Conjugated polymers or polymer dots as fluorescent labels
AssigneeIllumina, Inc.
InventorsNam Nguyen, Iuliana Petruta Maria, Andrew A. Brown, Wayne N. George, Xiaolin Wu, Xavier Von Hatten, Madushani Dharmarwardana
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Abstract
Embodiments of the present disclosure relate to compositions and methods for labeling of nucleotides, oligonucleotides, or polynucleotides with conjugated polymers or polymer dots. In particular, conjugated polymers or polymer dots can be used as detectable labels for nucleotides, oligonucleotides, or polynucleotides in sequencing by synthesis.
Claims30
A nucleotide, oligonucleotide or polynucleotide comprising a water-soluble conjugated polymer or a water-dispersible polymer dot attached thereto as a detectable label.
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A kit for sequencing application, comprising:
- an incorporation mixture composition comprising one or more of four different types of nucleotides each comprising a 3′ blocking group, wherein a first type of unlabeled nucleotide comprises a first reactive moiety covalently attached thereto;
- and
- a first labeling reagent comprising a water-soluble conjugated polymer or a water-dispersible polymer dot, each having a first functional moiety that is capable of reacting specifically with the first reactive moiety of the first type of unlabeled nucleotide to form covalent or non-covalent bonding.
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A method of determining the sequences of a plurality of different target polynucleotides, comprising:
- (a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon;
- and the sequencing primers are complementary to at least a portion of the target polynucleotides;
- (b) contacting the solid support with an aqueous solution comprising DNA polymerase and one or more of four different types of unlabeled nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides;
- wherein
- each of the four types of nucleotides comprises a 3′ blocking group;
- and
- the incorporation mixture comprises a first type of unlabeled nucleotide having a first reactive moiety covalently attached to the first type of unlabeled nucleotide;
- (c) contacting the extended copy polynucleotides with a first labeling reagent comprising a water-soluble conjugated polymer or a water-dispersible polymer dot comprising a functional moiety that reacts specifically with the first reactive moiety of the first type of unlabeled nucleotides to form covalent or non-covalent bonding;
- (d) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides;
- and
- (e) removing the 3′ blocking group of the incorporated nucleotides.
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Dependencies are inferred from the claim text.