Nucleic acid detection methods using universal priming
First Claim
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1. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
- a) providing a first probe comprising;
i) an upstream universal priming site (UUP);
ii) an adapter sequence;
iii) a first target-specific sequence comprising a first base at a readout position; and
iv) a downstream universal priming site (DUP);
b) contacting said first probe with said target sequence under conditions whereby only if said first base is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;
c) removing non-hybridized first probes;
d) denaturing said hybridization complex;
e) amplifying said first probe to generate a plurality of amplicons;
f) contacting said amplicons with an array of capture probes; and
g) determining the nucleotide at said detection position.
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Abstract
The present invention is directed to providing sensitive and accurate assays for genotyping with a minimum or absence of target-specific amplification.
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Citations
29 Claims
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1. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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a) providing a first probe comprising;
i) an upstream universal priming site (UUP);
ii) an adapter sequence;
iii) a first target-specific sequence comprising a first base at a readout position; and
iv) a downstream universal priming site (DUP);
b) contacting said first probe with said target sequence under conditions whereby only if said first base is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;
c) removing non-hybridized first probes;
d) denaturing said hybridization complex;
e) amplifying said first probe to generate a plurality of amplicons;
f) contacting said amplicons with an array of capture probes; and
g) determining the nucleotide at said detection position. - View Dependent Claims (2, 3, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 29)
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4. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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a) providing a plurality of readout probes each comprising;
i) an upstream universal priming site (UUP);
ii) an adapter sequence;
iii) a target-specific sequence comprising a unique base at a readout position; and
iv) a downstream universal priming site (DUP);
b) contacting said detection probes with said target sequence under conditions whereby only if said base at said readout position is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;
c) removing non-hybridized first probes;
d) denaturing said first hybridization complex;
e) amplifying said detection probes to generate a plurality of amplicons;
f) contacting said amplicons with an array of capture probes; and
g) determining the nucleotide at said detection position.
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5. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises:
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a) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising;
i) an upstream universal priming site (UUP); and
ii) a first target-specific sequence; and
b) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising;
i) a downstream universal priming site (DUP); and
ii) a second target-specific sequence comprising a first base at an interrogation position;
wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed and wherein at least one of said first and second ligation probes comprises an adapter sequence;
c) removing non-hybridized first probes;
d) providing a ligase that ligates said first and second ligation probes to form a ligated probe;
e) amplifying said ligated probe to generate a plurality of amplicons;
f) contacting said amplicons with an array of capture probes; and
g) determining the nucleotide at said detection position.
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6. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises:
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a) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising;
i) an upstream universal priming site (UUP); and
ii) a first target-specific sequence; and
b) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising;
i) a downstream universal priming site (DUP); and
ii) a second target-specific sequence comprising a first base at an interrogation position;
wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed and wherein at least one of said first and second ligation probes comprises an adapter sequence;
c) removing non-hybridized first probes;
d) providing a ligase that ligates said first and second ligation probes to form a ligated probe;
e) hybridizing said ligated probe to a rolling circle (RC) sequence comprising;
i) an upstream priming sequence; and
ii) a downstream priming sequence;
f)providing a ligase that ligates said upstream and downstream priming sites to form a circular ligated probe;
g) amplifying said circular ligated probe to generate a plurality of amplicons;
f) contacting said amplicons with an array of capture probes; and
g) determining the nucleotide at said detection position.
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7. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises:
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a) hybridizing a rolling circle (RC) probe to said target sequence, said RC probe comprising;
i) an upstream universal priming site (UUP); and
ii) a first target-specific sequence;
iii) a second target-specific sequence comprising a first base at an interrogation position; and
iv) an adapter sequence;
wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed;
c) providing a ligase that ligates said first and second ligation probes to form a ligated probe;
d) amplifying said ligated probe to generate a plurality of amplicons;
e) contacting said amplicons with an array of capture probes; and
f) determining the nucleotide at said detection position. - View Dependent Claims (8)
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17. A method of determining the identification of a nucleotide at a detection position in a genomic target sequence comprising:
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a) attaching a library of genomic target sequences to a solid support;
b) adding at least one probe and an enzyme to form an extended primer;
c) denaturing said extended primer from said target sequence;
d) hybridizing said extended primer to an array comprising capture probes; and
e) determining said nucleotide at said detection position.
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24. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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a) providing a support on which the target sequence is immobilized;
b) providing a first probe comprising;
i) an upstream universal priming site (UUP);
ii) an adapter sequence;
iii) a first target-specific sequence comprising a first base at a readout position; and
iv) a downstream universal priming site (DUP);
c) contacting said first probe with said target sequence under conditions whereby only if said first base is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;
d) removing non-hybridized first probes;
e) denaturing said hybridization complex;
f) amplifying said first probe to generate a plurality of amplicons;
g) contacting said amplicons with an array of capture probes; and
h) determining the nucleotide at said detection position
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25. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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a) providing a support on which the target sequence is immobilized;
b) providing a plurality of readout probes each comprising;
i) an upstream universal priming site (UUP);
ii) an adapter sequence;
iii) a target-specific sequence comprising a unique base at a readout position; and
iv) a downstream universal priming site (DUP);
c) contacting said detection probes with said target sequence under conditions whereby only if said base at said readout position is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;
d) removing non-hybridized first probes;
e) denaturing said first hybridization complex;
f) amplifying said detection probes to generate a plurality of amplicons;
g) contacting said amplicons with an array of capture probes; and
h) determining the nucleotide at said detection position.
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26. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises:
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a) providing a support on which the target sequence is immobilized;
b) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising;
i) an upstream universal priming site (UUP); and
ii) a first target-specific sequence; and
c) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising;
i) a downstream universal priming site (DUP), and ii) a second target-specific sequence comprising a first base at an interrogation position;
wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed and wherein at least one of said first and second ligation probes comprises an adapter sequence;
d) removing non-hybridized first probes;
e) providing a ligase that ligates said first and second ligation probes to form a ligated probe;
f) amplifying said ligated probe to generate a plurality of amplicons;
g) contacting said amplicons with an array of capture probes; and
h) determining the nucleotide at said detection position.
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27. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises:
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a) providing a support on which the target sequence is immobilized;
b) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising;
i) an upstream universal priming site (UUP); and
ii) a first target-specific sequence; and
c) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising;
i) a downstream universal priming site (DUP); and
ii) a second target-specific sequence comprising a first base at an interrogation position;
wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed and wherein at least one of said first and second ligation probes comprises an adapter sequence;
d) removing non-hybridized first probes;
e) providing a ligase that ligates said first and second ligation probes to form a ligated probe;
f) hybridizing said ligated probe to a rolling circle (RC) sequence comprising;
i) an upstream priming sequence; and
ii) a downstream priming sequence;
g) providing a ligase that ligates said upstream and downstream priming sites to form a circular ligated probe;
h) amplifying said circular ligated probe to generate a plurality of amplicons;
i) contacting said amplicons with an array of capture probes; and
j) determining the nucleotide at said detection position.
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28. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises:
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a) providing a support on which the target sequence is immobilized;
b) hybridizing a rolling circle (RC) probe to said target sequence, said RC probe comprising;
i) an upstream universal priming site (UUP); and
ii) a first target-specific sequence;
iii) a second target-specific sequence comprising a first base at an interrogation position; and
iv) an adapter sequence;
wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed;
c) providing a ligase that ligates said first and second ligation probes to form a ligated probe;
d) amplifying said ligated probe to generate a plurality of amplicons;
e) contacting said amplicons with an array of capture probes; and
f) determining the nucleotide at said detection position.
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Specification