Multiplex nucleic acid reactions
First Claim
Patent Images
1. A method of detecting target sequences in a sample comprising:
- a) providing a first solid support comprising at least a first and a second target sequence;
b) contacting said first and second target sequences with first and second probes, respectively, wherein each of said first and second probes comprise;
i) a first universal priming site;
ii) a target specific domain substantially complementary to at least a portion of said target sequence;
to form first and second hybridization complexes, respectively;
c) removing unhybridized probes;
d) contacting said first and second hybridization complexes with a first enzyme to form modified first and second probes, respectively;
e) contacting said modified first and second probes with;
i) at least a first primer that hybridizes to said universal priming site;
ii) NTPs; and
iii) an extension enzyme;
wherein said first and second modified probes are amplified to form first and second amplicons, respectively; and
f) detecting said amplicons.
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Abstract
The invention is directed to a variety of multiplexing methods used to amplify and/or genotype a variety of samples simultaneously.
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Citations
11 Claims
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1. A method of detecting target sequences in a sample comprising:
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a) providing a first solid support comprising at least a first and a second target sequence;
b) contacting said first and second target sequences with first and second probes, respectively, wherein each of said first and second probes comprise;
i) a first universal priming site;
ii) a target specific domain substantially complementary to at least a portion of said target sequence;
to form first and second hybridization complexes, respectively;
c) removing unhybridized probes;
d) contacting said first and second hybridization complexes with a first enzyme to form modified first and second probes, respectively;
e) contacting said modified first and second probes with;
i) at least a first primer that hybridizes to said universal priming site;
ii) NTPs; and
iii) an extension enzyme;
wherein said first and second modified probes are amplified to form first and second amplicons, respectively; and
f) detecting said amplicons. - View Dependent Claims (2, 3, 7, 8, 9)
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4. A method of detecting target sequences in a sample comprising:
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a) providing a first solid support comprising at least a first and a second target sequence;
b) contacting said first and second target sequences with first and second probes, respectively, wherein each of said first and second probes comprise;
i) a first universal priming site;
ii) a target specific domain substantially complementary to at least a portion of said target sequence;
to form first and second hybridization complexes, respectively;
c) removing unhybridized probes;
d) contacting said first and second probes with;
i) at least a first universal primer that hybridizes to said universal priming site;
ii) NTPs; and
iii) an extension enzyme;
wherein said first and second probes are extended to form first and second modified probes, respectively;
e) contacting said first and second modified probes with;
i) at least third and fourth probes, respectively, wherein said modified first and second probes comprise a detection position, said third and fourth probes each comprise an interrogation position; and
ii) a second enzyme, wherein said second enzyme only modifies said third and fourth probes if there is perfect complementarity between the bases at said interrogation position and said detection position, forming third and fourth modified probes; and
f) detecting said third and fourth modified probes.
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5. A method comprising:
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a) providing a plurality of target nucleic acid sequences each comprising from 3′
to 5′
a first, second and third target domain, said first target domain comprising a detection position, said second target domain being at least one nucleotide;
b) contacting said target nucleic acid sequences with sets of probes for each target sequence, each set comprising;
i) a first probe comprising from 5′
to 3′
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1) a first domain comprising a first universal priming sequence; and
2) a second domain comprising;
A) a sequence substantially complementary to said first target domain of a target sequence; and
B) an interrogation position within the 3′
four terminal bases;
ii) a second probe comprising a first domain comprising a sequence substantially complementary to said third target domain of a target sequence;
to form a set of first hybridization complexes;
c) contacting said first hybridization complexes with;
i) an extension enzyme; and
ii) dNTPs;
under conditions whereby if the base at said interrogation positions is perfectly complementary with the bases at said detection positions, extension of said first probes occurs through said second target domains to form second hybridization complexes;
d) contacting said second hybridization complexes with a ligase to ligate the extended first probes to said second probes to form amplification templates. - View Dependent Claims (6)
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10. A multiplex reaction method comprising:
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a. providing a sample comprising at least first and second targets;
b. hybridizing said first and second targets with first and second probes, respectively forming first and second hybridization complexes, respectively;
c. immobilizing said first and second hybridization complexes;
d. washing to remove unhybridized nucleic acids;
e. contacting said first and second hybridization complexes with an enzyme, whereby said first and second probes are modified forming modified first and second probes, respectively, whereby said modified first and second probes are modified to contain first and second interrogation nucleotides that are complementary to first and second detection nucleotides in said first and second targets, respectively;
f. contacting said modified first and second probes with;
i. first and second allele specific primers, respectively, whereby said first and second allele specific primers hybridize to said modified first and second probes, respectively, 5′
to said first and second interrogation nucleotides;
ii. dNTPs iii. polymerase, whereby said first and second allele specific primers are modified when a target domain of said allele specific primers is perfectly complementary to said modified target probes to form modified first and second allele specific probes;
g. amplifying said modified first and second allele specific probes to form first and second amplicons; and
h. detecting said first and second amplicons.
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11. A method comprising:
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a) providing a plurality of target nucleic acid sequences each comprising from 3′
to 5′
a first, second and third target domain, said first target domain comprising a detection position, said second target domain being at least one nucleotide;
b) contacting said target nucleic acid sequences with sets of probes for each target sequence, each set comprising;
i) a first probe comprising from 5′
to 3′
;
1) a first domain comprising a first universal priming sequence; and
2) a second domain comprising;
A) a sequence substantially complementary to said first target domain of a target sequence; and
B) an interrogation position within the 3′
six terminal bases;
ii) a second probe comprising a first domain comprising a sequence substantially complementary to said third target domain of a target sequence;
to form a set of first hybridization complexes;
c) contacting said first hybridization complexes with;
i) at least a first universal primer that hybridize to said first universal priming sequence;
ii) an extension enzyme; and
iii) dNTPs;
under conditions whereby if the base at said interrogation positions are perfectly complementary with the bases at said detection positions, extension of said first probes occurs through said second target domains to form second hybridization complexes;
d) contacting said second hybridization complexes with a ligase to ligate the extended first probes to said second probes to form amplification templates.
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Specification