Highly multiplexed digital nucleic acid determination using melting temperature
AssigneePresident and Fellows of Harvard College
InventorsDavid A. Weitz, Xing Zhao
Life of this patent
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Abstract
Methods, systems, and articles for multiplexed determination of target nucleic acids using thermal barcodes are provided. The thermal barcodes may, in some cases, be used along with one or more signaling entities for improved multiplexing. At least some methods described herein may be performed with a relatively high throughput, without sequencing of the nucleic acids, which can be expensive and slow. In some cases, the methods may be implemented using a system or article comprising a plurality of droplets comprising the target nucleic acids.
Claims72
A method, comprising:
- amplifying a target nucleic acid using a barcoded primer comprising a primer sequence and a thermal barcode sequence to produce a barcoded nucleic acid, the barcoded nucleic acid comprising a sequence identical to the thermal barcode sequence and a sequence identical to the target nucleic acid,
- wherein the thermal barcode sequence exhibits a first peak melting point and the target nucleic acid exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1° C.
A method, comprising:
- amplifying a target nucleic acid using a barcoded primer comprising a primer sequence and a thermal barcode sequence to produce a barcoded nucleic acid, the barcoded nucleic acid comprising a sequence identical to the thermal barcode sequence and a sequence identical to the target nucleic acid,
- wherein the thermal barcode sequence has a first ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy and the target nucleic acid has a second ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy, wherein the first ratio differs from the second ratio by at least 1% of the second ratio.
A method, comprising:
- amplifying a target nucleic acid using a barcoded primer comprising a primer sequence and a thermal barcode sequence to produce a barcoded nucleic acid, the barcoded nucleic acid comprising a sequence identical to the thermal barcode sequence and a sequence identical to the target nucleic acid,
- wherein the thermal barcode sequence comprises less than or equal to 500 nucleotides and wherein at least 55% of the nucleotides of the thermal barcode sequence are A or T or wherein at least 45% of the nucleotides of the thermal barcode are G or C, and
- wherein the thermal barcode sequence exhibits a peak melting temperature first peak melting point and the target nucleic acid exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1° C.
A method as in any preceding claim, wherein determining the barcoded nucleic acid comprises determining the presence or the absence of the target nucleic acid within a sample.
A method as in any preceding claim, wherein determining the barcoded nucleic acid comprises using one or more auxiliary barcodes configured to bind to at least a portion of the thermal barcode sequence or at least a portion of the target nucleic acid.
A method as in any preceding claim, wherein the presence of the barcoded nucleic acid is indicative of a disease or condition.
A method as in any preceding claim, wherein the absence of the barcoded nucleic acid is indicative of a disease or condition.
A method as in any preceding claim, wherein determining the barcoded nucleic acid comprises determining a subject from which the target nucleic acid originated.
A method as in any preceding claim, wherein the target nucleic acid is a first target nucleic acid of a plurality of target nucleic acids.
The method of any preceding claim, wherein a compartment comprises the nucleic acid.
The method of any preceding claim, wherein a droplet comprises the nucleic acid.
The method of any preceding claim, wherein a microwell comprises the nucleic acid.
A method as in any preceding claim, wherein the target nucleic acid is amplified in a droplet comprising a plurality of barcoded primers comprising primer sequences and thermal barcode sequences.
A method as in any preceding claim, wherein the method comprises amplifying a plurality of target nucleic acids using a plurality of primers comprising a plurality of thermal barcode sequences to produce a plurality of barcoded nucleic acids, and determining at least some of the plurality of barcoded nucleic acids at least in part by measuring a peak melting temperature or a temperature-dependent melting profile of the thermal barcode sequences.
A method as in any preceding claim, wherein the method further comprises forming a first plurality of droplets, at least 90% of which contain either only one target nucleic acid or no target nucleic acid, and at least 90% of which contain at least one primer.
A method as in any preceding claim, wherein the method further comprises breaking the first plurality of droplets to mix the amplified nucleic acids.
A method as in any preceding claim, wherein the method further comprises forming a second plurality of droplets, at least 90% of which contains either one of the amplified nucleic acids or no amplified nucleic acid, and at least 90% of which contain at least one primer.
A method as in any preceding claim, wherein determining the nucleic acid further comprises exposing detecting the target nucleic acid and/or the barcoded nucleic acid using an optical signaling entity.
A method as in any preceding claim, wherein the optical signaling entity comprises a fluorophore.
A method as in any preceding claim, wherein the optical signaling entity comprises a fluorescent quencher.
An article, comprising:
- a plurality of droplets comprising a first droplet and a second droplet, wherein:
- the first droplet comprises a first target nucleic acid and a first barcoded primer comprising a first primer sequence for the first target nucleic acid and a first thermal barcode sequence;
- and
- the second droplet comprises a second target nucleic acid and a barcoded primer comprising a second primer sequence for the second target nucleic acid and a second thermal barcode sequence;
- wherein a peak melting temperature of the first thermal barcode sequence differs from a peak melting temperature of the second thermal barcode sequence by at least 0.1° C.
An article, comprising:
- a plurality of droplets comprising a first droplet and a second droplet, wherein:
- the first droplet comprises a first target nucleic acid and a first barcoded primer comprising a first primer sequence for the first target nucleic acid and a first thermal barcode sequence;
- and
- the second droplet comprises a second target nucleic acid and a second barcoded primer comprising a second primer sequence for the second target nucleic acid and a second thermal barcode sequence;
- wherein the first thermal barcode sequence has a first ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy and the second thermal barcode sequence has a second ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy, wherein the first ratio differs from the second ratio by at least 1% of the second ratio.
A method or article as in any preceding claim, wherein a barcoded primer comprises a plurality of thermal barcode sequences.
A method or article as in any preceding claim, wherein the first thermal barcode sequence has a peak melting temperature that differs by at least 1° C. from a peak melting temperature of all other thermal barcode sequences of the plurality of thermal barcode sequences.
A method or article as in any preceding claim, wherein the plurality of thermal barcode sequences each have distinct temperature-dependent melting profiles.
A method or article as in any preceding claim, wherein the temperature-dependent melting profile of the thermal barcode sequence differs from a temperature-dependent melting profile of the target nucleic acid.
A method or article as in any preceding claim, wherein the thermal barcode sequence melts within a first temperature range and the target nucleic acid melts within a second temperature range different from the first temperature range.
A method or article as in any preceding claim, wherein the first temperature range is separated from the second temperature range by greater than or equal to 2° C.
A method or article as in any preceding claim, wherein the first temperature range is totally below the second temperature range.
A method or article as in any preceding claim, wherein the first temperature range is totally above the second temperature range.
A method or article as in any preceding claim, wherein the article comprises a first plurality of droplets, at least 90% of which contain either only one target nucleic acid or no target nucleic acid, and at least 90% of which contain at least one primer.
A method of determining a target substrate, the method comprising:
- exposing a library of barcoded nucleic acids to the target substrate such that at least some of the barcoded nucleic acids are able to bind to the target substrate;
- removing barcoded nucleic acids that do not bind to the target substrate;
- and
- identifying at least some of the barcoded nucleic acids that are able to bind to the target substrate using a melting profile of at least one thermal barcode of the barcoded nucleic acids.
An article, comprising:
- a plurality of compartments comprising a first compartment and a second compartment, wherein:
- the first compartment comprises a first target nucleic acid and a first barcoded primer comprising a first primer sequence for the first target nucleic acid and a first thermal barcode sequence;
- and
- the second compartment comprises a second target nucleic acid and a barcoded primer comprising a second primer sequence for the second target nucleic acid and a second thermal barcode sequence;
- wherein a peak melting temperature of the first thermal barcode sequence differs from a peak melting temperature of the second thermal barcode sequence by at least 0.1° C.
An article, comprising:
- a plurality of compartments comprising a first compartment and a second compartment, wherein:
- the first compartment comprises a first target nucleic acid and a first barcoded primer comprising a first primer sequence for the first target nucleic acid and a first thermal barcode sequence;
- and
- the second compartment comprises a second target nucleic acid and a second barcoded primer comprising a second primer sequence for the second target nucleic acid and a second thermal barcode sequence;
- wherein the first thermal barcode sequence has a first ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy and the second thermal barcode sequence has a second ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy, wherein the first ratio differs from the second ratio by at least 1% of the second ratio.
A method, comprising:
- selectively barcoding a target nucleic acid using a thermal barcode to form a barcoded target nucleic acid, and
- amplifying the barcoded target nucleic acid,
- wherein the thermal barcode exhibits a first peak melting point and the target nucleic acid exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1° C.
A method, comprising:
- selectively barcoding a target nucleic acid using a thermal barcode to form a barcoded target nucleic acid, and
- amplifying the barcoded target nucleic acid,
- wherein the thermal barcode sequence has a first ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy and the target nucleic acid has a second ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy, wherein the first ratio differs from the second ratio by at least 1% of the second ratio.
A method, comprising:
- selectively barcoding a target nucleic acid using a thermal barcode to form a barcoded target nucleic acid, and
- amplifying the barcoded target nucleic acid,
- wherein the thermal barcode sequence comprises less than or equal to 500 nucleotides and wherein at least 55% of the nucleotides of the thermal barcode sequence are A or T or wherein at least 45% of the nucleotides of the thermal barcode are G or C, and
- wherein the thermal barcode sequence exhibits a peak melting temperature first peak melting point and the target nucleic acid exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1° C.
Dependencies are inferred from the claim text.