The overall goal of the following experiment is to generate R-N-A-D-N-A hybrids at the chromosomal level in live yeast cells by transforming the yeast cells with RNA containing oligos. This is achieved by introducing a single strand, RNA, containing oligo into dividing yeast cells. The oligo will then an kneel with the complementary region of a mutated marker gene located on a yeast chromosome to form an R-N-A-D-N-A hybrid as a second step cells transformed by the RNA containing oligo are plated on the selective media.
This allows for growth of cells in which the RNA portion of the oligo serves as a template for the correction of the mutated marker gene. Next yeast colonies growing on the selective media accounted in order to ascertain the frequency of gene correction by the RNA containing oligo results are obtained that reveal the transfer of genetic information from the RNA tract of the oligo to the mutated marker gene in the yeast genome. This is evident from restriction, digestion analysis, sequence analysis, and the alkaline liability assay.
Hi, I'm HIN founder, the laboratory of Dr.Francesca in the school of biology at the Georgia Institute of Technology. Today we'll show you a procedure for generating R-N-A-D-N-A hybrids at the chromosomal level in live yeast cells. We use this procedure in our laboratory to study how the presence of RNA embedded in genomic DNA is tolerated by cells and to determine which factors affect R-N-A-D-N-A hybrid stability in vivo.
So let's get started. Prior to the start of this procedure, wipe all materials that will be used for the experiment, including oligo tubes, pipettes, vortex racks, experimental area, and gloves worn by the investigator with RNAs decontamination solution. To remove any potential RNAs contamination, use RNAs free water, chemical reagents, tubes, and pipette tips throughout the procedure.
Obtain an RNA containing oligo to transfer genetic information to genomic DNA of yeast cells. The yeast strain used here contains a mutant trip five gene with a two based deletion and one nonsense mutation. Such a yeast strain is a tryptophan atrophic mutant and does not form colonies on media without tryptophan.
The RNA containing oligo is a 65 MER with a two based DNA insertion designed to correct the deletion mutation. In addition, the oligo has one ribonucleotide to correct the nonsense mutation of the trip five allele once obtained, prepare the RNA containing oligo to a 250 micromolar stock solution as described in the written protocol. 16 hours before the transformation with the RNA containing oligo inoculate.
Five milliliters of rich yeast peptide dextrose or YPD liquid medium with the trip five mutant yeast cells and grow the cells at 30 degrees Celsius overnight. Following overnight incubation transfer 1.5 milliliters of the overnight culture into 50 milliliters of YPD liquid medium. Incubate the cells in a 30 degree Celsius shaker at 225 rotations per minute for four hours immediately before the transformation thaw.
The RNA containing oligos and corresponding DNA only oligos on ice. The corresponding DNA only oligos are used as a positive control. Dilute the oligos to 50 micromolar with RNAs free water in RNAs free tubes.
Prepare 70 microliters of each oligo to allow enough sample for three transformations of one mol with some excess denature, the RNA containing and DNA only oligos on the 100 degrees Celsius heat block for two minutes to eliminate secondary structures of the oligos immediately after denaturation. Place the tubes on ice, keeping them there until the transformation Prepare solution one and solution two immediately before transformation in RNAs free tubes. Transfer the cell culture to a 50 milliliter RNAs free tube and spin at 3000 rotations per minute corresponding to 1, 562 times gravity for two minutes.
Remove the supinate and wash cells with 50 milliliters of RNAs free water. Spin the cells again at 3000 rotations per minute for two minutes. Repeat this process five times to get rid of the culture medium and any RN ais that could be present after the final spin.
Remove the supinate and re suspend the cells in five milliliters of solution.One. Spin the sample at 3000 rotations per minute for two minutes, discard the s supernatant and add 250 microliters of solution one to the cells. This amount of cells is sufficient for seven to eight transformations.
Aliquot 50 microliters of the cell suspension into each of seven RNAs free micro centrifuge tubes. Next, add 20 of the 50 micromolar RNA containing oligo in three tubes. 20 microliters of the 50 micromolar DNA only oligo in three tubes and 20 microliters of sterile water with no oligo for the negative control in one tube.
Then add 300 microliters of solution two for each of the seven transformation reactions. Vortex the samples vigorously to mix the components. Incubate the transformation reactions at 30 degrees Celsius for 30 minutes in a shaker following incubation.
Heat shock the samples at 42 degrees Celsius for 15 minutes. Then spin down the cells at 5, 000 rotations per minute corresponding to 2, 236 times gravity for four minutes. Remove the SUP agent and resuspend the cells in 100 microliters of sterile water for many of three tubes.
Take an aliquot of this cell suspension and dilute each aliquot with sterile water by 100, 000 fold. Add about 15 sterile glass beads per plate and plate 100 microliters of the 100, 000 fold diluted cells on three YPD plates. Plate 100 microliters of the resuspended cells from each transformation reaction on one Petri dish of synthetic complete solid medium without tryptophan and shake the plates evenly.
Then remove the beads. Incubate the cells on YPD plates at 30 degrees Celsius for two days and incubate the cells plated on synthetic complete solid medium without tryptophan at 30 degrees Celsius for four to five days. Count and compare the number of colonies grown on the selective medium as well as on YPD medium to calculate the gene correction frequency for the RNA containing oligo for the DNA only oligo and for the no oligo control.
All no colony formation is expected on the selective medium when no oligos are added to the cells streak out several randomly picked transformed colonies onto YPD medium to obtain single colony isolates. Incubate the cells for two days at 30 degrees Celsius for colony growth. Following incubation, pick several single colonies and make patches on YPD and on the selective medium.
Synthetic complete solid medium without tryptophan following one day incubation growth is observed for the cells transformed with the oligos, but not for the no oligo control strain on the selective synthetic complete solid medium without tryptophan. Next, design a pair of primers to amplify the region targeted by the RNA containing oligo using colony PCR to begin colony PCR. Re suspend the cells taken from the individual patches in 50 microliters of water containing one unit of liase is incubate the samples at room temperature for 10 minutes, followed by incubation in a heat block at 100 degrees Celsius for five minutes to break the cells and release genomic DNA into solution.
Prepare the PCR reactions in PCR tubes, adding 10 microliters of the cell suspension to each tube set up and perform the amplification reaction as described in the written protocol. Run the resulting samples on a 1%arose gel to observe the PCR product. Since the genetic information transferred by the RNA containing oligo in this experiment generates a new VAN 91 1 restriction site in the yeast genomic target region, it is possible to verify the correct transfer of information by digesting the PCR product with the van 91 1 restriction enzyme.
Set up the digestion reaction to include six microliters of PCR product buffer BSA 0.5 microliters of the restriction enzyme and sterile water to 15 microliters. Incubate the samples for one hour at 37 degrees Celsius. Run an undigested sample together with the digested samples on the same row of a 2%arose gel to observe the genetic modification transferred by the RNA tract of the RNA containing oligo.
Purify the PCR products by using a PCR purification kit and prepare them for DNA sequencing. Submit the samples for sequencing with the same primers used to amplify the product. Finally, analyze the DNA sequencing results using software that allows alignment of multiple sequences with a chosen reference sequence to perform alkali treatment of the RNA containing oligo for each reaction at one mol of the RNA containing oligo or the DNA only oligo into a 1.5 milliliter tube.
Then add four microliters of one molar sodium hydroxide for hydrolysis or four microliters of water for the negative control. Incubate the samples at 65 degrees Celsius in a water bath for one hour following incubation. Transfer the reaction to ice.
Neutralize the hydrolysis reaction with two microliters of 1.2 molar hydrochloric acid, four microliters of one molar tris hydrochloride and four microliters of water for the negative control. Add six microliters of water and four microliters of one molar tris hydrochloride. Keep the samples on ice until the transformation which can be performed.
As just described, yeast cells transformed with no oligo do not form any colonies on the synthetic complete solid medium without tryptophan fan. Conversely, cells grown on synthetic complete solid medium without trytophan after cells are transformed with one ole of the RNA containing oligo. Now show colonies.
Yeast colonies also grow on synthetic complete solid medium without trytophan after cells are transformed with one nanomole of the corresponding DNA only control oligo the detection of genetic information transfer from the RNA containing oligo to yeast chromosomal DNA is evident by VAN 91 1 restriction digestion of the PCR product amplifying the targeted genomic region as shown the PCR product of the trip. Five locus amplified from the genomic DNA of the trip. Five mutant strain can be seen.
Also shown is the PCR product amplified from genomic DNA derived from a trip plus colony targeted by the DNA only oligo. The PCR products amplified from genomic DNA derived from trip plus colonies targeted by the RNA containing oligo were also run on the gel. The results of the van 91 1 restriction digestion of the same PCR products reveal a 278 base pair amplified PCR band and the digestion product bands by van 91 1 of 177 base pairs and 101 base pairs.
DNA sequencing reveals the gene correction by the RNA containing oligo here. A DNA electropherogram of the genomic region targeted by the RNA containing oligo is shown. The g in the DNA sequence derives from the RG on the RNA containing oligo go.
Also boxed is the insertion of the CG basis sequences of the trip five region of the trip plus transformants targeted by the DNA only oligo and the RNA containing oligo match in the consensus sequence and are compared with that of the trip. Five mutant cells before targeting by the oligos. The repair RNA containing oligo on the bottom has a two base DNA insertion and a one base RNA substitution.
The region's boxed in blue with yellow shade show that the RNA containing oligo as well as the DNA only oligo precisely corrected the deletion mutation and nonsense mutation in all tested samples, the dash lines mark the position of the RNA containing oligo sequence. Alkali treatment prevents gene correction by the RNA containing oligo the transformation frequency by the RNA containing oligo and the DNA only oligo is shown. The aerobars rep represent the standard error of the mean for three independent transformations for each oligo.
The DNA only oligo displays similar transformation frequency without and with sodium hydroxide treatment. Conversely, transformation frequency by the RNA containing oligo drops to zero following treatment with sodium hydroxide. Therefore, the preparation with the RNA containing oligo is not contaminated with DNA only oligo.
Thus, the observed transformation frequency is specific to the RNA containing oligo. We've just shown you how to form an R-N-A-D-N-A hybrid at the chromosomal level and the review transfer of genetic information from RNA to genomic DNA in yeast cells. When doing this procedure, it's important to remember to have excellent quality of the RNA containing oligos and to work in RNA free conditions.
So that's it. Thanks for watching and good luck with your experiments.
