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US 2021/0241857 A1Published application
Method and device for digital high resolution melt
AssigneeThe Regents of the University of California
InventorsStephanie I Fraley, Sinead Charpentier, Daniel Ortiz Velez, Mridu Sinha, Benjamin Albert Yang
Life of this patent
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Abstract
Methods are provided for nucleic acid analysis via a platform which incorporates a digital sample partitioning platform such as a microfluidic chip or digital droplet platform and instrumentation to accomplish universal amplification, High Resolution Melting (HRM), and machine learning within reactions simultaneously.
Claims21
-62. (canceled)
A method of profiling a sequence of a nucleic acid in a sample, the method comprising:
- combining a sample comprising a nucleic acid with
- one or more amplification primers,
- one or more DNA intercalating dyes;
- a reference dye;
- and
- reagents for amplifying a nucleic acid to form a reaction mixture, wherein the reagents for amplifying a nucleic acid comprise a polymerase;
- partitioning the reaction mixture into 10,000 or greater partitions, wherein each partition has a volume of 1 nL or less of the reaction mixture;
- amplifying the nucleic acid in each partition to produce an amplification product, wherein the amplification product comprises zero, one, or more amplicons;
- performing melt analysis of the amplification product in each partition by simultaneously heating and imaging the partitions by performing imaging at an imaging rate that is synchronized with the heating rate, whereby a melt curve, a melting temperature (Tm), or a combination thereof, for the amplification product in each partition is produced;
- and
- profiling the sequence of the nucleic acid by comparing the melt curve shape for the amplification product in each partition to a plurality of melt curves from different nucleic acid sequences, or by comparing the melting temperature (Tm) for the amplification product in each partition to a plurality of melting temperatures from different nucleic acid sequences.
8 more elements
A system comprising:
- a digital sample partitioning platform configured to partition a reaction mixture into 10,000 or more partitions;
- a heating device configured to apply heating or cooling uniformly to each partition;
- a temperature controller;
- an imagining device configured to capture images from each partition in one or more light channels;
- and
- a processor configured to perform machine learning for automated melt curve classification,
- wherein the system is configured to perform imaging at an imaging rate that is synchronized with a heating rate of the temperature controller.
4 more elements
Dependencies are inferred from the claim text.