Patent DE-10045521-A1 (DE)
Title: Determining amplification efficiency of a target nucleic acid comprises measuring real-time amplification of diluted series, setting signal threshold value, and determining cycle number at which threshold is exceeded
Inventor: SAGNER GREGOR
IPC: C12Q1/68
Filed: 2000-09-13
Abstract: Determining (M1) amplification efficiency (AE) of target nucleic acid (I) comprises: (a) preparing a dilution series of (I); (b) amplifying (I) under defined reaction conditions and measuring real-time amplification; (c) setting a defined signal threshold value; (d) determining for each dilution, the cycle number at which the signal threshold value is exceeded; and (e) determining AE as a function of the original amount of (I).  Independent claims are also included for the following:  (1) absolute quantification (M2) of (I) in a sample comprising: (a) determining the amplification efficiencies of (I) and of an internal or external standard under defined amplification conditions; (b) amplifying (I) contained in the sample and of the internal or external standard under the same defined reaction conditions; (c) measuring the amplification of (I) and standard in real time; and (d) calculating the original copy number in the sample by correction of the copy number derived from steps (a-d) with the aid of the amplification efficiencies determined in the first step. (2) quantification (M3) of (I) relative to a reference nucleic acid in a sample comprising: (a) determining the amplification efficiencies of (I) and of the reference nucleic acid under defined amplification conditions; (b) amplifying (I) contained in the sample as well as of the reference nucleic acid contained in the sample under the same defined amplification conditions; (c) measuring the amplification of (I) and of the reference nucleic acid in real-time; and (d) calculating the original ratio of (I) and reference nucleic acid in the sample by correction of the ratio derived from steps (a-d) with the aid of the amplification efficiencies determined in the first step. (3) relative quantification (M4) of (I) relative to a reference nucleic acid and standardized with a calibrator sample comprising: (a) preparing a common or two separate dilution series of (I) and reference nucleic acid; (b) amplifying various dilutions of (I) and reference nucleic acid under defined reaction conditions, where the amplification of the nucleic acid is measured in real-time; (c) setting defined signal threshold values for (I) and reference nucleic acid; (d) determining the cycle number (Cp) to which the signal threshold values defined for (I) and reference nucleic acid are exceeded in each dilution; (e) determining a continuously differentiable function of the Cp values as a function of the logarithm of the amounts used of (I) and determining a continuously differentiable function of the determined Cp values as a function of the logarithm of the amounts used of reference nucleic acid or vice versa; (f) determining the Cp values of (I) and reference nucleic acid in the sample to by analyzed as well as in a calibrator sample; (g) assigning the Cp values measured in steps (a-f) to particular function values of the functions determined; (h) calculating the quotients of the function values of (I) and reference nucleic acid from steps (a-f) for the sample to be analyzed as well as for the calibrator sample; and  (i) determining the ratio of the two quotients as a measure for the original amount of (I) contained in the sample.