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US 2014/0370507 A1Published application
Nucleic acid melting analysis with saturation dyes
AssigneeUniversity of Utah Research Foundation and BioFire Defense, LLC
InventorsCarl T. Wittwer, Luming Zhou
Life of this patent
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Abstract
Methods are provided for nucleic acid analysis wherein a target nucleic acid is mixed with a dsDNA binding dye to form a mixture. Optionally, an unlabeled probe is included in the mixture. A melting curve is generated for the target nucleic acid by measuring fluorescence from the dsDNA binding dye as the mixture is heated. Dyes for use in nucleic acid analysis and methods for making dyes are also provided.
Claims20
A PCR reaction mixture for analyzing a target nucleic acid, comprising:
- oligonucleotide primers configured for amplifying at least a portion of the target nucleic acid to generate an amplicon, the oligonucleotide primers comprising a first primer and a second primer, the first primer operable to hybridize to the amplicon at a first locus and the second primer operable to hybridize to the amplicon at a second locus, the amplicon comprising at least one nucleic acid strand;
- a thermostable polymerase;
- an unlabeled probe having a nucleic acid sequence and configured to hybridize to the at least one nucleic acid strand of the amplicon between the first and second primers or between the first locus and second locus of the amplicon, the unlabeled probe blocked at its 3′-end to prevent extension during amplification, the unlabeled probe operable to hybridize along its entire length to one allele of the amplicon and to hybridize with at least one mismatch to a second allele of the amplicon such that the unlabeled probe binds differentially to the first and second alleles of the amplicon;
- and
- a dsDNA binding dye having a percent saturation of at least 90%, the dsDNA binding dye operable to bind a homoduplex comprising the unlabeled probe hybridized to the first allele of the amplicon and a heteroduplex comprising the unlabeled probe hybridized to the second allele of the amplicon, the dsDNA binding dye provided at a concentration suitable for distinguishing between the homoduplex and the heteroduplex by melting curve analysis of a signal produced by the dsDNA binding dye during melting of the homoduplex and heteroduplex,
- wherein the amplicon, the unlabeled probe, and the dsDNA binding dye are all free in solution.
3 more elements
A kit for analyzing a target nucleic acid, comprising:
- an unlabeled probe blocked at its 3′-end to prevent extension during amplification, the unlabeled probe configured to hybridize complementarily to a portion of at least one allele of the target nucleic acid;
- and
- a dsDNA binding dye having a percent saturation of at least 90%, the dsDNA binding dye operable to bind a homoduplex comprising the unlabeled probe hybridized with a first allele of the target nucleic acid and a heteroduplex comprising the unlabeled probe hybridized with a second allele of the target nucleic acid, the dsDNA binding dye provided at a concentration suitable for distinguishing between the homoduplex and the heteroduplex by melting curve analysis of a signal produced by the dsDNA binding dye during melting of the homoduplex and heteroduplex.
A kit for analyzing a target nucleic acid, the target nucleic acid comprising a chain of nucleotide bases, the kit comprising:
- an unlabeled probe blocked at its 3′-end to prevent extension during amplification of the target nucleic acid, the unlabeled probe comprising a chain of nucleotide bases, the unlabeled probe configured to hybridize to a first allele of the target nucleic acid by base-pairing interactions, with each nucleotide base of the unlabeled probe base-pairing with a corresponding nucleotide base of the first allele of the target nucleic acid;
- and
- a dsDNA binding dye having a percent saturation of at least 90%,
- wherein when combined with the target nucleic acid in a mixture, the unlabeled probe and dsDNA binding dye are operable for detecting a double-stranded nucleic acid with the dsDNA binding dye without immobilizing the unlabeled probe or the target nucleic acid, the double-stranded nucleic acid comprising a strand of the target nucleic acid hybridized with the unlabeled probe.
1 more element
Dependencies are inferred from the claim text.