Extract genomic DNA from all the mutant samples of a large (approximately 500 chromosomes, see Appendix 1, Note 12) F2 mapping population.
PpcAM58 mutant samples of about 140 μM for fully-reduced studies and 70 μM for redox titrations were prepared in 80 mM phosphate buffer with NaCl (250 mM final ionic strength) in H2O.
Interestingly, the non-mutant sample of the father (AG15693) revealed an increase of variability compared with the two daughters (AG15694, AG15695), likely reflecting the fact that, besides presenting the HGP phenotype, the father was 28 y older at sample acquisition.
The average (AVG) expression ratio of the mutant sample to the wild-type control was calculated from 12 replicates together with the respective standard error (SE), a standard t-test result (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; *****, P < 0.00001 probability in Student's t-test).
The ratios of mutant samples to the wild-type control were normalized by a trimmed geometric mean [ 11, 32, 33].
We recorded 2D C C correlation spectra of the mutant samples to verify that the protein refolded and was properly reconstituted into lipid bilayers.
We also performed simulations, using random mutant sampling, to determine the minimum number of mutants per samples that would be sufficient to achieve a consistent mutation spectrum in the BaP group.
That is, although some variation exists within each mutant sample group, all of the samples fall along the normal differentiation pathway.
This program compares the ratio of a target sequence to a reference DNA sequence in the mutant sample with the ratio of these sequences in a wild type sample.
Genomic DNA of the KRAS mutant sample 81K (G12D Ht) was mixed with wild type DNA of sample 75K in dilutions containing 50%, 25 %, 10 %, 5 2.5% and 0% mutant alleles.
