Home › Cell Biology › Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides
Cell Biology JoVE (Open Access) Citable · DOI

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

DOI: 10.3791/2152-v
What you'll learn
  • ✓ Prepare RNA-containing oligonucleotides for yeast transformation
  • ✓ Perform yeast transformation with RNA-DNA hybrid sequences
  • ✓ Analyze gene correction events via phenotypic assays
  • ✓ Apply alkali treatment to confirm RNA-mediated effects
Protocol

Biopharma Insights This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Difficulty
intermediate
Total time
~2 days (including yeast growth and analysis)
Model organism
Saccharomyces cerevisiae
Biosafety
BSL-1

Steps

1
Prepare RNA-containing oligo and yeast cells

Prepare the RNA-containing oligonucleotide and grow yeast cells to the appropriate stage for transformation.

▶ 01:53
2
Transform yeast with RNA-containing oligo

Introduce the RNA-containing oligonucleotide into yeast cells using a standard transformation protocol.

▶ 04:40
3
Analyze gene correction by RNA oligo

Assess the efficiency of gene correction by plating and scoring for the corrected phenotype.

▶ 07:47
4
Alkali treat RNA-containing oligo

Perform alkali treatment of the oligonucleotide to degrade RNA and confirm the RNA-dependent effect.

▶ 11:10
5
Review representative transformation results

Examine representative results from transformation experiments using RNA-containing oligonucleotides.

▶ 12:20
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