Mapping of LOH events by RFLP-SNP assay.
*The location and sequences of the SNP markers in YPH45 or YJM789 strain backgrounds are listed in Table S2. For URA3 and G418R, PCR and agarose gel electrophoresis is carried out to verify the presence or the absence of the corresponding sequences (N- PCR product absent; Y – PCR product present). For StyI, BsrBI, SpeI, NarI, and HindIII, PCR products are digested with the corresponding restriction enzyme and run on agarose gel to verify presence of YJM789-SNP or YPH45-SNP. For example, 700 nt region including the StyI-SNP site is amplified and digested with StyI. If both YJM789 and YPH45 are present, there will be three bands – 700 nt uncut band from YJM789 and 450 nt and 250 nt bands resulting from enzyme digest of the PCR product amplified from YPH45 sequence. If the region of YPH45 containing the StyI-SNP site is lost due to recombination, PCR/restriction digest will only result in the 700 nt uncut band from YJM789 sequence. YJM – YJM789 sequence present; YPH/YJM- both YPH45 and YJM789 sequences present. Graphical representation of LOH events are shown in Figure 3A and Figure S4.
Mapping of LOH events by RFLP-SNP assay.
