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US 2003/0143604 A1Published application
Real-time monitoring of PCR amplification using nanoparticle probes
OwnerJames J. Storhoff, Brett Fritz +1inventors
InventorsJames J. Storhoff, Brett Fritz, Mark Herrmann
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Abstract
The present invention relates to the use of nanoparticle detection probes to monitor amplification reactions, especially polymerase chain reactions (“PCR”). More specifically, the present invention involves the use of nanoparticles oligonucleotide conjugates treated with a protective agent such as bovine serum albumin in an homogeneous assay format in order to quantitatively and qualitatively detect a target polynucleotide.
Claims91
A method for detecting the presence of a target polynucleotide in a sample comprising:
- (a) providing a reaction and detection mixture comprising in combination:
- (1) a sample;
- (2) a nucleic acid amplification system;
- and
- (3) a nanoparticle detection system comprising one or more types of nanoparticles having one or more types of oligonucleotides bound thereto, the oligonucleotides bound to the nanoparticles have a sequence that is complementary to at least a portion of the sequence of the amplified target polynucleotide;
- (b) amplifying said target polynucleotide through at least one amplification cycle;
- (c) allowing the binding of said oligonucleotides bound to the nanoparticle to said amplified target polynucleotide under conditions effective to allow hybridization between said oligonucleotides bound to the nanoparticle and said amplified target polynucleotide;
- (d) determining the amount of signal generated as a result of the binding of the oligonucleotide bound to the nanoparticle to said amplified target polynucleotide;
- (e) optionally repeating steps (b)-(d);
- and
- (f) detecting the presence of said target polynucleotide by analyzing for the amount of signal produced after at least one amplification cycle.
8 more elements
A method for detecting the presence of a target polynucleotide in a sample, the target polynucleotide comprising a first and a second complimentary strand, said method comprising:
- (a) providing a reaction and detection mixture comprising in combination:
- (1) a sample,
- (2) a thermostable DNA polymerase,
- (3) 2′ deoxynucleoside-5′-triphosphates,
- (4) a forward-primer capable of binding to the first complimentary strand,
- (5) a reverse-primer capable of binding to the second complimentary strand in a position that will direct DNA synthesis toward the site of annealing of the forward-priming oligonucleotide, and
- (6) a nanoparticle detection system comprising one or more types of nanoparticles having one or more types of oligonucleotides bound thereto, the oligonucleotides bound to the nanoparticles have a sequence that is complementary to at least a portion of the sequence of the amplified target polynucleotide;
- (b) denaturing said target polynucleotide for an initial denaturation period;
- (c) denaturing said target polynucleotide for a cycle denaturation period;
- (d) incubating the reaction and detection mixture to allow binding of said nanoparticle-labeled oligonucleotides and said amplified target polynucleotide under conditions effective to allow hybridization between said oligonucleotides bound to the nanoparticle and said amplified target polynucleotide;
- (e) determining the amount of signal generated by the binding of said nanoparticle-labeled oligonucleotide with said amplified target polynucleotide;
- (f) annealing said forward priming and reverse priming oligonucleotides to the target polynucleotide;
- (g) synthesizing polynucleotide strands complementary to said first and second complementary strands of said target polynucleotide, said synthesis being catalyzed by the thermostable DNA polymerase;
- (h) optionally repeating steps (c)-(h);
- and
- (i) detecting the presence of said target polynucleotide by analyzing the amount of signal generated after at least one amplification cycle.
13 more elements
A method for detecting the presence of a target polynucleotide in a sample, the target polynucleotide comprising a first and a second complimentary strand, said method comprising:
- (a) providing a reaction and detection mixture comprising in combination:
- (1) sample,
- (2) a thermostable DNA polymerase,
- (3) 2′ deoxynucleoside-5′-triphosphates,
- (4) a forward-primer comprising a nanoparticle-labeled DNA primer sequence capable of binding to the first complimentary strand,
- (5) a reverse-primer capable of binding to the second complimentary strand in a position that will direct DNA synthesis toward the site of annealing of the forward-priming oligonucleotide,
- (6) a nanoparticle detection system comprising one or more types of nanoparticles having one or more types of oligonucleotides bound thereto, the oligonucleotides bound to the nanoparticles have a sequence that is complementary to at least a portion of the sequence of the extension product of the nanoparticle labeled DNA primer sequence, and
- (b) denaturing said target polynucleotide for an initial denaturation period;
- (c) denaturing said target polynucleotide for a cycle denaturation period;
- (d) annealing said forward priming and reverse priming oligonucleotides to the target polynucleotide;
- (e) determining the amount of signal generated by the binding of the extended DNA sequence bound through the nanoparticle labeled primer to the complementary nanoparticle probe and the nanoparticles having oligonucleotides bound thereto;
- (g) synthesizing polynucleotide strands complementary to said first and second complementary strands of said target polynucleotide, said synthesis being catalyzed by the thermostable DNA polymerase;
- (h) optionally repeating steps (c)-(h);
- and
- (i) detecting the presence of said target polynucleotide by analyzing the amount of signal generated after at least one amplification cycle.
12 more elements
A method for detecting the presence of a target polynucleotide in a sample, the target polynucleotide comprising a first and a second complimentary strand, said method comprising:
- (a) providing a reaction and detection mixture comprising in combination:
- (1) sample,
- (2) a thermostable DNA polymerase,
- (3) 2′ deoxynucleoside-5′-triphosphates,
- (4) a forward-primer capable of binding to the first complimentary strand,
- (5) a reverse-primer comprising a nanoparticle-labeled DNA primer sequence capable of binding to the second complimentary strand in a position that will direct DNA synthesis toward the site of annealing of the forward-priming oligonucleotide,
- (6) a nanoparticle detection system comprising one or more types of nanoparticles having one or more types of oligonucleotides bound thereto, the oligonucleotides bound to the nanoparticles have a sequence that is complementary to at least a portion of the sequence of the extension product of the nanoparticle labeled DNA primer sequence, and
- (b) denaturing said target polynucleotide for an initial denaturation period;
- (c) denaturing said target polynucleotide for a cycle denaturation period;
- (d) annealing said forward priming and reverse priming oligonucleotides to the target polynucleotide;
- (e) determining the amount of signal generated by the binding of the extended DNA sequence bound through the nanoparticle labeled primer to the complementary nanoparticle probe;
- (g) synthesizing polynucleotide strands complementary to said first and second complementary strands of said target polynucleotide, said synthesis being catalyzed by the thermostable DNA polymerase;
- (h) optionally repeating steps (c)-(h);
- and
- (i) detecting the presence of said target polynucleotide by analyzing the amount of signal generated after at least one amplification cycle.
12 more elements
A method for detecting the presence of a target polynucleotide in a sample, the target polynucleotide comprising a first and a second complimentary strand, said method comprising:
- (a) providing a reaction and detection mixture comprising in combination:
- (1) sample,
- (2) a thermostable DNA polymerase,
- (3) 2′ deoxynucleoside-5′-triphosphates,
- (4) a forward-primer comprising a nanoparticle-labeled DNA primer sequence capable of binding to the first complimentary strand,
- (5) a reverse-primer comprising a nanoparticle-labeled DNA primer sequence capable of binding to the second complimentary strand in a position that will direct DNA synthesis toward the site of annealing of the forward-priming oligonucleotide, and
- (b) denaturing said target polynucleotide for an initial denaturation period;
- (c) denaturing said target polynucleotide for a cycle denaturation period;
- (d) annealing said forward priming and reverse priming oligonucleotides to the target polynucleotide;
- (e) determining the amount of signal generated by the binding of the amplified DNA sequences attached to the nanoparticle labeled primers;
- (g) synthesizing polynucleotide strands complementary to said first and second complementary strands of said target polynucleotide, said synthesis being catalyzed by the thermostable DNA polymerase;
- (h) optionally repeating steps (c)-(h);
- and
- (i) detecting the presence of said target polynucleotide by analyzing the amount of signal generated after at least one amplification cycle.
11 more elements
A method for detecting the presence of a target polynucleotide in a sample comprising:
- (a) providing a reaction and detection mixture comprising in combination:
- (1) a sample;
- (2) a nucleic acid amplification system;
- and
- (3) a nanoparticle detection system comprising one or more types of nanoparticles having one or more types of oligonucleotides bound thereto, the oligonucleotides bound to the nanoparticles have a sequence that is complementary to at least a portion of the sequence of the amplified target polynucleotide;
- (b) amplifying said target polynucleotide through at least one amplification cycle;
- (c) allowing the binding of said oligonucleotides bound to the nanoparticle to said amplified target polynucleotide under conditions effective to allow hybridization between said oligonucleotides bound to the nanoparticle and said amplified target polynucleotide;
- (d) observing a detectable change.
5 more elements
A method for detecting the presence of a target polynucleotide in a sample comprising:
- (a) providing a reaction and detection mixture comprising in combination:
- (1) a sample;
- (2) a nucleic acid amplification system;
- and
- (3) a nanoparticle detection system comprising one or more types of nanoparticles having one or more types of oligonucleotides bound thereto, the oligonucleotides bound to the nanoparticles have a sequence that is complementary to at least a portion of the sequence of the amplified target polynucleotide;
- (b) amplifying said target polynucleotide through at least one amplification cycle;
- (c) allowing the binding of said oligonucleotides bound to the nanoparticle to said amplified target polynucleotide under conditions effective to allow hybridization between said oligonucleotides bound to the nanoparticles and said amplified target polynucleotides;
- (d) observing a detectable change resulting from the hybridization of the oligonucleotides on the nanoparticles with the amplified target polynucleotide.
5 more elements
A kit comprising:
- (a) a nucleic acid amplification system;
- and
- (b) a nanoparticle detection system comprising one or more types of nanoparticles having one or more types of oligonucleotides bound thereto, said nanoparticles produced by a process comprising contacting a nanoparticle having oligonucleotides bound thereto with a protective agent in aqueous solution in amounts sufficient to substantially prevent interference of said nucleic acid amplification reaction in the presence of said nanoparticle.
A nanoparticle having oligonucleotides bound thereto for use as a detection probe in a nucleic acid amplification reaction, said nanoparticle produced by contacting a nanoparticle having oligonucleotides bound thereto with a protective agent in aqueous solution in amounts sufficient to substantially prevent interference of said nucleic acid amplification reaction in the presence of said nanoparticle.
Dependencies are inferred from the claim text.