Bias in Estimations of DNA Content by Competitive

MARIANO J. ALVAREZ; AMAICHA M. DEPINO; OSVALDO L. PODHAJCER; FERNANDO J. PITOSSI

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2000 vol. 287 p. 87 - 94

0003-2697

Competitive PCR   is a  highly sensitive  method for specific DNA  quantification. Despite the lack of  stud- ies related to  the accuracyof  the method it has been widely used. Here we  present asimulation model for competitive PCR, whichtakes into accountthe effi- ciency decay asa linear relationship ofthetotal prod- uct yield. The  model helped us  to  study the kind and magnitude of  errors that arise from quantitative and semiquantitative competitive PCR   protocols and to findways to  minimize them. The  simulation data sug- gest that differences in amplification efficiency be- tween target and standard templates induce stronger biases in quantitative than in semiquantitative com- petitive PCR. Quantitative competitive PCR  can only be  used when both efficiencies are equal. In  contrast, semiquantitative competitive PCR  can be  used even when the target is amplifiedwith a  higher efficiency thanthe standard, since under such conditions the method tends to  underestimate the differences in ini- tial DNA  content. These predictions have been con- firmed with experimental data and show that the esti- mation  of   the  amplification efficiencies  is  a prerequisite for the use of quantitative and semiquan- titative  competitive  PCR. A  simple methodfor   this estimation is alsopresented.