Transformation with Oligonucleotides Creating Clustered Changes in the Yeast Genome

Department of Biology, Emory University, Atlanta, Georgia, USA.
PLoS ONE (Impact Factor: 3.23). 08/2012; 7(8):e42905. DOI: 10.1371/journal.pone.0042905
Source: PubMed


We have studied single-strand oligonucleotide (oligo) transformation of yeast by using 40-nt long oligos that create multiple base changes to the yeast genome spread throughout the length of the oligos, making it possible to measure the portions of an oligo that are incorporated during transformation. Although the transformation process is greatly inhibited by DNA mismatch repair (MMR), the pattern of incorporation is essentially the same in the presence or absence of MMR, whether the oligo anneals to the leading or lagging strand of DNA replication, or whether phosphorothioate linkages are used at either end. A central core of approximately 15 nt is incorporated with a frequency of >90%; the ends are incorporated with a lower frequency, and loss of the two ends appears to be by different mechanisms. Bases that are 5-10 nt from the 5' end are generally lost with a frequency of >95%, likely through a process involving flap excision. On the 3' end, bases 5-10 nt from the 3' end are lost about 1/3 of the time. These results indicate that oligos can be used to create multiple simultaneous changes to the yeast genome, even in the presence of MMR.

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    • "Transformation by electroporation was performed essentially as described previously[28,67]. An overnight culture of yeast cells (0.5 ml) was inoculated into 25 ml of YPAD[68], incubated with shaking at 30u to an A 600 of 1.3–1.5, washed twice with cold H 2 O, and once with cold 1 M sorbitol. "
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