The overall goal of this procedure is to produce sufficient DNA amplicon from a single micro dissected polytan chromosome arm to use directly for fish experiments. This is accomplished by first preparing polytan chromosome squashes on specially designed membrane slides for micro dissection. The second step of the procedure is to isolate and capture individual polytan chromosome arms by laser micro dissection.
The third step is to isolate DNA from the micro dissected sample and amplify the DNA using two rounds of whole genome amplification. The final steps are to label the amplified DNA and to use them in fish experiments. Ultimately, results can yield copious fluorescently, labeled DNA for a region of interest.
The main advantage of this technique over existing methods like fish with back clones or PCR fragments, is that large portions of chromosomes can be painted fluorescently with these labeled probes. Also, no sequence needs to be known for the chromosome paints as they're mechanically isolated from intact chromosome arms. First isolate the chromosomes in this case from the ovaries of about five mosquitoes fixed in fresh car noise solution.
Once a good chromosome spread is confirmed, the chromosomes are flattened by denaturation, followed by cold overnight incubation. The next day, the chromosomes are dehydrated and washed to prepare them for microdissection. Be sure to clean the microdissection scope with ethanol and sterilize gloves and tubes with uv.
Then microdisect the chromosomes following a secondary video provided in the text protocol. Collect the DNA using a cogen kit with a modification to accommodate an inverted tube. Follow this with column purification and elution into water.
There are two routes for whole genome amplification of a single chromosome arm. The ley g root provides an initially larger yield, but necessitates NIC translation to label the DNA. The genome plex root as the advantage of allowing for multiple rounds of replication as well as direct labeling of the end result probes.
Be sure to use the most sterile conditions possible as amplification of contaminants is highly probable. For the genome plex root First, allow the single cell WGA four kit to produce the first batch of amplified DNA. Second purify the DNA using the genome clean and concentrator kit.
Do not forget to elute the wash DNA using water and not TE buffer. Third, reify the DNA and fourth label it for fish both steps. Use the WGA three re amplification kit.
Labeling the DNA requires the use of a special DNTP mix that incorporates a specific ratio of nucleotides as well as the labeled DUTP. When executing the rege kit, root first used their single cell WGA kit using freshly sterilized water and recently made D two buffer. Next, follow the NIC translation protocol to label the amplified DNA.
Be sure to check the DNA fragment size on a gel. The fragments should be approximately 200 to 500 base pairs. To finish either root ethanol precipitate the label DNA and centrifuge the sample at four degrees Celsius for 10 minutes.
Then air dry the recovered pellet and add 40 microliters of hybridization buffer. Once dried in advance, prepare slides with square walls of nail polish onto which the cover slips will rest, so the nuclei will not be squashed by the cover slip. Now dissect fresh ovaries from Christopher's stage three half grave female mosquitoes.
Transfer the ovaries to a tube with 150 to 250 microliters of buffer, a plus 0.5%digit toin in the tube. Use a large dissection needle to destroy the follicular membranes. Then vortex the tube for five to 10 minutes to further disturb the follicles.
Scrape down any large follicular pieces and centrifuge the tube for 30 seconds at low speed, about 500 RPM. Transfer the supernatants to a new tube and add 100 microliters of buffer A to the original tube containing the broken up follicles. Now, repeat the nuclear isolation steps on the broken up follicles until the visible tissue is just small particles.
Collect all the supernat into the same tube as they will all contain mostly nuclei. Next, spin down the tube containing the supernat and the tube containing the remaining tissue, which is processed as a safeguard. Discard the supernat and add 200 microliters of buffer A with 0.1%Triton vortex the tubes vigorously until the pellets are broken up, and then incubate the samples overnight at four degrees Celsius.
The next day, centrifuge the tubes for five minutes At 10, 000 RPM, remove the supernatants and add 200 microliters of 4%paraform aldehyde in PBS. Then incubate the tubes in a thermo mixer for 30 minutes, mixing them at 450 RPM. Remove the fixative using a five minute spin at 5, 000 RPM, followed by discarding the super names.
Then add buffer A with 0.1%Triton and incubate the tubes for five minutes in a thermo mixer at 450 RPM centrifuge, the wash cells for another five minutes. At 5, 000 RPM, have the labeled probe ready warmed to 37 degrees Celsius. When the cells are pelleted, replace the super names with at least 20 microliters of labeled probe.
Don't worry about its concentration. Next, denature the DNA at 95 degrees Celsius in the Thermo mixer at 450 RPM for 10 minutes and continue the denaturation at 80 degrees Celsius for 15 minutes with sustained mixing. Then drop the temperature to 37 degrees Celsius and keep the sample mixing overnight.
The next day, recover the nuclei by centrifuging for five minutes. At 5, 000 RPM 2, 655 Gs.Replace the SUPERNAT with a wash of buffer, a containing 0.1%tritton. Allow the cells to wash for five minutes with agitation at approximately 500 RPM at room temperature.
Remove the wash with another centrifugation and repeat the wash process twice more. After removing the third wash directly resuspend the nuclei into a drop of prolonged anti fade with DPI. Finish by transferring the nuclei carefully, avoiding bubbles to a prepared slide and apply a cover slip genome plex and ee.
Single cell WGA kits were both used as described on mosquito tissue. The resultant DNA was compared by gel electrophoresis. Clearly the yield varied significantly and this was confirmed by spectroscopy as well.
Fish was performed using five probes for micro dissected material on mosquito polytan chromosomes. Four autosome alarm were labeled with three fluoro fours using the WGA three kit. The three R chromosome is labeled in green fluorescein.
The three L chromosome is in a mixture of red and yellow. The two R chromosome is in yellow and the two L chromosome is in red. The X chromosome was labeled in orange using NIC translation of the ley G material and a separate experiment to establish the correspondence between e chromatic portions of polytan and mitotic chromosome arms.
Chromosome paints were hybridized to interphase chromosomes. The two R arm is labeled in green. The three R arm is in pink, and the three L arm is labeled in orange.
Chromatin is stained in blue by DPI prophase chromosomes were observed. The X chromosome has a red label corresponding to the 18 S-R-D-N-A probe Pro metaphase chromosomes were viewed next. Metaphase chromosomes were also found.
The brightly blue stained regions correspond to heterochromatin to visualize the 3D organization of a single polytan chromosome arm in the cell nucleus. Whole mount fish was performed on sue strain. Mosquito ovarian nurse cells chromosome arm territories are clearly seen on the polytan chromosomes.
Likewise, distinct chromosome arm territories are clearly seen in nuclei at interface. While attending this procedure, it is important to perform all steps with contamination risks in mind. It is very easy to contaminate the samples resulting in low amplification yield of the intended DNA.
