Method of quantifying mutant allele burden of target gene

A method of quantifying a mutant allele burden of a target gene in a subject, comprising:

• providing a first plasmid that includes a mutant allele sequence located at a first region of the target gene, and an internal control sequence derived from the target gene and located at a second region of the target gene, the second region being different from the first region;
• providing a second plasmid that includes a wild-type allele sequence located at the first region of the target gene, and the internal control sequence;
• subjecting genomic DNA of the subject to a first quantitative polymerase chain reaction using a reaction mixture containing a mutant allele-specific primer pair and a first detectable probe for detecting the mutant allele sequence, and an internal control sequence-specific primer pair and a second detectable probe for detecting the internal control sequence, so as to measure a mutant allele expression level of the target gene;
• subjecting standard diluents of the first plasmid formed from serial dilution with the second plasmid to a second quantitative polymerase chain reaction using the reaction mixture, so as to create a standard curve of the mutant allele burden of the target gene;
• and
• correlating the measured mutant allele expression level of the target gene to the standard curve, so as to determine the mutant allele burden of the target gene in the subject.