DNA测序实验方法步骤
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Polyacrylamide gels for fluorescent DNA sequencing are prepared as described above except that the gel mix is filtered prior to polymerization. Optically-ground, low fluorescence glass plates are carefully cleaned with hot water, distilled water, and ethanol to remove potential fluorescent contaminants prior to taping.
Denaturing 6% polyacrylamide gels are poured into 0.3 mm X 89 cm X 52 cm taped plates and fitted with 36 well forming combs.
After polymerization, the tape and the comb are removed from the gel and the outer surfaces of the glass plates are cleaned with hot water, and rinsed with distilled water and ethanol.
The gel is assembled into an ABI sequencer, and the checked by laser-scanning. If baseline alterations are observed on the ABI-associated Macintosh computer display, the plates are recleaned. Subsequently, the buffer wells are attached, electrophoresis buffer is added, and the gel is pre-electrophoresed for 10-30 minutes at 30 W.
Prior to sample loading, the pooled and dried reaction products are resuspended in formamide/EDTA loading buffer by vortexing and then heated at 90degC. A sample sheet is created within the ABI data collection software on the Macintosh computer which indicated the number of samples loaded and the fluorescent labeled mobility file to use for sequence data processing. After cleaning the sample wells with a syringe, the odd-numbered sequencing reactions are loaded into the respective wells using a micropipettor equipped with a flat-tipped gel-loading tip. The gel then is electrophoresed for 5 minutes before the well are cleaned again and the even numbered samples are loaded. The filter wheel used for dye-primers and dye-terminators is specified on the ABI 373A CPU, also where electrophoresis conditions are adjusted. Typically electrophoresis and data collection are for 10 hours at 30W on the ABI 373A that is fitted with a heat-distributing aluminum plate in contact with the outer glass gel plate in the region between the laser stop and the sample loading wells (26).
After data collection, an image file is created by the ABI software which related the fluorescent signal detected to the corresponding scan number. The software then determined the sample lane positions based on the signal intensities. After the lanes are tracked, the cross-section of data for each lane are extracted and processed by baseline subtraction, mobility calculation, spectral deconvolution, and time correction. On the Macintosh computer, the collected data can be viewed in several formats. The overall graphics image of the gel can be displayed to assess the accuracy of lane tracking, and the data from each sample lane can be viewed as either a four-color raw fluorescent signal versus scan number, as a chromatogram of processed sequence data, or as a string of nucleotides. After processing, the sequence data files are transferred to a SPARCstation 2 using NFS Share.
Protocol
1. Prepare 8 M urea, 4.75% polyacrylamide gels, as described above, using a 36-well forming comb. Alternatively, the recipe can be scaled up to one liter.
2. Prior to loading, remove the tape from around the entire gel and carefully clean the outer surface of the gel plates with hot water. Rinse the glass with distilled water and then with ethanol, and allow the ethanol to evaporate.
3. Assemble the gel plates into an ABI 373A DNA Sequencer by placing the plates on the ledge in the bottom buffer well and clamping the gel into place with the black clamps attached to the laser stop.
4. Check the glass plates by closing the ABI lid and selecting "Start Pre-run" and then "Plate Check" from the ABI display. Adjust the PMT on the ABI display ("Calibration", "PMT") so that the lower scan (usually the blue) line corresponds to an intensity value of 800-1000 as displayed on the Macintosh computer data collection window. If the baseline of four-color scan lines is not flat, reclean the glass plates.
4. Attach the top buffer and the alignment brace, and fill both buffer wells with 1X MTBE electrophoresis buffer. Affix the aluminum heat distribution plate by setting it on the laser stop against the glass plates.
5. Pre-electrophorese the gel for 10-30 minutes by choosing "Start Pre-run" and "Pre-run Gel".
6. Use MakeSampleSheetOU to create a sample sheet or do this from within the ABI data collection software by entering the names and the fluorescent mobility file ("b920_21.mob" for fluorescent-labeled M13 -21 universal forward primer, "DyePrimer{M13RP1}" for fluorescent-labeled M13 universal reverse primer, "DyeTerm {any primer}" for AmpliTaq Terminators, and "DyeTerm{T7}-SetB" for Sequenase[TM] fluorescent-labeled dye terminators) to use for analysis. This Macintosh program and the related files are available from our ftp site at ftp://ftp.genome.ou.edu/ as a stuffit 1.5.1, binhexed file.
7. Prepare the samples for loading. Add 3 ul of FE to the bottom of each tube, vortex, heat at 90degC for 3 minutes, and centrifuge to reclaim condensation.
