Quantitative determination of nucleic acid, by competitive polymerase chain reaction in presence of known amount of competitor very similar to the target

摘要

Method for quantitative determination of nucleic acid (I) in a biological sample. A competitive, quantitative polymerase chain reaction (PCR) is performed using (i) forward and reverse primers that bind to a fragment of (I) suitable for specific detection; (ii) a known amount of competitor DNA (Ia), generated in an earlier PCR, that has the same size and base composition as (I) and differs only minimally from (I); (iii) usual PCR auxiliaries and (iv) the sample. The individual strands of both amplicons (from (I) and (Ia)) are analyzed, directly or after a standard cleavage reaction, for mass and amount, so that they are unequivocally identified from mass and, from the ratio of their amounts and the known amount of (Ia), the amount of (I) is calculated.