Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)_n and (A/T)_n microsatellites

摘要

During microsatellite polymerase chain reaction (PCR), insertion-deletion mutations produce stutter products differing from the original template by multiples of the repeat unit length. We analyzed the PCR slippage products of (CA)_n and (A)_n tracts cloned in a pUC18 vector. Repeat numbers varied from two to 14 (CA)_n and four to 12 (A)_(n~·) Data was generated on approximately 10 single molecules for each clone type using two rounds of nested PCR. The size and peak areas of the products were obtained by capillary electrophoresis. A quasi-likelihood approach to the analysis of the data estimated the mutation rate/repeat/PCR cycle. The estimated the mutation rate/repeat/PCR cycle. The rate for (CA)_n tracts was 3.6 * 10~(-3) with contractions 14 times greater than expansions. For (A)_n tracts the rate was 1.5 * 10~(-2) and contractions outnumbered expansions by 5-fold. The threshold for detecting 'stutter' products was computed to be four repeats for (CA)_n and eight repeats for (A)_n or ～8 bp in both cases. A comparison was made between the computationally and experimentally derived threshold values. The threshold and expansion to contraction ratios are explained on the basis of the active site structure of Taq DNA polymerase and models of the energetics of slippage events, respectively.