Roche Molecular Systems, Inc.

Roche Diagnostics Operations, Inc.

Roche Diagnostics Gmbh

Idaho Technology, Inc.

Richie Soong

Karim Tabiti

Martin Gutekunst

Gregor Sagner

Randy P Rasmussen

Deepika Marine deSilva

Hallegh Page Millward

Gisela Betzl

John G Ward

The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

Method for real-time quantification of nucleic acids

Method for the efficiency-corrected real-time quantification of nucleic acids

The present invention concerns a method for determining the efficiency of the amplification of a target nucleic acid comprising the following steps: (i) preparation of a dilution series of the target nucleic acid, (ii) amplifying the target nucleic acid under defined reaction conditions, the amplification being measured in real-time (iii) setting a defined signal threshold value, (iv) determining the cycle number at which the signal threshold value is exceeded for various dilutions, (v) determining the amplification efficiency as a function of the amount of original target nucleic acid. The present invention also concerns a method for the quantification of a target nucleic acid in a sample in which the efficiency of the amplification reaction is determined in this manner and is taken into account in the quantification.

Method for determining the efficiency of nucleic acid amplifications

The invention provides a method for quantitative multiplex PCR of two targets whereby one target is present in at least 100-fold molar excess over that of the other target. The method further comprises the addition of a thermostable DNA polymerase with a final concentration of at least 0.5 units/&#x3bc;l.

Quantitative multiplex PCR with high dynamic range

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Tutzing, Germany

Richie Soong