Bst DNA polymerase-catalyzed radiolabeled two-step sequencing reactions (26) are modified from those presented earlier (25) by altering the absolute amounts and the relative deoxy/dideoxynucleotide ratios in the termination mixes. Two separate termination mixes provided optimal overlap for sequence data starting in the polylinker and extending to approximately 600 bases from the priming site. This two-step format eliminated the need for the chase required in the Bst one-step reaction (25).
Each extension reaction contained 500-750 ng of Biomek isolated single-stranded DNA, reaction buffer, nucleotide extension mix, oligonucleotide primer (typically M13 (-40) universal sequencing primer, see Appendix D), either [a-32-P]dATP or [a-35-S]dATP and Bst polymerase. After the reactions are extended for 2 minutes at 67deg C and briefly centrifuged, four aliquots are removed and added to the appropriate base-specific termination mix. All nucleotide mixes contained the guanosine nucleotide analog, 7-deaza-dGTP, but differed in their deoxy/dideoxynucleotide ratios to yield fragments ranging in size from the beginning of the polylinker to greater than 300 bases from the primer, or fragments from about 150 to greater than 600 bases from the primer for "short" or "long" mixes, respectively. Following an incubation at 67degC for 10 min and a brief centrifugation, the reactions are stopped by the addition of dye/formamide/EDTA, and incubated at 100degC. When desired, sequencing reactions are stored at -70degC prior to the addition of loading dye.
When double-stranded pUC-based subclones are used as templates, the amount of primer is doubled and a denaturing/annealing step is added. Here, 3 ug of plasmid DNA, isolated by either the mini- or midi-prep diatomaceous earth method, is mixed with primer, placed in a boiling-water bath, and rapidly cooled by plunging into an ethanol/dry-ice bath (28). Following an incubation on ice, the remaining sequencing extension reagents (reaction buffer, nucleotide extension mix, either [a-32-P]dATP or [a-35-S]dATP, and Bst polymerase) are added. Reactions are performed as described above for single-stranded sequencing.
Protocol
For single-stranded DNA sequencing:
1. Prepare the following extension reaction in a microcentrifuge tube:
750 ng M13 template DNA 2 ul Bst reaction buffer 2 ul Bst nucleotide extension mix 1 ul oligonucleotide primer (2.5 ng/ul) 0.5-1 ul [alpha]-32-P-dATP or [alpha]-35-S-dATP 1 ul diluted Bst polymerase (0.1 U/ul) q.s. sterile ddH2O 12 ul[alpha]-32-P-dATP (PB 10384) and [alpha]-35-S-dATP (SJ 1304) from Amersham.
Dilute the Bst polymerase (BioRad 170-3406) in Bst dilution buffer.
2. Incubate the reactions for 2 minutes at 67degC, and briefly centrifuge to reclaim condensation.
3. Remove 2.5 ul aliquots for each reaction into the four base-specific termination mixes (either short or long), already pipetted into a V-bottomed microtiter plate (Dynatech).
4. Incubate the reactions for 10 minutes at 67degC, and briefly centrifuge to reclaim condensation. It is possible to store the reactions at -70degC at this stage.
5. Stop the reactions by the addition of 4 ul of dye/formamide/EDTA and incubate for 5-7 minutes at 100degC.
For double-stranded DNA sequencing:
1. To denature the DNA and anneal the primer, incubate the following reagents in a boiling water bath for 4-5 minutes and rapidly cool the reaction by plunging into an ethanol/dry ice bath.
3 ug plasmid DNA 5 ng oligonucleotide primer q.s. sterile ddH2O 9 ul2. Incubate the reaction in an ice-water bath for 5 minutes, and then add the following reagents:
2 ul Bst reaction buffer 2 ul Bst nucleotide extension mix 0.5-1 ul [alpha]-32-P-dATP or [alpha]-35-S-dATP 1 ul diluted Bst polymerase (0.1 U/ul) 15 ul[alpha]-32-P-dATP (PB 10384) and [alpha]-35-S-dATP (SJ 1304) from Amersham.
Dilute the Bst polymerase (BioRad 170-3406) in Bst dilution buffer.
3. Proceed with the sequencing reaction as described above in steps 2-5 for single-stranded templates
To prepare polyacrylamide gels for DNA sequencing, the appropriate amount of urea is dissolved by heating in water and electrophoresis buffer, the respective amount of deionized acrylamide-bisacrylamide solution is added, and ammonium persulfate and TEMED are added to initiate polymerization. Immediately after the addition of the polymerizing agents, the gel solution is poured between two glass plates, taped together and separated by thin spacers corresponding to the desired thickness of the gel, taking care to avoid and eliminate air bubbles. Prior to taping, these glass plates are cleaned with Alconox detergent and hot water, are rinsed with double distilled water, and dried with a Kimwipe. Typically, the notched glass plate is treated with a silanizing reagent and then rinsed with double distilled water. After pouring, the gel immediately is laid horizontally and a well forming comb is inserted into the gel and held in place by metal clamps. The polyacrylamide gels are allowed to polymerize for at least 30 minutes prior to use. After polymerization, the comb and the tape at the bottom of the gel are removed. The vertical electrophoresis apparatus is assembled by clamping the top and bottom buffer wells onto the gel, and adding running buffer to the buffer chambers. The wells are cleaned by circulating buffer into the wells with a syringe and, immediately prior to the loading of each sample, the urea in each well is suctioned out with a mouth pipette.
Each base-specific sequencing reaction terminated with the short termination mix is loaded using a mouth pipette onto a 0.15 mm X 50 cm X 20 cm, denaturing 5% polyacrylamide gel and electrophoresed for 2.25 hours at 22 mA. The reactions terminated with the long termination mix typically are divided in half and loaded onto two 0.15 mm X 70 cm X 20 cm denaturing 4% polyacrylamide gels. One gel is electrophoresed at 15 mA for 8-9 hours and the other is electrophoresed for 20-24 hours at 15 mA. After electrophoresis, the glass plates are separated and the gel is blotted to Whatman paper, covered with plastic wrap, dried by heating on a Hoefer vacuum gel drier, and exposed to X-ray film. Depending on the intensity of the signal and whether the radiolabel is 32-P or 35-S, exposure times varied from 4 hours to several days. After exposure, the films are developed by processing in developer and fixer solutions, rinsed with water, and air dried. The autoradiogram then is placed on a light-box and the sequence is manually read and the data typed into a computer.
Protocol
1. Prepare 8 M urea, polyacrylamide gels according to the following recipe (100 ml), depending in the desired percentage:
4% 5% 6% urea 48 g 48 g 48 g 40% A & B 10 ml 12.5 ml 15 ml 10X MTBE 10 ml 10 ml 10 ml ddH2O 42 ml 39.5 ml 37 ml 15% APS 500 ul 500 ul 500 ul TEMED 50 ul 50 ul 50 ulUrea (5505UA) is from Gibco/BRL.
2. Combine the urea, MTBE buffer, and water and incubate for 5 minutes at 55deg C and then stir to dissolve the urea.
3. Cool briefly, add the A & B, mix, and then filter the gel mix through a 0.2 micron filter.
4. While stirring, add the APS and TEMED polymerization agents and then immediately pour in between two taped glass plates with 0.15 mm spacers. (Prior to taping, the notched, front glass plate should be treated with a small amount of silanizing reagent and then rinsed with ddH2O).
5. Insert the well forming comb, clamp, and allow the gel to polymerize for at least 30 minutes.
6. Prior to loading, remove the tape around the bottom of the gel and the well-forming comb. Assemble the vertical electrophoresis apparatus by clamping the upper and lower buffer chambers to the gel plates, and add 1X MTBE electrophoresis buffer to the chambers.
7. Flush the sample wells with a syringe containing running buffer, and immediately prior to loading each sample, flush the well with running buffer using the mouth pipette.
9. Load 1-2 ul of sample into each well using a mouth pipettor equipped with drawn capillary tube tips, and then electrophorese according the following guidelines (during electrophoresis, cool the gel with a fan):
termination electrophoresis reaction polyacrylamide gel conditions short 5%, 0.15 mm X 50 cm X 20 cm 2.25 hours at 22 mA long 4%, 0.15 mm X 70 cm X 20 cm 8-9 hours at 15 mA long 4%, 0.15 mm X 70 cm X 20 cm 20-24 hours at15 mA10. After electrophoresis, remove the buffer wells, the tape, and pry the gel plates apart. The gel should adhere to back plate. Blot the gel to a 40 cm X 20 cm sheet of 3MM Whatman paper, cover with plastic wrap, and dry on a Hoefer gel fryer for 25 minutes at 80degC
11. Place the dried gel in a cassette and expose to Kodak XRP-1 film.
12. Develop the film for 1-5 minutes in Kodak GBX developer, rinse in distilled water for 30 seconds, fix in Kodak GBX fixer for 5 minutes, and then rinse again in distilled water for 30 seconds. Allow the film to air dry.
Each base-specific fluorescent-labeled cycle sequencing reaction routinely included approximately 100 or 200 ng Biomek isolated single-stranded DNA for A and C or G and T reactions, respectively. Double-stranded cycle sequencing reactions similarly contained approximately 200 or 400 ng of plasmid DNA, isolated using either the standard alkaline lysis or the diatomaceous earth modified alkaline lysis procedures. All reagents except template DNA are added in one pipetting step from a premix of previously aliquotted stock solutions stored at -20degC (see Appendix B). To prepare the reaction premixes, reaction buffer is combined with the base-specific nucleotide mixes. Prior to use, the base-specific reaction premixes are thawed and combined with diluted Taq DNA polymerase and the individual fluorescent end-labeled universal primers (see Appendix C) to yield the final reaction mixes, that are sufficient for 24 template samples.
Once the above mixes are prepared, four aliquots of single or double-stranded DNA are pipetted into the bottom of each 0.2 ml thin-walled reaction tube, corresponding to the A, C, G, and T reactions, and then an aliquot of the respective reaction mixes is added to the side of each tube. These tubes are part of a 96-tube/retainer set tray in a microtiter plate format, which fits into a Perkin Elmer Cetus Cycler 9600. Strip caps are sealed onto the tube/retainer set and the plate is centrifuged briefly. The plate then is placed in the cycler whose heat block had been preheated to 95deg C, and the cycling program immediately started. The cycling protocol consisted of 15-30 cycles of seven-temperatures:
At this stage, the reactions frequently are frozen and stored at -20degC for up to several days. Prior to pooling and precipitation, the plate is centrifuged briefly to reclaim condensation. The primer and base-specific reactions are pooled into ethanol, and the DNA is precipitated and dried. These sequencing reactions could be stored for several days at -20degC.
Protocol
1. Pipette 1 or 2 ul of each DNA sample (100 ng/ul for M13 templates and 200 ng/ul for pUC templates) into the bottom of the 0.2 ml thin-walled reaction tubes (Robbins Scientific). Use the 1 ul sample for A and C reactions, and the 2 ul sample for G and T reactions. Meanwhile, preheat the PE Cetus Thermocycler 9600 to 95degC (Program #2).
2. Prepare the Taq polymerase dilution. AmpliTaq polymerase (N801-0060) is from Perkin-Elmer Cetus.
30 ul AmpliTaq (5U/ul) 30 ul 5X Taq reaction buffer 130 ul ddH20 190 ul diluted Taq for 24 clones3. Prepare the A, C, G, and T base specific mixes by adding base-specific primer and diluted Taq to each of the base specific nucleotide/buffer premixes:
A,C/G,T 60/120 ul 5X nucleotide extn/term/5X Taq reaction buffer premix 30/60 ul diluted Taq polymerase 30/60 ul respective fluorescent end-labeled primer 120/240 ul4. Seal the reaction tubes carefully with the strip caps, and centrifuge briefly at 2500 rpm. Place the tube/retainer set in the 9600 Cycler, abort the soak file program, and run program #11. This program will cycle the sequencing reactions for 30 cycles, and then will link to a 4degC soak file until that program is aborted. (It is possible to freeze the reactions at -20degC after cycling, prior to the pooling step).
5. Briefly centrifuge the plate to reclaim condensation. Pool the four base specific reactions into 250 ul of 95% ethanol.
6. Precipitate the sequencing reactions, and store the dried samples at -20deg C.
Single-stranded dye-terminator reactions required approximately 2 ug of phenol extracted M13-based template DNA. The DNA is denatured and the primer annealed by incubating DNA, primer, and buffer at 65degC. After the reaction cooled to room temperature, alpha-thio-deoxynucleotides, fluorescent-labeled dye-terminators, and diluted Sequenase[TM] DNA polymerase are added and the mixture is incubated at 37degC. The reaction is stopped by adding ammonium acetate and ethanol, and the DNA fragments are precipitated and dried. To aid in the removal of unincorporated dye-terminators, the DNA pellet is rinsed twice with ethanol. The dried sequencing reactions could be stored up to several days at -20degC.
Double-stranded dye-terminator reactions required approximately 5 ug of diatomaceous earth modified-alkaline lysis midi-prep purified plasmid DNA. The double-stranded DNA is denatured by incubating the DNA in sodium hydroxide at 65degC, and after incubation, primer is added and the reaction is neutralized by adding an acid-buffer. Reaction buffer, alpha-thio-deoxynucleotides, fluorescent-labeled dye-terminators, and diluted Sequenase[TM] DNA polymerase then are added and the reaction is incubated at 37degC. Ammonium acetate is added to stop the reaction and the DNA fragments similarly are precipitated, rinsed, dried, and stored.
Protocol
For Single-stranded reactions:
1. Add the following to a 1.5 ml microcentrifuge tube:
4 ul ss DNA (2 ug) 4 ul 0.8 uM primer 2 ul 10x MOPS buffer 2 ul 10x Mn[2+]/isocitrate buffer 12 ul2. To denature the DNA and anneal the primer, incubate the reaction at 65-70deg C for 5 minutes. Allow the reaction to cool at room temperature for 15 minutes, and then briefly centrifuge to reclaim condensation.
3. To each reaction, add the following reagents and incubate for 10 minutes at 37degC. (For more than one reaction, a pot of the reagents should be made).
7 ul ABI terminator mix (401489) 2 ul diluted Sequenase[TM] (3.25 U/ul) 1 ul 2 mM a-S dNTPs 22 ulThe undiluted Sequenase[TM] (70775) from United States Biochemicals is 13 U/ul and should be diluted 1:4 with USB dilution buffer prior to use resulting in a working dilution of 3.25 U/ul.
4. Add 20 ul 9.5 M ammonium acetate and 100 ul 95% ethanol to stop the reaction and vortex.
5. Precipitate the DNA in an ice-water bath for 10 minutes. Centrifuge for 15 minutes at 12,000 rpm in a microcentrifuge at 4degC. Carefully decant the supernatant, and rinse the pellet by adding 300 ul of 70-80% ethanol. Vortex and centrifuge again for 15 minutes, and carefully decant the supernatant.
6. Repeat the rinse step to insure efficient removal of the unincorporated terminators. (Alternatively, after the first rinse step, droplets of supernatant can be removed by carefully absorbing them with a Q-tip cotton swab or a rolled up Kimwipe).
7. Dry the DNA for 5-10 minutes (or until dry) in the Speedy-Vac, and store the dried reactions at -20degC.
For double-stranded reactions:
1. Add the following to a 1.5 ml microcentrifuge tube:
5 ul ds DNA (5 ug) 4 ul 1 N NaOH 3 ul ddH2O 12 ul2. Incubate the reaction at 65-70degC for 5 minutes, and then briefly centrifuge to reclaim condensation.
3. Add the following reagents to each reaction, vortex, and briefly centrifuge:
3 ul 8 uM primer 9 ul ddH2O 4 ul MOPS-Acid buffer 28 ul4. To each reaction, add the following reagents and incubate for 10 minutes at 37degC. (For more than one reaction, a pot of the reagents should be made).
4 ul 10X Mn[2+]/isocitrate buffer 6 ul ABI terminator mix 2 ul diluted Sequenase[TM] (3.25 U/ul) 1 ul 2 mM [alpha]-S-dNTPs 22 ulThe undiluted Sequenase[TM] from United States Biochemicals is 13 U/ul and should be diluted 1:4 with USB dilution buffer prior to use resulting in a working dilution of 3.25 U/ul.
5. Add 60 ul 8 M ammonium acetate and 300 ul 95% ethanol to stop the reaction and vortex.
6. Precipitate the DNA in an ice-water bath for 10 minutes. Centrifuge for 15 minutes at 12,000 rpm in a microcentrifuge at 4degC. Carefully decant the supernatant, and rinse the pellet by adding 300 ul of 70-80% ethanol. Vortex and centrifuge again for 15 minutes, and carefully decant the supernatant.
7. Repeat the rinse step to insure efficient removal of the unincorporated terminators. (Alternatively, after the first rinse step, droplets of supernatant can be removed by carefully absorbing them with a Q-tip cotton swab or a rolled up Kim-wipe).
8. Dry the DNA for 5-10 minutes (or until dry) in the Speedy-Vac.
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 wells 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, 6% 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, and "DyeTerm{T7}-SetB" for Sequenase[TM] fluorescent-labeled dye terminators) to use for analysis.
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.
8. Abort the pre-electrophoresis, and flush the sample wells with electrophoresis buffer with a syringe. Using flat-tipped gel loading pipette tips, load each odd-numbered sample. Pre-electrophorese the gel for at least 5 minutes, flush the wells again, and then load each even-numbered sample.
9. Begin the electrophoresis (30 W for 10 hours) run by selecting "Start Run" on the ABI display and by choosing "Begin Data Collection" from the controller box within the ABI data collection software on the Macintosh.
10. After data collection, the ABI software will automatically open the data analysis software, which will create the imaged gel file, extract the data for each sample lane, and process the data. Check the imaged gel file for sample tracking, and then transfer the results folder containing the sequence trace files to a SPARCstation 2 where the hard disk is mounted on the ether-netted Macintosh computer via NFS Share.