Patent ID: 8304194

Claim:
A method for quantifying the absolute amount of at least two target nucleic acid molecules in a biological sample, the method comprising the steps of: a) preparing a sample by combining a biological sample comprising the at least two target nucleic acid molecules and a known amount of at least two standard nucleic acids, wherein said at least two standard nucleic acids have a nucleotide sequence that is one base different than the respective target nucleic acid molecule sequence; b) amplifying the nucleic acids in the sample of step a); c) using a single base primer extension reaction at the site of differentiation between each of the standard and the target nucleic acid to produce at least two extended target-specific nucleic acids and at least two extended respective standard-specific nucleic acids; d) detecting the at least two extended target-specific nucleic acids and the at least two extended respective standard-specific nucleic acids in a single multiplex mass spectrometry assay to obtain a peak area for each of the extended target-specific and extended respective standard-specific nucleic acids; and e) quantifying the absolute amount of at least the two target nucleic acids by measuring a ratio of the peak area representing the extended target-specific nucleic acids and the extended respective standard-specific nucleic acids and calculating the absolute amount based on the amount of each standard initially added to the sample.