Multiplex nucleic acid reactions
DC CAFCFirst Claim
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1. A multiplex method for determining whether a sample contains at least 100 different target sequences, comprising:
- a) providing a sample which may contain at least 100 different single-stranded target sequences attached to a first solid support;
b) contacting said target sequences with a probe set comprising more than 100 different single-stranded probes, wherein each of said more than 100 different probes comprises;
i) a first universal priming site, wherein each of said more than 100 different probes has identical universal priming sites, andii) a target specific domain, such that different double-stranded hybridization complexes are formed, each of the different hybridization complexes comprising one of said more than 100 different single-stranded probes and one of the different single-stranded target sequences from the sample;
c) removing unhybridized probes;
d) contacting said probes of the hybridization complexes with a first enzyme and forming different modified probes;
e) contacting said modified probes with;
i) at least a first primer that hybridizes to said universal priming site;
ii) NTPs; and
iii) an extension enzyme;
wherein said different modified probes are amplified and forming different amplicons;
f) immobilizing said different amplicons to a second solid support, andg) detecting said different amplicons immobilized to said second solid support, thereby determining whether the sample contains at least 100 different target sequences.
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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.
323 Citations
22 Claims
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1. A multiplex method for determining whether a sample contains at least 100 different target sequences, comprising:
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a) providing a sample which may contain at least 100 different single-stranded target sequences attached to a first solid support; b) contacting said target sequences with a probe set comprising more than 100 different single-stranded probes, wherein each of said more than 100 different probes comprises; i) a first universal priming site, wherein each of said more than 100 different probes has identical universal priming sites, and ii) a target specific domain, such that different double-stranded hybridization complexes are formed, each of the different hybridization complexes comprising one of said more than 100 different single-stranded probes and one of the different single-stranded target sequences from the sample; c) removing unhybridized probes; d) contacting said probes of the hybridization complexes with a first enzyme and forming different modified probes; e) contacting said modified probes with; i) at least a first primer that hybridizes to said universal priming site; ii) NTPs; and iii) an extension enzyme; wherein said different modified probes are amplified and forming different amplicons; f) immobilizing said different amplicons to a second solid support, and g) detecting said different amplicons immobilized to said second solid support, thereby determining whether the sample contains at least 100 different target sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification