Amplicon melting analysis with saturation dyes

A method of PCR analysis comprising the steps of:

• mixing a dsDNA binding dye having a percent saturation of at least 50% with a sample comprising a target nucleic acid and primers configured for amplifying the target nucleic acid,
• amplifying the target nucleic acid in the presence of the dsDNA binding dye, and
• monitoring fluorescence of the dsDNA binding dye, wherein the monitoring step comprises melting the amplified target nucleic acid to generate a melting curve, and
• identifying the genotype using a shape of the melting curve, and wherein the dsDNA binding dye is a compound having the formula:
• wherein
• the moiety represents an optionally-substituted fused monocyclic or polycyclic aromatic ring or an optionally-substituted fused monocyclic or polycyclic nitrogen-containing heteroaromatic ring;
• X is oxygen, sulfur, selenium, tellurium or a moiety selected from C(CH3)2 and NR1, where R1 is hydrogen or C1-6 alkyl;
• R2 is selected from the group consisting of C1-6 alkyl, C3-8 cycloalkyl, aryl, aryl(C1-2 alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, alkylsulfonate, optionally substituted cyclic heteroatom-containing moieties, and optionally substituted acyclic heteroatom-containing moieties;
• t=0 or 1;
• Z is a charge selected from 0 or 1;
• R3 is selected from the group consisting of hydrogen, C1-6 alkyl, and —C(O)Ph;
• R9 and R10 are each independently selected from the group consisting of hydrogen and C1-6 alkyl;
• n=0, 1, or 2;
• indicates a single bond that is in a tautomeric relationship with an adjacent double bond, and
• Q is an heterocycle selected from the group of structures consisting of:
• wherein R4, R5, R6, R7, and R8 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, alkenylalkynyl, aryl, heteroaryl, alkoxy, alkylthio, and dialkylamino, each of which may be optionally substituted;
• an acyclic heteroatom-containing moiety or a cyclic heteroatom-containing moiety;
• a BRIDGE-DYE;
• and a reactive group;
• each of which optionally includes a quaternary ammonium moiety.

A method of PCR analysis comprising the steps of:

• providing a mixture of a dsDNA binding dye having at least 90% saturation, a target nucleic acid, and primers configured for amplifying the target nucleic acid,
• amplifying the target nucleic acid in the presence of the dsDNA binding dye,
• monitoring fluorescence of the dsDNA binding dye,
• generating a melting curve for the target nucleic acid,
• normalizing magnitude differences of the melting curve,
• repeating the providing, amplifying, generating and normalizing steps with at least one additional target nucleic acid,
• comparing the magnitude difference normalized melting curves, and plotting the fluorescence difference between the magnitude difference normalized curves, wherein the melting curve of one selected target nucleic acid is selected as the standard and is plotted as standard across melting temperatures and the melting curve for each additional target nucleic acid is plotted as a difference from the standard across the melting temperatures.

A method of PCR analysis comprising the steps of:

• providing a mixture of a dsDNA binding dye having at least 90% saturation, a target nucleic acid, and primers configured for amplifying the target nucleic acid,
• amplifying the target nucleic acid in the presence of the dsDNA binding dye,
• monitoring fluorescence of the dsDNA binding dye,
• generating a melting curve for the target nucleic acid,
• normalizing magnitude differences of the melting curve,
• repeating the providing, amplifying, generating and normalizing steps with at least one additional target nucleic acid,
• comparing the magnitude difference normalized melting curves, and plotting the fluorescence difference between the magnitude difference normalized curves, wherein the melting curve of one selected target nucleic acid is selected as the standard and is plotted as standard across melting temperatures and the melting curve for each additional target nucleic acid is plotted as a difference from the standard across the melting temperatures wherein the standard is plotted as zero across all melting temperatures.