Digital assays with a reporter for amplicon length
Digital assay system, includes methods, apparatus, and compositions, for distinguishing and measuring different types of templates according to the different lengths of corresponding amplicons, which may be amplified by the same pair of primers. The system may include a length-sensitive reporter generating luminescence that varies according to amplicon length. The system may, for example, be utilized to identify, distinguish, and/or quantify wild-type and mutant/variant templates, processed and unprocessed template, a target template and a primer dimer, or the like.
1. A method of performing a digital assay, the method comprising:
partitioning a same reaction mixture into a plurality of droplets, the reaction mixture containing a first template for a first amplicon, a second template for a second amplicon, two or more primers to generate the first and second amplicons from their corresponding templates, and an intercalating dye, wherein only a subset of the droplets contain a copy of the first template and only a subset of the droplets contain a copy of the second template;
amplifying nucleic acid in the droplets to generate the first amplicon from the first template and the second amplicon from the second template, wherein the first amplicon and the second amplicon are of different length from one another;
detecting an intensity of light emitted by the intercalating dye from individual droplets while the intercalating dye remains bound to the first amplicon and the second amplicon; and
comparing the intensity of light detected from each individual droplet to one or more threshold values to classify each individual droplet as negative for both amplicons, positive only for the first amplicon, or positive for the second amplicon.
2. The method of claim 1 , wherein the first amplicon and the second amplicon represent distinct first and second alleles of a same gene.
3. The method of claim 1 , wherein the first amplicon represents at least a portion of a messenger RNA and the second amplicon represents at least a portion of a precursor of the messenger RNA that contains an intron.
4. The method of claim 1 , wherein each of the first amplicon and the second amplicon contains the same repeated element of two or more nucleotides, and wherein the repeated element is repeated a different number of times in the first amplicon relative to the second amplicon.
5. The method of claim 1 , wherein the step of amplifying nucleic acid is performed with a same pair of primers to generate the first amplicon and the second amplicon.
6. The method of claim 1 , further comprising (i) a step of determining a number of partitions positive or negative for the second amplicon and (ii) a step of determining a level of the second template based on the number of partitions, wherein each of steps (i) and (ii) is performed with a processor.
7. The method of claim 6 , wherein the step of determining a level includes a step of calculating a concentration of the second template based on the number using the processor.
8. The method of claim 6 , further comprising a step of determining a ratio of the first template to the second template using the processor.