The overall goal of this procedure is to map DNA probes to mosquito polytan chromosomes using a high pressure chromosome preparation, followed by automated fluorescent in C two hybridization and automated imaging. This is accomplished by first preparing polytan chromosome spreads using a high pressure method. The next step of the procedure is to prepare fluorescent probes by labeling genomic back DNA with the fluorochrome using a Nick translation protocol.
The third step is to perform fluorescent in C two hybridization using automated slide staining systems. The final step is to scan and photograph labeled chromosomes using an automatic fluorescent imaging system. Ultimately, images can show the localization of a fluorescently labeled DNA probe on the poly chromosome.
The main advantage of this technique or existing methods, like a standard manual in situ hybridization protocol is that the highest throughput physical mapping protocol produces more consistent results and dramatically reduces hands-on time. This protocol begins by dissecting half gravity and oly females at about 25 hours post blood feeding to collect their ovaries when they are at Christopher's stage three. At this stage, the transparent area with nurse cells within follicles should have a round shape, collect ovaries from five females and fix them in 500 microliters of freshly made modified car noise solution.
They can then be stored at room temperature for up to 24 hours or for longer at minus 20 degrees Celsius. Next, just prior to making ovary dissection slides, prepare car noise solution and 50%propionic acid. Place each pair of ovaries in a drop of fresh car noise solution on a dust-free slide under a dissection microscope.
Split the ovaries with the dissecting needle. Transfer all of the ovary pieces to six clean slides with 50%propionic acid. Separate the follicles from each other using dissecting needles.
Then wipe away any remaining tissues with a paper towel. Add another drop of 50%proponic acid and wait for three to five minutes. Legend, the follicles sit in 50%Proponic acid for three to five minutes significantly improves the chromosome spread.
Next, apply a cover slip and wait another minute to spread the nuclei. Wrap the slide with filter paper and plastic. Set a Dremel between three and 5, 000 RPM, and for about one minute, lightly swirled a spinning dremel tip against the cover slip.
Confirm the spread quality with a phase microscope and apply the dremel longer if needed, to flatten the chromosomes. Place a second cover slip adjacent to the first, then sandwich the cover slips with another microscope. Slide next, wrap the slides with a plastic sheet and with filter paper, place the wrap slides in a vice to squeeze the slides to between 85 and 120 inch pounds.
After flattening the chromosomes on all six slides, unwrap the slides and remove the extra slides to further flatten the chromosomes. Incubate the slides at 55 degrees Celsius for 10 to 15 minutes at a slide denaturation hybridization system. Next, dip a slide in liquid nitrogen for at least 15 seconds or until the bubbling has stopped.
Then quickly remove the cover slip with a razor blade and immediately place the slide in cold, 50%ethanol for five minutes. Repeat this process for each slide sequentially. Dehydrate the slides in a 70%a 90%and a 100%ethanol bath.
Submerge them in each bath for five minutes. Finally, after the slides air dry, they are ready for fish for staining. An automated device performs most of the steps in the procedure except the probe labeling.
So before loading the device, prepare the fluorescent probe by labeling back DNA with the fluorochrome using a commercial NIC translation protocol with the probe prepared, load the slides and reagents into the automated slides. Staining system and program the system to perform the following steps. First, supply 800 microliters of one XPBS for 20 minutes.
Second, blow dry the slides. Third, fix the slides with 450 microliters of 4%formalin in one XPBS for one minute. Then wash the slides with 100%ethanol for one second, twice and for two minutes.
Once after the ethanol bath, blow dry the slides again. Now heat the slides at 45 degrees Celsius for two minutes to avoid bubbling. Then apply 20 microliters of the DNA probe, followed by a drop of mineral oil and a cover slip next in nature, the chromosomes at 90 degrees Celsius for 10 minutes.
Then set the slides to hybridize at 42 degrees Celsius for 14 hours. After the hybridization set the stringency washes for two minutes at 42 degrees Celsius, followed by four consecutive one second applications of two XSSC and drying. Then apply 800 microliters of 0.4 XSSC at 42 degrees Celsius for 10 minutes twice, followed by one more wash and two XSSC at 25 degrees Celsius for 10 minutes.
Now apply the stain 50 microliters of yo-yo one followed by mineral oil and cover slips. Incubate the slides at 25 degrees Celsius for 10 minutes. Then remove the cover slips.
Wash slides in two XSSC four times for one second per wash and blow the slides dry. Finally, apply 15 microliters of prolonged gold anti fade reagent to each slide and attach new cover slips. Power up the Accord plus automated scanning system, beginning with the Olympus U-R-F-L-T power supply for a halogen bulb.
Then the computer, and lastly the microscope with a camera attachment. Now open the duet software package first, set up a 10 x pre-scan. Click the online button, enter a new case ID and assign a slide id.
Click the dot labeled bf. This is the brightfield option. Select a scan choice to 10 x pre-scan.
Select 2, 500 x circle, 10, 000 x circle or rectangle. Click set and run. And then, okay, follow the prompts to adjust the scanning properly and then click finish.
To start the scan, press the main tab to go back to the main screen. Now click on the offline button, find the case ID and slide ID that was assigned. And click offline.
Scan in the black box at the top left area. Click on an arrow and select 10 x pre-scan. Use the arrows to go through the scanned images after finding an image of interest.
Double click on the screen in the middle of the targeted region and press snap. This will target an image for capture later on after selecting all of the targets. Right click each image, click classify select 10 x pre-scan and select polytan.
Return to the main menu and set up the 40 x pre-scan. Select online again and select a slide. Change BF to ffl and change the task name to revisit dash X 40 dash rg.
Change the section right below the last setting to revisit dash all. Then run the program and follow the prompts. To set up the automation, click the start matching views button to match 10 x and 40 x images.
Click finish to start the scan. Once done, click classify and look at the images. Preparations of ovarian nurse cell polytan chromosomes from female anomaly Gambia were made using the high pressure technique.
This method does not damage or change most of the chromosome structure. It flattens bent chromosomes and thus reveals hidden fine bands that are not seen on regular preparations. The chromosome was probed with back DNA clones obtained from the anomaly S Gambia nd Tam back library.
The library was generated from male and female first in star larva of the anomaly S Gambia pest strain. Following this procedure, an automated high throughput physical mapping can be performed in order to facilitate the rapid development of chromosome based genome assemblies.
