From Fred Cross, The Rockefeller University, August 2018
This collection of ts-lethal mutations and null alleles was generated by UV mutagenesis as previously described (Tulin and Cross 2014; Breker et al 2016). The identification of the causative mutations for lethality was determined by MAPS-SEQ, a multiplexed genetic/bioinformatics strategy (Breker et al 2018). Caveats for gene identification are discussed in that paper. It is highly recommended that the co-segregation of the indicated mutation (for example by allele-specific PCR) and ts-lethality be confirmed as a first step. The strains provided were not backcrossed and harbor up to many hundreds of non-lethal mutations; these ‘passenger’ mutations in some cases are mildly deleterious, so we recommend at least two rounds of backcrossing before detailed experimentation. Please note, when one causative mutation is indicated, that is the only coding-sequence changing mutation identified at the causative region. if more than one is indicated, they are both potential causative mutations and further genetic validation should be performed. Non-coding-sequence changing mutations in the causative region are not indicated.
Among the passenger (non-lethal) mutations, some were identified as early nonsense or severe splicing mutations and therefore may be potentially null alleles for non-essential genes. In order to isolate the mutation and determine its phenotype, it should be separated from the ts-lethal mutation by back-crossing and verified by sequence analysis (for example by allele-specific PCR).
For allele-specific PCR, we have had a very high success rate with the competitive amplification assay using oligos designed according to the principles described in Onishi, Pringle and Cross 2015.
Breker M, Lieberman K, Cross FR (2018) Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii. Plant Cell. 30(6): 1178-1198
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