The overall goal of this procedure is to determine the DNA methylation status of imprinted genes in endosperm. This is accomplished by first emasculating, the female parent. The female parent is then pollinated with pollen of the male parent.
Next, the endosperm is dissected out of the cross pollinated seed. The final step of the procedure is to perform bi sulfate sequencing of endosperm DNA. Ultimately, results can be obtained that show the DNA methylation status of the maternal or paternal allele of an imprinted gene through DNA sequencing polymorphism in different ecotypes and bi sulfate sequencing.
Hi, I am Dr.Yang Shaw from the Department of Biology at the St.Louis University. Hi, I'm Matthew Ray in the Dr.J Lab and the Department of Biology at St.Louis University. I'm Diman Panu from Dr.J's lab.
Today we'll show you a procedure for dissecting a rugby officer and determine DNA meth status by by sulf FAR sequencing. We use this procedure in our laboratory to study gene imprinting regulated by DNA methylation and demethylation. So let's get started.
To begin this procedure, choose two different Arabidopsis ecotypes to serve as female and male parents in order to distinguish the maternal and paternal alleles by using DNA sequence polymorphism. Here Columbia zero and Landsberg Erecta are chosen as female and male parents respectively. Take care to choose young and healthy plants.
Emasculate the female parent with the aid of a dissecting microscope. Locate stage 12 flowers and remove any flowers or LICs above and below them by clipping the base of the pedestal with scissors. Sterilize forceps by dipping the base of the tip gently into a beaker of 95%ethanol.
This will remove any pollen grains on the forceps and kill the pollen. Gently pry apart the flower buds using forceps and carefully remove four seels, four petals and six stamen leaving the pistol bare and intact. Try to avoid damage to the carpals during this process.
The next step is to pick the pollen donor and carry out pollination the best pollen Don are and thd Open flowers at stage 14 with the petals extending at a 90 degree angle to the pistol in which a lot of pollen is shedding. Grab the flower at the base and just above the pedestal, which will cause the flower to spread open dust. The stigma of the prepared pistol with the anther.
After the pollination, the stigma will be covered with yellow pollen, which can be easily observed under a microscope. After pollinating all the emasculated pistols on a plant labeled the cross with a jewelry tag. Include the date and information of female and male parents.
Place a stake in the soil close to the plant. Use a string to tie the stem of the in fluorescence to the stake and cover the pollinated pistols with a plastic bag. At seven or eight days after pollination or DAP, the seed should be ready to be harvested at the mid to late torpedo stage of embryogenesis and dissected.
For endosperm and embryo, place an eight DAP salic under the dissecting microscope. Using a pair of forceps, hold the EK P cell and use the tip of another pair of forceps to slide open the sole in the margin where the two carpals fuse. Use a pair of BPS to pick up one seed and place it on a glass slide.
Then make a small cut at the micro pile end to slide out the embryo. Squeeze the uncut end to push out the endosperm and separate it from the seed coat. Next, place the embryo and endosperm into separate micro tubes and liquid nitrogen.
Continue this until enough embryo or endosperm is accumulated from 10 to 15 CLICs. Then store the tubes in a minus 80 degree Celsius freezer prior to the bisulfite treatment. Isolate genomic endosperm or embryo DNA using a subtle trimethyl ammonium bromide or CTAB procedure.
Once the endosperm genomic DNA has been isolated, digest 100 nanograms in a 20 microliter total volume reaction with restriction enzymes that cut outside the region to be analyzed. For the ME promoter restriction enzymes, xh, O one ND one and PST one are used. Denature the restriction enzymes by boiling the DNA for five minutes.
Then quench the reaction on ice. Add one ninth volume or 2.2 microliters of three molar sodium hydroxide and incubate at 37 degrees Celsius for 15 minutes. Following incubation, transfer the solution to a 250 microliter PCR tube.
Next, make a 6.24 molar urea. Four molar sodium by sulfite solution by dissolving 7.5 grams of urea in 10 milliliters of sterile distilled water, slowly at 7.6 grams of sodium meta by sulfite over one to two hours while the solution is heated and stirred. Once dissolved, adjust the pH to five with freshly made 10 molar sodium hydroxide.
Add sterile distilled water to a final volume of 20 milliliters. Transfer 208 microliters of this solution to 22.2 microliters of the denatured endosperm genomic DNA. Then add 12 microliters of freshly made 10 millimolar hydroquinone to the DNA.
Conduct the bisulfite treatment in a PCR machine with 30 cycles of 55 degrees Celsius for 15 minutes, followed by 95 degrees Celsius for 30 seconds. Next, desalt the bisulfite treated DNA using the wizard, DNA cleanup system from Promega. As per manufacturer's instructions, measure the exact volume of TE recovered from the column after desalting.
Then at 6.3 molar sodium hydroxide to a final concentration of 0.3 molar. Incubate the mixture at 37 degrees Celsius for 15 minutes. Following incubation, add 10 molar ammonium acetate to a final concentration of three molar, two microliters of 20 micrograms per microliter TRNA and three volumes of 100%ethanol.
Mix the sample and centrifuge for 15 minutes at 14, 000 RPM. Wash the pellet once with 70%ethanol, followed by a short centrifuge to remove extra ethanol. Finally dry the pellet in a speed back for five minutes and resuspend in 25 microliters of TE buffer.
The sodium BI cite treated DNA is now ready for PCR analysis. To sequence the four kilobase Madea promoter Design many sets of primers and amplify 14 overlapping fragments to cover the entire region. Begin by designing a forward primer to sequence the top strand.
First, choose a guine rich region in order to have a higher annealing temperature without extra long nucleotides in the primers. Next, change cytosine to perine at CG and C NG contexts. Then change any other cytosine to thymine.
In designing a reverse primer, choose a cytosine rich region. Change guine to purine at CG and CNG context and any other guine to adenine. To sequence the bottom strand again, start by designing a forward primer.
Choose a cytosine rich region and change guine to purine at CG and CNG contexts. Change the other guines to adenine. In designing the reverse primer, choose AINE rich region.
Change cytosine to paridine at CG and CNG contexts and change the remaining cytosines tot thymine. Once primers have been designed, prepare a PCR reaction using two microliters of the sodium bi sulfite treated DNA as a template for each PCR amplification. Run the PCR reaction and analyze the product by gel electrophoresis to confirm the correct size of the fragment.
Next gel. Purify the PCR product and clone it into the in vitrogen topo TA cloning vector PCR 2.1. As an insert and sequence individual clones, the principle of bi sulfite sequencing is that unmethylated cytosine will be converted to uracil due to hydrolytic deamination by high concentration of sodium by sulfite at pH 5.0.
Conversely, five methyl cytosine will not be modified by sodium by sulfite. Uracil will be amplified as thymine in the PCR product, whereas methylated cytosine will remain cytosine after PCR amplification. After obtaining the sequencing result, compare it with the strand specific template that is used for PCR amplification.
If a cytosine residue in the template reads as a thymine in the sequencing result, it indicates that the cytosine is not methylated. If a cytokine residue in the template remains a cytosine in the sequencing result, it means that the cytosine is methylated the methylation status of the maternal MEA allele in the minus 500 base pair region of the MEA promoter for five methylated CPG sites in the sequenced clones of Columbia, g Clarus crossed with RLD endosperm is shown methylated cytosines are indicated by black filled circles and unmethylated cytosines by white unfilled circles. Similarly, the methylation status for the maternal Madea allele of Columbia, Gladys crossed with RLD embryo is shown.
We've just shown you how to isolate endosperm and perform by sulfite sequencing when doing this procedure. It's important to remember to follow the protocol correctly. So that's it.
Thanks for watching and good luck with your experiments.
