Amplicon melting analysis with saturation dyes

Abstract

Methods are provided for nucleic acid analysis wherein a target nucleic acid that is at least partially double stranded is mixed with a dsDNA binding dye having a percent saturation of at least 50% to form a mixture. In one embodiment, the nucleic acid is amplified in the presence of the dsDNA binding dye, and in another embodiment 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.

1 - 24 . (canceled) 
     
     
         25 . A method for detecting the presence or absence of heteroduplex DNA in a sample, the method comprising:
 (a) mixing a target nucleic acid with a saturating dsDNA binding dye to form a target nucleic acid mixture,   (b) amplifying the target nucleic acid mixture to generate an amplicon,   (c) melting the amplicon while measuring fluorescence from the saturating dsDNA binding dye to generate a melting curve for the amplicon,   (d) detecting the presence or absence of heteroduplex DNA by a shape of the melting curve that is indicative of the presence or absence of heteroduplex DNA.   
     
     
         26 . The method of  claim 25 , further comprising:
 (e) denaturing the amplicon, and   (f) renaturing the amplicon,   wherein steps (e) and (f) occur prior to step (c).   
     
     
         27 . The method of  claim 26 , wherein step (f) includes cooling at a rate of at least −2° C./s. 
     
     
         28 . The method of  claim 27 , wherein step (c) includes heating at a rate of 0.2 to 0.4° C./s. 
     
     
         29 . The method of  claim 26 , further comprising:
 prior to step (e), generating a second melting curve by melting the amplicon while measuring fluorescence from the saturating dsDNA binding dye, and   subsequent to step (c), comparing the melting curve to the second melting curve.   
     
     
         30 . The method of  claim 25 , wherein the step (d) includes
 identifying a first melting transition in the melting curve, and   identifying the presence or absence of a second melting transition in the melting curve, wherein the second melting transition is at a temperature lower than the first melting transition, the presence of the second melting transition is indicative of heteroduplex DNA, and the absence of the second melting transition is indicative of the absence of heteroduplex DNA.   
     
     
         31 . The method of  claim 30 , wherein a negative first derivative is used to convert the first melting transition and second melting transition into a first Tm peak and a second Tm peak. 
     
     
         32 . The method of  claim 25 , wherein the step (a) occurs prior to step (b). 
     
     
         33 . The method of  claim 32 , wherein the saturating dsDNA binding dye has a percent saturation of at least 90%. 
     
     
         34 . The method of  claim 25 , further comprising:
 repeating steps (a) through (d) on a second sample, and   comparing the melting curve from the sample to the melting curve from the second sample to determine if a genotype of the first sample is the same as a genotype of a second sample.   
     
     
         35 . The method of  claim 34 , wherein step (d) for both the sample and the second sample indicate a presence of a heterozygote, and the melting curve from the sample and the melting curve from the second sample are different, indicating that the sample and the second sample are different heterozygotes. 
     
     
         36 . The method of  claim 34 , wherein step (d) for both the sample and the second sample indicate a presence of a homozygote, and the melting curve from the sample and the melting curve from the second sample are different, indicating that the sample and the second sample are different homozygotes. 
     
     
         37 . The method of  claim 36 , wherein the different homozygotes are A vs T homozygotes.