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US 2011/0033846 A1Published application
Concurrent Analysis of Multiple Patient Samples Using Solid Phase Addressable Multiplex Test with High Signal-to-Noise Ratio
AssigneeAutogenomics, Inc.
InventorNanibhushan Dattagupta
Life of this patent
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Abstract
Contemplated systems and methods allow analysis of multiple and distinct patient samples using a labeling scheme that entirely avoids carry-over of a label or reagent to the analytic platform, typically an addressable solid phase. In preferred aspects, a hybridization portion, a fluorophore, and/or a quencher are removed by a 5′-3′-exonuclease activity of a polymerase from a reporter oligonucleotide to so remove the oligonucleotide from the pool of molecules that bind to the solid phase and/or or to provide signal differentiation by removal of a fluorophore or quencher.
Claims20
A method of assisting execution of a multiplexed diagnostic assay on a solid phase at high signal-to-noise ratio, comprising:
- providing a plurality of first pairs of oligonucleotides suitable for amplification of a first nucleic acid that is suitable as a diagnostic marker for a first condition;
- providing a plurality of separate first labeled oligonucleotides, wherein each of the first labeled oligonucleotides has a common first portion that is suitable for hybridization with the first nucleic acid, and wherein each of the first labeled oligonucleotides has a distinct second portion;
- wherein each of the first labeled oligonucleotides has a unique identifier associated with the distinct second portion;
- providing instructions to separately perform a plurality of amplification reactions in a plurality of distinct patient samples, wherein each reaction uses one pair of the plurality of first pairs of oligonucleotides and one labeled oligonucleotide of the plurality of first labeled oligonucleotides;
- wherein the amplification reaction is performed using a DNA polymerase having 5′->3′-exonuclease activity under conditions that allow removal of (a) a quencher from the labeled oligonucleotide or (b) the distinct second portion from the labeled oligonucleotide;
- providing instructions to pool the plurality of amplification reactions, and to hybridize the pooled labeled oligonucleotides to a solid phase using the respective second portions;
- and
- providing a deconvolution table that associates a single test result for each of the different patient samples based on the first diagnostic marker and the unique identifier.
5 more elements
A data storage medium comprising:
- software programmed to establish a deconvolution table that includes
- (a) associative data of a plurality of diagnostic marker-specific common first portions of a plurality of labeled oligonucleotides, respectively, with a plurality of distinct second portions present in each of the plurality of labeled oligonucleotides;
- (b) associative data of the distinct second portions of the plurality of oligonucleotides with a solid phase characteristic;
- and
- (c) associative data of each of the plurality of labeled oligonucleotides with a patient identifier such that the table correlates a plurality of diagnostic tests for at least two distinct patients;
- wherein the associative data (a) and (b) are preprogrammed;
- and
- wherein the software is further programmed to acquire the patient identifier from an operator or operating system to thereby generate associative data (c).
5 more elements
A kit comprising:
- a plurality of labeled oligonucleotides having a structure of either
- (a) 5′-(X)n1Q-(X)n2-M-(X)n3-F-(X)n4-(Y)-(X)n5-(Z)-(X)n6-3′ or 5′-(X)n1-(Z)-(X)n2-M-(X)n3-F-(X)n4-(Y)-(X)n5-3′, wherein X is a nucleotide, n1-n6 are independently an integer between 0 and 50, Q is a fluorescence quenching portion, M is a modified nucleotide, F is a fluorophore, Y is the common first portion, and Z is the second portion, or
- (b) 5′-(X)n1-F-(X)n2-(Z)-(X)n3-M-(X)n4-Q-(X)n5-(Y)-(X)n6-3′ or 5′-(X)n1-(Y)-(X)n2-Q-(X)n3-M-(X)n4-(Z)-(X)n5-F-(X)n6-3′, wherein X is a nucleotide, Q is an optional fluorescence quenching portion;
- n1-n5 are independently an integer between 0 and 50, M is a modified nucleotide, F is a fluorophore, Y is the common first portion, and Z is the second portion;
- and
- a deconvolution table in which (a) sequence information of the first portion is correlated with a condition, (b) sequence information of the second portion is correlated with a physical characteristic of a solid phase, and (c) a unique identifier is correlated with an individual oligonucleotide of the plurality of labeled oligonucleotides.
3 more elements
A method of performing a multiplexed analytic test, comprising:
- separately performing a plurality of amplification reactions using a DNA polymerase having 5′->3′-exonuclease activity on a plurality of patient samples in which a condition-specific pair of oligonucleotides is employed to produce a plurality of amplicons, respectively;
- wherein each of the separate amplification reactions further includes an individual and distinct labeled oligonucleotide having a structure of
- (a) 5′-(X)n1Q-(X)n2-M-(X)n3-F-(X)n4-(Y)-(X)n5-(Z)-(X)n6-3′ or 5′-(X)n1-(Z)-(X)n2-M-(X)n3-F-(X)n4-(Y)-(X)n5-3′, wherein X is a nucleotide, n1-n6 are independently an integer between 0 and 50, Q is a fluorescence quenching portion, M is a modified nucleotide, F is a fluorophore, Y is the common first portion, and Z is the second portion, or
- (b) 5′-(X)n1-F-(X)n2-(Z)-(X)n3-M-(X)n4-Q-(X)n5-(Y)-(X)n6-3′ or 5′-(X)n1-(Y)-(X)n2-Q-(X)n3-M-(X)n4-(Z)-(X)n5-F-(X)n6-3′, wherein X is a nucleotide, Q is an optional fluorescence quenching portion;
- n1-n6 are independently an integer between 0 and 50, M is a modified nucleotide, F is a fluorophore, Y is the common first portion, and Z is the second portion;
- pooling the separate amplification reactions, and hybridizing the labeled oligonucleotides to a solid phase using distinct anchor molecules at predetermined positions on the solid phase to thereby bind the labeled oligonucleotides in predetermined positions;
- and
- using a deconvolution table to assign a test result to each plurality of patient samples using sequence information of the second portion.
5 more elements
Dependencies are inferred from the claim text.