The expected number of neutral mutations produced per generation is equal to the rate at which mutations are produced, multiplied by the population size.
Various synthetic peptides incorporating the designed mutations were produced and their helical content estimated by circular dichroism.
So long as mutations are produced at a steady rate, then the neutral substitutions that we observe will be produced at a steady rate as well.
Mutations were produced through 15 cycles of PCR, where each cycle consisted of 95°C for 1 minute, 58°C for 30 seconds, and 70 C for 10 minutes.
Mutations were produced by a single-step PCR reaction using the following PCR amplification reaction (50 µL) contained Phusion DNA polymerase GC buffer, 200 µM each of the four deoxynucleoside triphosphates, 2 mM MgCl2, 3% DMSO, 80 ng of template DNA (Ets-C31 pET28a vector), 0.5 µM primers forward and reverse, and 1 µL of Phusion DNA polymerase (Finnzymes, USA, Product # F-530-S).
All mutations were produced by site-directed mutagenesis.
Sequence analysis of selected clones was carried out to confirm that only the desired chimeras or mutations were produced.
Therefore, to estimate the rate at which mutations are produced, a correction for selection needs to be implemented.
Consequently, more new mutations were produced in more aggregated landscapes which facilitated the emergence of new pathogen genotypes able to thrive on the crop.
However, for the great majority of chemical carcinogens, these mutations are produced almost exclusively through errors of DNA replication (Preston, 1991).
