Estimation of the mutation rate during error-prone polymerase chain reaction

Error-prone polymerase chain reaction (PCR) is widely used to introduce poi nt mutations during in vitro evolution experiments, Accurate estimation of the mutation rate during error-prone PCR is important in studying the diver sity of error-prone PCR product. Although many methods for estimating the m utation rate during PCR are available, all the existing methods depend on t he assumption that the mutation rate is low and mutations occur at differen t places whenever they occur. The available methods may not be applicable t o estimate the mutation rate during error-prone PCR, We develop a mathemati cal model for error-prone PCR and present methods to estimate the mutation rate during error-prone PCR without assuming low mutation rate. We also dev elop a computer program to simulate error-prone PCR, Using the program, we compare the newly developed methods with two other methods, We show that wh en the mutation rate is relatively low (< 10(-3) per base per PCR cycle), t he newly developed methods give roughly the same results as previous method s. When the mutation rate is relatively high (> 5 x 10(-3) per base per PCR cycle, the mutation rate for most error-prone PCR experiments), the previo us methods underestimate the mutation rate and the newly developed methods approximate the true mutation rate.