleotide comprising a sequence at least partially complementary to the second tag portion of the second oligonucleotide probe and hybridizes to the second tag portion to form a duplex, wherein said second quenching oligonucleotide comprises a fourth quenching moiety which quenches the detectable signal generated by the fluorescent moiety on the second tag portion when the second quenching oligonucleotide is hybridized to the second tag portion;
wherein the duplex between the second quenching oligonucleotide and the second tag portion of the second oligonucleotide probe has a higher melting temperature (Tm) value than the duplex between the first quenching oligonucleotide and the first tag portion of the first oligonucleotide probe;
(b) amplifying the first and second target nucleic acid sequences by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5′ to 3′ nuclease activity such that during an extension step of each PCR cycle, the 5′ to 3′ nuclease activity of the nucleic acid polymerase allows
(i) cleavage and separation of the fluorescent moiety on the first tag portion from the first quenching moiety on the first annealing portion of the first oligonucleotide probe, wherein at the extension step the first quenching oligonucleotide remains hybridized to the first tag portion, and
(ii) cleavage and separation of the fluorescent moiety on the second tag portion from the third quenching moiety on the second annealing portion of the second oligonucleotide probe, wherein at the extension step the second quenching oligonucleotide remains hybridized to the second tag portion;
(c) increasing temperature to a first temperature at which the first quenching oligonucleotide is not hybridized to the first tag portion from the first oligonucleotide probe and the second quenching oligonucleotide remains hybridized to the second tag portion from the second oligonucleotide probe;
(d) measuring a fluorescent signal at the first temperature;
(e) increasing temperature to a second temperature which is higher than the first temperature, at which the second quenching oligonucleotide is not hybridized to the second tag portion from the second oligonucleotide probe;
(f) measuring a fluorescent signal at the second temperature;
(g) obtaining a calculated signal value by subtracting the fluorescent signal detected at the first temperature from the fluorescent signal detected at the second temperature;
(h) repeating steps (b) through (g) in multiple PCR cycles to produce desired quantity of amplification products from the first and second target nucleic acid sequences;
(I) determining the presence of the first target nucleic acid sequence from the fluorescent signals detected at the first temperature from the multiple PCR cycles and the presence of the second target nucleic acid sequence from the calculated signal values from the multiple PCR cycles.

16 more elements

Dependencies are inferred from the claim text.

Description16,144 words

Summary7,440 words

#This application contains a Sequence Listing submitted as an electronic text file named “33677_US3.txt”, having a size in bytes of 2 kb, and created on Oct. 7, 2019. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

#The present invention relates to methods for polymerase chain reaction (PCR) particularly to methods for performing multiplexed real-time PCR.

#The polymerase chain reaction (PCR) has become a ubiquitous tool of biomedical research, disease monitoring and diagnostics. Amplification of nucleic acid sequences by PCR is described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188. PCR is now well known in the art and has been described extensively in the scientific literature. See PCR Applications, ((1999) Innis et al., eds., Academic Press, San Diego), PCR Strategies, ((1995) Innis et al., eds., Academic Press, San Diego); PCR Protocols, ((1990) Innis et al., eds., Academic Press, San Diego), and PCR Technology, ((1989) Erlich, ed., Stockton Press, New York). A “real-time” PCR assay is able to simultaneously amplify and detect and quantify the starting amount of the target sequence. The basic TaqMan real-time PCR assay using the 5′-to-3′ nuclease activity of the DNA polymerase is described in Holland et al., (1991) Proc. Natl. Acad. Sci. 88:7276-7280 and U.S. Pat. No. 5,210,015. The real-time PCR without the nuclease activity (a nuclease-free assay) has been described in a U.S. application Serial No. filed on Ser. No. 12/330,694 filed on Dec. 9, 2008. The use of fluorescent probes in real-time PCR is described in U.S. Pat. No. 5,538,848.

Brief description of the drawings14 figures

#FIG. 1 is a graphical description of one embodiment of the oligonucleotide probe used to perform the methods of the invention.

#FIG. 2 is a graphical representation of the method of the invention that shows the separation of the tag portion and subsequent dissociation of the quenching oligonucleotide.

#FIG. 3 is a description of one embodiment of the methods of the present invention.

Detailed description8,012 words

#The term “sample” as used herein includes a specimen or culture (e.g., microbiological cultures) that includes nucleic acids. The term “sample” is also meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin. Biological samples include whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchioalveolar, gastric, peritoneal, ductal, ear, arthroscopic), biopsy sample, urine, feces, sputum, saliva, nasal mucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, embryonic cells and fetal cells. In a preferred embodiment, the biological sample is blood, and more preferably plasma. As used herein, the term “blood” encompasses whole blood or any fractions of blood, such as serum and plasma as conventionally defined. Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants. Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.

#The terms “target” or “target nucleic acid” as used herein are intended to mean any molecule whose presence is to be detected or measured or whose function, interactions or properties are to be studied. Therefore, a target includes essentially any molecule for which a detectable probe (e.g., oligonucleotide probe) or assay exists, or can be produced by one skilled in the art. For example, a target may be a biomolecule, such as a nucleic acid molecule, a polypeptide, a lipid, or a carbohydrate, which is capable of binding with or otherwise coming in contact with a detectable probe (e.g., an antibody), wherein the detectable probe also comprises nucleic acids capable of being detected by methods of the invention. As used herein, “detectable probe” refers to any molecule or agent capable of hybridizing or annealing to a target biomolecule of interest and allows for the specific detection of the target biomolecule as described herein. In one aspect of the invention, the target is a nucleic acid, and the detectable probe is an oligonucleotide. The terms “nucleic acid” and “nucleic acid molecule” may be used interchangeably throughout the disclosure. The terms refer to oligonucleotides, oligos, polynucleotides, deoxyribonucleotide (DNA), genomic DNA, mitochondrial DNA (mtDNA), complementary DNA (cDNA), bacterial DNA, viral DNA, viral RNA, RNA, message RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), siRNA, catalytic RNA, clones, plasmids, M13, P1, cosmid, bacteria artificial chromosome (BAC), yeast artificial chromosome (YAC), amplified nucleic acid, amplicon, PCR product and other types of amplified nucleic acid, RNA/DNA hybrids and polyamide nucleic acids (PNAs), all of which can be in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides and combinations and/or mixtures thereof. Thus, the term “nucleotides” refers to both naturally-occurring and modified/nonnaturally-occurring nucleotides, including nucleoside tri, di, and monophosphates as well as monophosphate monomers present within polynucleic acid or oligonucleotide. A nucleotide may also be a ribo; 2′-deoxy; 2′,3′-deoxy as well as a vast array of other nucleotide mimics that are well-known in the art. Mimics include chain-terminating nucleotides, such as 3′-O-methyl, halogenated base or sugar substitutions; alternative sugar structures including nonsugar, alkyl ring structures; alternative bases including inosine; deaza-modified; chi, and psi, linker-modified; mass label-modified; phosphodiester modifications or replacements including phosphorothioate, methylphosphonate, boranophosphate, amide, ester, ether; and a basic or complete internucleotide replacements, including cleavage linkages such a photocleavable nitrophenyl moieties.

#The presence or absence of a target can be measured quantitatively or qualitatively. Targets can come in a variety of different forms including, for example, simple or complex mixtures, or in substantially purified forms. For example, a target can be part of a sample that contains other components or can be the sole or major component of the sample. Therefore, a target can be a component of a whole cell or tissue, a cell or tissue extract, a fractionated lysate thereof or a substantially purified molecule. Also a target can have either a known or unknown sequence or structure.

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Claims22

Abstract

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Claims22

Description16,144 words

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Life of this patent

Description16,144 words