Once the samples have undergone PCR, they can be sequenced on the ABI 373 Stretch sequencers. The samples are run out on a 4.75% acrylamide gel which will separate the DNA fragments according to their length. This will be achieved by running an electrical current through the system. Because DNA is charged, it will migrate through the gel and the gel matrix will impede rate of DNA migration in proportion to the length of the DNA fragment. Thus, the smaller fragments will travel faster through the gel and pass the sensor first. The ABI 373 sequencer is able to differentiate between the bases via fluorescent dyes. Each base pair corresponds to a particular dye (For more information, refer to catalyst section). When subjected to a particular wavelength of light (a laser), these dyes will emit light at a frequency characteristic of each dye. A sensor will be able to determine the frequency and thus will know which base has just passed by. The computer will then store this data and at the end of the run, will interpret these series of fluorescence into a sequence file which can then viewed by the user. The ABI 373 can run thirty-two samples simultaneously through the use of a horizontally scanning laser/sensor unit. Thus, as the multiple samples travel through the gel, they reach the level of the scanning unit. As the laser passes by it will excite each lane which contains a specific sample. The sensor will detect the resultant emission from each lane and store it in one of thirty-two separate data files.
Pouring the 4.75% Acrylamide Gels
1-Take two clean glass plates, one notched and one back plate. Lay the flat plate on the right end of the apparatus, scratched side facing down. Place two white spacers along the edges of the back plate and secure with small black clips. Place the notched plate at the far left end of the apparatus and let it overlap on the back plate, scratch mark facing up.
4.75% Acrylamide Mix
Add urea, water, and acrylamide and stir into solution over a warm water bath. Once in solution, add 2 grams of DOWEX MR-3 resin. Con tinue to stir for 10 minutes. Vacuum filter the slurry and collect solu tion. Add 10X TBE to solution. Make up difference to 1 liter with dH20. Store at 4 °C.
2-Mix 40 ml of the 4.75% acrylamide mix with 226.8 µl of 10% ammonium persulfate and 226.8 µl of 0.66 M TEMED. Mix the solutions together and then draw it up in a 60-cc syringe.
The TEMED and ammonium persulfate catalyze a polymerization reaction causing the urea and acrylamide to form a gel matrix.
10% Ammonium Persulfate
Add 1.00 g of ammonium persulfate with dH2O to 10 ml. Aliquot into 1 ml Eppendorf tubes and store at -20 °C until use.
3-Slowly dispense the acrylamide mix onto the back plate and push the notched plate over it. Gently push the notched plate forward, removing the clips as you encounter them. Make sure the spacers do not move. Add the acrylamide and move the notched plate until the two plates are flush. Insert the top spacer and secure it with three large clips. Place two clips on each side of the plates to hold them together.
Allowing the formation of air bubbles within the gel not recommended, even along the sides of the gels. Be especially careful of introducing air at the spacer/gel interface at the top of the plate. The spacers may rise while pouring, simply push them back down.
4-Let the gel polymerize for at least four hours. Attach a piece of tape with your initials and the date the gels were poured on the gel plate.
Preparing the Samples
1-Load 4 µl of gel loading dye into each sample well. Place the sample plate on the 80 °C hot plate for about 5 min. Keep the cover on the plate to prevent evaporation.
The loading dye contains formamide. Combined with the heat, this will cause the DNA to dissociate for sequencing.
Loading Dye
Aliquot 55 µl of buffer into each well of a cetus plate. Store at -70 °C, for immediate use may store at -20 °C
Installing Gels on 373 Sequencers
1-Wash a gel with dH2O. Dry the gel plate first with paper towels and then lens paper.
When washing the gel plates be sure to get as much acrylamide off as possible. Generally, the cleaner the plate the easier it will be to set up. Be especially meticulous about the region the laser will be scanning. Using slightly wetted lens paper will help get rid of dust which interferes with the machines.
2-Place the gel in a 373, with notched plate facing away from you.
3-Close the door and select either Main Menu and Pre-run or simply Pre-run on the ABI pad. Subsequently, press Plate Check function. You should be able to hear the stage motor begin moving.
4-On the sequencer's Mac open the Data Collection application. Once the application is open, open the Scan and Map windows.
5-Observe the scan of the gel. There should be four colors, red, yellow, green, and blue. The red should be the highest and the blue the lowest. All blue peaks must be eliminated from the scan; however, small peaks can be tolerated. Select the Map function on the Mac and a plot of scan over time will appear. If there are no blue peaks on the map display then the glass plates are clean enough. Otherwise, slowly open the door and using wetted lens paper, wipe the glass clean and scan execute a plate check again.
The plate check/scan determines the level of cleanliness of the gel plates. Blue peaks will result in an inability to read the samples. A baseline of noise is normal; therefore, checking the scan display against the map dis play will provide the most complete image.
Hint: even the tiniest amount of acrylamide will smear when wiped with a paper towel which will cause a large blue peak. Therefore, it is advisable to clean the plate thoroughly the first time before wiping the plate dry.
Also, if the peak simply will not go away, use the little plastic chips found near each machine and insert them at the bottom of the plate. These spacers will raise the gel, which may just be enough to clear a defect in a glass plate.
6-Check to see the value of the blue trace. It should be somewhere between 900 and 1100. If they are not then select Main Menu and then the Calibration mode. Keep on scrolling through until you reach the screen displaying the PMT Wattage. Adjust the PMT setting by 5-10 watts until the yellow and green lines reach the proper range.
The PMT setting determines the sensitivity of the machine to the various wavelengths emitted by the dyes when they fluoresce. Therefore, the higher the setting the more sensitive the machine. This has both advan tages and disadvantages.
7-From the Main Menu on the 373 select the Set-up Run option. Check to make sure that the duration of the run matches the duration you programmed into the Mac. Also, check to see that the power setting is correct. Change as needed.
8-Attach the upper reservoir buffer chamber and the T-bar across the top of the gel.
9-Fill both the upper and lower buffer chambers with 1X TBE. Ensure that the bottom of the gel is submersed in buffer.
1X TBE is the salt solution needed to carry the current. It has a conductiv ity of around 1050-1150 µS. When filling the upper chamber, do not hesitate to fill the buffer right up to the rim as buffer will evaporate during the course of the run. If enough buffer evaporates, the run will terminate prematurely.
10-Using a 60-cc syringe, wash out the top of the gel. Insert the shark's tooth comb into the top of the gel with the well-numbers facing you.
When inserting the shark's tooth comb, do not insert so far as to make the gel surface in each well appear convex. This can interfere with the accurracy or basecalling.
11-Pre-run the gel for a few seconds to ensure all the electrical conncections are working.
Sample in a well will diffuse into the buffer if not promptly run into the gel. Discovering electrical problems after you have loaded samples
results in losing those samples.
1 X TBE Buffer
Loading Samples and Running the Gel
1-With the formamide dye loaded, place the 96-well plate in the 85 °C heat block for 5 min. Remember to keep the lid on top to prevent evaporation.
The heat block will cause the dissociation of the DNA and its subsequent binding to the formamide dye.
2-It is necessary to pre-run the gel. Using a wide-barrel 50 ml syringe, wash out the wells with buffer (urea will leach out of the gel into the well and interefere with loading). First load the samples in the odd columns into the odd lanes of the sequencing gel. After having loaded all the gels, close the lid and then select the Pre-run function. Then select the Pre-run Gel function. Let the samples pre-run between three to five minutes.
The base calling software requires that half of the samples be pre-run in order for optimal results. The spacing of the Hamilton syringe permits loading eight lanes at a time in this format.
3-After pre-running the gel for 3 min, wash out the wells with the 60-cc sy ringe. Load the samples in the even columns into the even rows of the se quencing gel and pre-run again for 3 min. After the second pre-run, wash out the wells again with the 60-cc syringe, close the lid and select the Choose Run function from the Main Menu screen on the 373. Select the Sequencing Run function and for the Full Scan option.
The movement of the stage motor within the sequencer should be
audible after several seconds.
4-Return to the Mac and open StackSS .
This Hypercard document helps the operator create the Sample Sheet and adjust the Settings needed for proper Data Collection and Analysis.
5-Choose the project number from the project pull-down menu matching the project number on the sample plate. The window below the pull-down menus should list all the available plates from that project. If the matching plate number is listed in that window, select it, click on " Choose Plate", and skip to step 7. If the desired plate is not listed, click on " Make Plate".
6-Create the desired sequencing plate number by using the project, plate and run number pull-down menus. Once all numbers are correct, click on " Save in Database". Once the plate has been created in the database, click on " Return to StackSS".
7-Once the correct plate has been chosen, use the remaining pull-down menus to specify the following:
8-Click on "Start Data Collection ." The subsequent window asks if you wish to begin running the gel, click on " yes".
9-The Data Collection control window should appear on the left side of the screen. Double check that the 373 is running and start collection by clicking on the green "Collect" button. That button should blink indicating that col lection has begun, and the timer below the button should begin counting down from the run length entered at step 7.
If the computer states that there is insufficient memory, the program will fail to commence collecting. Simply return to the Finder and erase the oldest results folder from the "Transferred" Folder. Then return to the Data Collection program and press collect.
10-Move the collection window to the far right side so that no one can acci dentally press the "Stop" button. As a quick double check, press -6. The correct sample sheet should appear on the screen. If this doesn't happen, press "Stop" and elect to Discard and Quit . Restart the Data Collection program and re-start the process.
Post-Sequencing Processing
1-Turn off the 373 Sequencer.
Every 373 sequencer needs to be turned off for at least ten minutes between each run. Sequencers can be shut down as soon as Data Col lection finishes, allowing gel take down and set up during Analysis.
2-Move the Sample sheet file and the gel file into the results folder for your gel run.
3-Double click on the Move Results application. A window will appear listing the results folders which need to be transferred. Select a results folder and click on either To Be Tracked or Needs Analysis depending on whether analysis was already run on that gel. When the transfer is complete quit from the Move Results application.
The option to move results folders to Needs Analysis exists because it is possible to stop Analysis from launching. At the end of Data
Collection, a window appears stating that in one minute Analysis will
automatically be launched. During this minute you may cancel the launch of Analysis, after which you will need to move unanalyzed files to the Needs Analysis folder on Transfer. This maneuver expedites starting the subsequent gel run because one does not have to wait for Analysis to finish on the Mac attached to the 373.
Sequencer Take-Down
1-Once the power is off, open the door on the 373 and insert the vacuum line into the top buffer reservoir. Turn on the vacuum source and close both the bleeder and drain valves. Open the valve adjacent to each sequencer to bein buffer removal.
2-When the top reservoir is empty, move the hose to the bottom reservoir.
3-Unscrew the clamps at the right and left ends of the top reservoir. Place the spacer on top of the sequencer and carry the reservoir to the sink.
The reservoirs should be rinsed out with dH 2O between runs. Dry the face of each reservoir to ensure the orange grommet forms a watertight seal for the next run.
4-Remove the gel and place in the storage rack in the sink.
5-Wipe down the sequencer wherever buffer may have spilled.
6-Using a thin metal spatula, pry open the gel plates and discard the gel in the acrylamide waste container.
Pry at either bottom corner of the gel (bottom according to its orienta tion in the sequencer). Pry gently. The plate retaining the gel may be laid flat on a washcloth (to prevent scatching) while a paper towel can be laid across the gel and pressed flat. Roll off the paper towel and the gel wil come with it.
7-Wash the plates with hot water and Alconox soap.
Wash thoroughly; acrylamide particles left on the plates increase the likelihood bubbles will form when pouring gels.
8-Place the clean plates in a rack to dry.