Nucleic acid melting analysis with saturation dyes
AssigneesBioFire Defense, LLC, University of Utah Research Foundation
InventorsLuming Zhou, Carl T. Wittwer, Philip Seth Bernard, Virginie Dujols
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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.
Claims19
A method of quantifying an amount of a target nucleic acid, comprising:
- (a) providing a PCR reaction mixture comprising:
- a pair of oligonucleotide primers configured for amplifying at least a portion of the target nucleic acid to generate an amplicon;
- a thermostable polymerase;
- a fluorescent dye;
- and
- a probe comprising a nucleic acid having a nucleic acid sequence complementary to at least a portion of the amplicon such that hybridizing forms a probe:target duplex, the probe being blocked at its 3′-end to prevent extension of the probe during amplification;
- (b) thermocycling the reaction mixture through a specified number of cycles;
- (c) monitoring fluorescence during a melting transition of the probe:target duplex to generate a fluorescence curve;
- and
- (d) quantifying the amount of the target nucleic acid using the fluorescence curve.
A method of quantifying an amount of a target nucleic acid, comprising:
- (a) providing a PCR reaction mixture comprising:
- a pair of oligonucleotide primers configured for amplifying at least a portion of the target nucleic acid to generate an amplicon;
- a thermostable polymerase;
- a probe comprising a nucleic acid having a nucleic acid sequence complementary to at least a portion of the amplicon, the probe being operable to hybridize to the amplicon so as to form a first duplex, the probe being blocked at its 3′-end;
- and
- a fluorescent dye having a percent saturation of at least 90%;
- (b) thermocycling the reaction mixture through a specified number of cycles;
- (c) monitoring fluorescence of the fluorescent dye during a melting transition of the first duplex to generate a fluorescence curve;
- and
- (d) quantifying an amount of the target nucleic acid using the fluorescence curve.
A method of quantifying an amount of a target nucleic acid, comprising:
- (a) thermocycling a PCR reaction mixture through a specified number of cycles to produce one or more amplicon, the reaction mixture comprising:
- a pair of oligonucleotide primers configured for amplifying at least a portion of the target nucleic acid to generate the one or more amplicon;
- a thermostable polymerase;
- an unlabeled probe comprising a nucleic acid having a nucleic acid sequence completely complementary to at least a portion of a first amplicon generated by amplification of a first allele of the target nucleic acid, the unlabeled probe being blocked at its 3′-end, the unlabeled probe being operable to hybridize to the first amplicon so as to form a first duplex;
- and
- a dsDNA binding dye having a percent saturation of at least 90%, the dsDNA binding dye being operable to bind to the first duplex, wherein at the concentration provided, the dsDNA binding dye does not significantly inhibit amplification of the target nucleic acid;
- (b) monitoring fluorescence of the fluorescent dye during a melting transition of the first duplex to generate a fluorescence curve;
- and
- (c) quantifying an amount of the first allele of the target nucleic acid using the fluorescence curve.
Dependencies are inferred from the claim text.