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US 12,742,206 B2Granted
Multiplex drop-off digital polymerase chain reaction methods
AssigneeStilla Technologies
InventorsBarbara André, Rémi Dangla, Jordan Madic, Allison Mallory
Life of this patent
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Abstract
The present application provides multiplex digital polymerase chain reaction (dPCR) assays such as multiplex drop-off dPCR assays, methods, systems, and kits. The methods described herein are useful in a variety of applications, such as detection of microsatellite instability and quantification of site-specific genome-edited products.
Claims18
A method for quantification of wildtype and allelic sequences at a plurality of target regions in a sample comprising nucleic acid molecules distributed among a plurality of partitions of the sample,
- wherein 70% or more of the total number of partitions each comprises a plurality of probe sets corresponding to the plurality of target regions,
- wherein each probe set of the plurality of probe sets comprises:
- a drop-off probe comprising a drop-off label and an oligonucleotide drop-off sequence complementary to a wildtype sequence at a target region corresponding to the respective probe set;
- a reference probe comprising a reference label and an oligonucleotide reference sequence complementary to a wildtype sequence at an adjacent reference region upstream or downstream to the target region corresponding to the respective probe set;
- an allele-specific (AS) probe comprising an AS label and an oligonucleotide AS sequence complementary to an allelic sequence at the target region corresponding to the respective probe set,
- wherein the reference label, the drop-off label, and the AS label of each probe set of the plurality of probe sets are detectable via different detection channels;
- wherein reference labels of the plurality of probe sets are detectable via different detection channels with respect to each other;
- wherein at least one reference label of the plurality of probe sets and at least one drop-off label of the plurality of probe sets are detectable via the same detection channel;
- and
- wherein the set of reference labels of the plurality of probe sets, the set of drop-off labels of the plurality of probe sets, and the set of the AS labels of the plurality of probe sets have overlapping labels;
- the method comprising:
- detecting hybridization of reference probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the reference regions in the plurality of partitions;
- detecting hybridization of drop-off probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the target regions in the plurality of partitions;
- and
- detecting hybridization of AS probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising allelic sequences at the target regions in the plurality of partitions to provide quantification of wildtype sequences and allelic sequences at the plurality of target regions in the sample.
12 more elements
A method for quantification of wildtype and allelic sequences at a plurality of target regions in a sample comprising nucleic acid molecules distributed among a plurality of partitions of the sample,
- wherein 70% or more of the total number of partitions each comprises a plurality of probe sets corresponding to the plurality of target regions, wherein each probe set of the plurality of probe sets comprises:
- a drop-off probe comprising a drop-off label and an oligonucleotide drop-off sequence complementary to a wildtype sequence at a target region corresponding to the respective probe set;
- a reference probe comprising a reference label and an oligonucleotide reference sequence complementary to a wildtype sequence at an adjacent reference region upstream or downstream to the target region corresponding to the respective probe set;
- and
- an allele-specific (AS) probe comprising an AS label and an oligonucleotide AS sequence complementary to an allelic sequence at the target region corresponding to the respective probe set,
- wherein the reference label, the drop-off label, and the AS label of each probe set of the plurality of probe sets are detectable via different detection channels;
- wherein drop-off labels of the plurality of probe sets are detectable via different detection channels with respect to each other;
- wherein at least one reference label of the plurality of probe sets and at least one drop-off label of the plurality of probe sets are detectable via the same detection channel;
- and
- wherein the set of reference labels of the plurality of probe sets, the set of drop-off labels of the plurality of probe sets, and set of the AS labels of the plurality of probe sets have overlapping labels;
- the method comprising:
- detecting hybridization of reference probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the reference regions in the plurality of partitions;
- detecting hybridization of drop-off probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the target regions in the plurality of partitions;
- and
- detecting hybridization of AS probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising allelic sequences at the target regions in the plurality of partitions to provide quantification of wildtype sequences and allelic sequences at the plurality of target regions in the sample.
12 more elements
A method for quantification of wildtype and allelic sequences at a plurality of target regions in a sample comprising nucleic acid molecules distributed among a plurality of partitions of the sample,
- wherein 70% or more of the total number of partitions each comprises a plurality of probe sets corresponding to the plurality of target regions,
- wherein each probe set of the plurality of probe sets comprises:
- a drop-off probe comprising a drop-off label and an oligonucleotide drop-off sequence complementary to a wildtype sequence at a target region corresponding to the respective probe set;
- a reference probe comprising a reference label and an oligonucleotide reference sequence complementary to a wildtype sequence at an adjacent reference region upstream or downstream to the target region corresponding to the respective probe set;
- and
- an allele-specific (AS) probe comprising an AS label and an oligonucleotide AS sequence complementary to an allelic sequence at the target region corresponding to the respective probe set,
- wherein the reference label, the drop-off label, and the AS label of each probe set of the plurality of probe sets are detectable via different detection channels;
- wherein reference labels of the plurality of probe sets are detectable via different detection channels with respect to each other;
- wherein drop-off labels of the plurality of probe sets are detectable via different detection channels with respect to each other;
- wherein AS labels of the plurality of probe sets are detectable via different detection channels with respect to each other;
- wherein at least one reference label of the plurality of probe sets and at least one drop-off label of the plurality of probe sets are detectable via the same detection channel;
- and
- wherein the set of reference labels of the plurality of probe sets, the set of drop-off labels of the plurality of probe sets, and set of the AS labels of the plurality of probe sets have overlapping labels;
- the method comprising:
- detecting hybridization of reference probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the reference regions in the plurality of partitions;
- detecting hybridization of drop-off probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising wildtype sequences at the target regions in the plurality of partitions;
- and
- detecting hybridization of AS probes of the plurality of probe sets to nucleic acid molecules or amplicons thereof comprising allelic sequences at the target regions in the plurality of partitions to provide quantification of wildtype sequences and allelic sequences at the plurality of target regions in the sample.
15 more elements
Dependencies are inferred from the claim text.