Patent ID: 7846696

Claim:
A method for estimating a molar ratio of a target nucleic acid to a total amount of the target nucleic acid and a reference nucleic acid in a test sample, the method comprising: (a) preparing a plurality of standard nucleic acid samples, each having a different predetermined molar ratio of the target nucleic acid to the total amount of the target nucleic acid and the reference nucleic acid; (b) performing a Polymerase Chain Reaction (PCR) reaction on each of said plurality of standard nucleic acid samples using (1) one of said plurality of standard nucleic acid samples, (2) a primer pair including a detecting primer for the target nucleic acid, and (3) a primer pair including a detecting primer for the reference nucleic acid; (c) measuring an amount of amplified target nucleic acid product and an amount of amplified reference nucleic acid product in each of said plurality of standard nucleic acid samples; (d) calculating a first continuously differentiable function which approximates the relationship between the predetermined molar ratios and a consumption rate of the detecting primer for the target nucleic acid, in each of said plurality of standard nucleic acid samples after step (b); (e) calculating a second continuously differentiable function which approximates the relationship between the predetermined molar ratios and a consumption rate of the detecting primer for the reference nucleic acid in each of said plurality of standard nucleic acid samples after step (b); (f) performing a Polymerase Chain Reaction (PCR) reaction on a test sample in using (1) the test sample, (2) the primer pair including the detecting primer for the target nucleic acid, and (3) the primer pair including the detecting primer for the reference nucleic acid; (g) measuring an amount of amplified target nucleic acid product and an amount of amplified reference nucleic acid product in the test sample; (h) estimating the molar ratio of the target nucleic acid to the total amount of the target nucleic acid and the reference nucleic acid in the test sample based on (1) the first continuously differentiable function, (2) the second continuously differentiable function, (3) the primer consumption rate of the detecting primer for the target nucleic acid in the test sample, and (4) the primer consumption rate of the detecting primer for the reference nucleic acid product in the test sample.