The overall goal of this procedure is to analyze DNA methylation in a single oocyte. This is accomplished by first removing cumulus cells and soap palita from collected oocytes. The second step is to embed the oocyte in a two to one aros lysis solution to protect the minute amount of DNA from being lost or degraded.
Next bi sulfite mutagenesis is performed, followed by amplification through nested polymerase chain reaction. The final step is to ligate and clone the PCR product. Clones are then picked PCR amplified and sent for sequencing.
Ultimately, bi sulfite mutagenesis and sequencing is used to show DNA methylation at cytosine phosphate guine, or CPG dinucleotides of a region of interest in a single oocyte. The main advantage of this technique over existing methods like bi sulfite mutagenesis of large cell numbers, is that data can be obtained from a single oocyte without pooling cytes PCR bias and chemo cell contamination. On the day of oocyte collection, freshly prepare the solutions listed in the written protocol when making solutions reduce the chance of DNA contamination by changing gloves often and using filter tips also keep tubes angled away when open and recap all tubes when not in use.
Begin OSI collection by placing the dissected mouse ovaducts into M two media and ripping the M pulley to extract the cumulus cell complex. Then separate the cytes from the cumulus cell complex using 0.3 milligrams per milliliter of hyaluronidase solution in a 30 microliter drop of M two media. Keep the cytes in solution only as long as it takes to remove the cumulus cells as lengthy exposure may damage them.
Wash the oocytes in 30 microliter drops of M two media by transferring the oocytes from drop to drop. Removing the cumulus cells periodically for a total of three washes. Remove the Z of LUCITA using acidic tyro solution.
First place the oocytes in one 30 microliter drop of solution, then transfer to another 30 microliter drop. As any media carried along will dilute the acid and reduce its efficiency. Keep the cytes in solution only as long as it takes to remove the zona as lengthy exposure may damage them.
Following removal of the zona, wash the cytes again in a 30 microliter drop of M two media to perform agros embedding first place the lysis solution on a 70 degree Celsius heat block. Add the preheated low melting point, or LMP Agros to the lysis solution, producing a two to one AGROS to lysis solution. Then place a single oocyte onto a clean glass slide in minimal M two media.
The next step is to embed the single cyte into the Agros and lysis solution. This must be done quickly as the agros will harden at temperatures below 65 degrees Celsius. Take up 10 microliters of the Agros lysis solution into a pipette tip under a microscope.
Gently expel about one microliter or less of the agros lysis solution onto the glass slide, allowing it to mix with the minimal media. Gently pick up the oocyte into the pipette tip. Then put all 10 microliters into an einor tube with 300 microliters of mineral oil so that the resulting bead takes on a spherical shape.
Incubate the tube on ice for 10 minutes to perform lysis. Remove the 300 microliters of mineral oil and add 500 microliters of the SDS lysis buffer. Incubate the sample overnight in a water bath set to 50 degrees Celsius on the second day.
Prepare the bi sulfide mutagenesis solutions fresh as described in the written protocol. Begin mutagenesis by fully removing the 500 microliters of SDS lysis buffer. Keep in mind that any lysis buffer remaining will dilute the agros when it is heated and the bead will be more susceptible to dissolving in the subsequent steps.
Following complete removal of the lysis buffer, add 300 microliters of mineral oil. Incubate the sample for two and a half minutes on a 90 degree Celsius heat block. Take carat to mix or stir, extend, or fluctuate the temperature.
After two and a half minutes. Incubate the sample on ice for 10 minutes. To perform denaturation, remove the mineral oil and add one milliliter of 0.1 molar sodium hydroxide to the tube flick and invert the tube five to six times.
Incubate the sample for 15 minutes. In a water bath set to 37 degrees Celsius inverting every three to four minutes, the bead should float in the sodium hydroxide. Note that the bead is no longer at the bottom of the tube.
To perform by sulfite treatment first spin the tube gently following removal of the sodium hydroxide. Add 300 microliters of mineral oil and 500 microliters of BIS sulfite solution. Take care to avoid exposure to light and incubate the tube for three and a half hours in a 50 degrees Celsius water bath.
Now perform desination. First incubate the sample on ice for three minutes before removing the mineral oil and BIS sulfite solution. After a gentle spin, add one milliliter of 0.3 molar sodium hydroxide flick and invert the tube five to six times.
Incubate the sample for 15 minutes. In a 37 degree Celsius water bath inverting every three to four minutes, again, the bead should float in the sodium hydroxide prior to beginning PCR. Wash the samples by first spinning gently.
Then remove the sodium hydroxide and add one milliliter of one xte pH 7.5. Shake for five to 10 minutes at room temperature. Following a second gentle spin, remove the one XTE.
Repeat this washing process twice. Then add one milliliter of autoclave, double distilled water. Shake for five to 10 minutes at room temperature, spin gently for a third time and remove the water.
Repeat the double distilled water wash twice. Finally, remove all supernatant, leaving only the aros bead. Check that the pH of the supernatant is 5.0.
If the supernatant is still too basic, wash the beads again with double distilled water. Meanwhile, add the first round PCR mix to the PCR tube as described in the written protocol. Slide the solid agro speed to the lip of the einor tube.
Then carefully transfer the agro speed to the PCR tube heat to 70 degrees Celsius for one minute and mix then amplify is suggested in the written protocol. Following first round amplification, prepare the second round PCR reaction using five microliters of the first round product as a template. Be sure to bipa below the layer of mineral oil.
Proceed to perform the second round of amplification as directed in the text as a diagnostic test. Second round samples can be cut with a restriction enzyme that is methylation or strain specific electrophoresis the digestion products on an 8%acrylamide gel. Any heterogeneous bands that result represent more than one sequence to clone the second round product ligate into the vector and perform TA cloning as described in the manuscript accompanying this video following TA cloning plate, all of the reaction mix onto an agar and LB plate, supplemented with ampicillin, ex gal and IPTG and incubate the plate at 37 degrees Celsius overnight.
Then add the colony PCR master mix to the PCR tube as listed in the written protocol. Pick a white bacterial colony from the plate with a pipette tip and swirl it into 35 microliters of the colony PCR master mix in a PCR tube. Proceed to amplify the suspension as described in the text following PCR amplification electrophoresis four microliters of the PCR product on a 1.5%AGROS gel.
Finally, send approximately 30 microliters of the PCR product for sequencing of five colony PCRs per cyte. Once sequencing results are obtained, methylation patterns can be read any original cytosine guanine that remained as such was methylated. Conversely, any original cytosine guanine that is now a thiamine guanine was unmethylated following nested PCR amplification using bi sulfite converted primers.
It is possible to confirm a successful conversion by visualizing a correct fragment size on an aros gel individual metaphase. Two oocyte represents one parental allele and in theory has one imprinted methylation pattern As such. Second round PCR products can be tested for unintentional contamination.
A restriction enzyme sensitive to DNA methylation can be used to digest the second round PCR product to assess whether it contains a methylated or unmethylated allele. Here, a methylated cytosine within the enzyme recognition sequence is cleaved. While an unmethylated cytosine that is converted to thymine is no longer recognized by the enzyme and is uncut any metaphase two oocyte sample containing both methylated and unmethylated alleles should be discarded as it is indicative of cumulus cell contamination or polar body inclusion Following ligation and transformation.
Successful colony PCR amplification can be visualized on an AROS gel to ensure samples with the correct product size are sent for sequencing. For this example, the expected amplicon size following ligation is hundred 56 base pairs. Here, clone five has an incorrect amplicon size and should not be sent for sequencing.
The sequence of five individual clones from a metaphase two oocyte should produce five identical methylation patterns and identical non CPG conversion rates. Any samples that contain more than one pattern should be discarded. Since ovulated metaphase two cytes have two chromosome copies or an attached polar body, there is a possibility for obtaining two similar sequence patterns.
Data should be discarded from any cytes that have highly dissimilar methylation patterns. Since cumulus cell contamination cannot be ruled out When attempting this procedure, it is important to remember first to wash oocytes multiple times in fresh medium, following HI IDE treatment to remove as many cumul cells as possible. Secondly, the low melting point agros will harden at temperatures below 65 degrees Celsius, so work carefully but quickly.
And finally, sodium bis sulfate and hydroquinone are light sensitive, so reaction tubes and solutions should be wrapped in foil.
