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Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides
Published on: November 24, 2010
Transformation with oligonucleotides creating clustered changes in the yeast genome
Gina P Rodriguez1, Joseph B Song, Gray F Crouse
1Department of Biology, Emory University, Atlanta, Georgia, USA.
Plos One
|August 24, 2012
Summary
This study shows that short DNA sequences (oligonucleotides) can modify yeast genomes. Even with DNA mismatch repair (MMR) present, a central part of the oligonucleotide is reliably incorporated, enabling multiple genetic changes.
Area of Science:
- Molecular Biology
- Genetics
- Yeast Biology
Background:
- Single-strand oligonucleotide (oligo) transformation is a method for genetic modification.
- Understanding oligo incorporation is crucial for optimizing genome editing efficiency.
Purpose of the Study:
- To investigate the incorporation patterns of 40-nt oligos during yeast transformation.
- To determine the influence of DNA mismatch repair (MMR) on oligo incorporation.
- To identify mechanisms of oligo end loss during transformation.
Main Methods:
- Utilized 40-nt long oligos with multiple base changes to track incorporation.
- Performed yeast transformation experiments under varying conditions (MMR presence/absence, DNA strand annealing, phosphorothioate linkages).
- Quantified the incorporation frequency of different oligo segments.
Main Results:
- A central ~15 nt core of the oligo is incorporated with >90% frequency.
- Oligo incorporation patterns are largely unaffected by MMR, DNA replication strand, or phosphorothioate linkages.
- Distinct mechanisms govern the loss of 5' and 3' oligo ends, with >95% 5' end loss and ~33% 3' end loss for terminal bases.
Conclusions:
- Oligonucleotides can efficiently introduce multiple simultaneous genetic modifications in yeast.
- Oligo transformation is robust and effective even in the presence of the DNA mismatch repair system.
- Mechanisms of end-joining and repair influence the extent of oligo incorporation.
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