Calculation and verification of the ages of retroprocessed pseudogenes

To verify the existence of processed pseudogenes in different primates and their correlation with the estimated age of divergence, selected regions of processed pseudogenes of alpha-enolase, calmodulin II (CALMII), and argininosuccinate synthetase (AS) were amplified by the polymerase chain reaction (PCR) using DNA of blood samples. Published primate divergence times from the accepted paleontological records and the age of the pseudogenes based on molecular clock calculations were compared to data obtained by detection of PCR products exhibiting the expected amplicon size of the pseudogene region. For the alpha-enolase and the CALMII pseudogenes Psi(2), and Psi(3), calculated divergence times were 11, 19, and 36 Myr, respectively. For the AS pseudogenes Psi(1), Psi(3), and Psi(7), the divergence times were calculated to be 21, 25, and 16 Myr, respectively. Primer design and the annealing temperature are critical factors in the detection of pseudogenes in different species and impact greatly on the interpretation of the PCR analysis. The estimated divergence times of the selected pseudogenes utilizing calculations based on the molecular clock theory correlated well with experimental PCR detection of the selected pseudogenes represented in this study.