High speed nucleic acid melting analysis
A method and system have been provided to perform high speed nucleic acid melting analysis while still obtaining accurate melting curve sufficient for genotyping. This rapid ability to interrogate DNA should be useful whenever time to result is important, such as in molecular point of care testing. Specifically, microfluidics enables genotyping by melting analysis at rates up to 50° C./s, requiring less than is to acquire an entire melting curve. High speed melting reduces the time for melting analysis, decreases errors, and improves genotype discrimination of small amplicons.
1. A method for performing a nucleic acid high-speed melting analysis, the method comprising:
providing a microfluidic device having one or more nucleic acid samples therein;
providing an imaging system in communication with the one or more nucleic acid samples;
providing a thermal system in thermal communication with the one or more nucleic acid samples;
increasing the temperature of the one or more nucleic acid samples to achieve nucleic acid dissociation, wherein the temperature is increased by the thermal system at a ramp rate selected from a range of greater than 1° C./s to 50° C./s.
acquiring images of the one or more nucleic acid samples during nucleic acid dissociation to generate a melting profile for each nucleic acid sample; and
genotyping the nucleic acids based on the melting profile(s);
wherein genotypes are classified by using a discrimination value, wherein the discrimination value is calculated as a ratio of inter-class and intra-class distance for the melting rate being used, wherein the ratio is calculated as the mean of the absolute value of the difference between two inter-class curves at each value along the x-axis divided by the mean of the absolute value of the difference between two intra-class curves at each value along the x-axis; and wherein the inter-class distance reflects differences between genotypes and the intra-class distance reflects differences between duplicates of the same genotype.
2. The method of claim 1 , wherein the microfluidic device comprises a microfluidic cartridge and a reaction chip.
3. The method of claim 2 , wherein the reaction chip comprises one or more microchannels.
4. The method of claim 3 , wherein the melting analysis is performed when the one or more samples are in the one or more microchannels of the reaction chip.
5. The method of claim 1 , wherein the nucleic acid melting analysis is performed by increasing a temperature of the one or more nucleic acid samples at a rate selected from the range of greater than 1° C./s to 8° C./s.
6. The method of claim 1 , wherein the nucleic acid melting analysis is performed by increasing a temperature of the one or more nucleic acid samples at a rate selected from the range of 8° C./s to 16° C./s.
7. The method of claim 1 , wherein each of the nucleic acids in the one or more nucleic acid samples is less than 100 bp long.
8. The method of claim 1 , wherein the microfluidic device is primed prior to performing the nucleic acid melting analysis.
9. The method of claim 1 , wherein the nucleic acid melting analysis is preceded by amplification of the nucleic acids in the one or more nucleic acid samples.
10. The method of claim 1 , wherein each of the one or more nucleic acid samples includes at least one internal temperature control sequence having a melting temperature that is substantially greater than a melting temperature of the nucleic acids in the one or more nucleic acid samples.
11. The method of claim 1 , further comprising sequentially obtaining melting curves for the one or more nucleic acid samples at a plurality of ramp rates between 0.13° C./s and 32° C./s to determine an optimal ramp rate corresponding to the highest genotype discrimination, the genotype discrimination being calculated for the plurality of ramp rates between 0.13° C./s and 32° C./s.
12. The method of claim 1 , wherein the ramp rate used maximizes the ratio of inter-class to intra-class distance, which minimizes the number of no-call samples and the number of false positive and false negative samples among those called.
13. The method of claim 12 , wherein the ramp rate used results in a homozygote genotype discrimination value of greater than 6.
14. The method of claim 12 , wherein the ramp rate used results in a heterozygote genotype discrimination value of greater than 5.