B33 Preparing and Using a Sequencing Gel
To determine the base sequence of a DNA fragment.
A piece of DNA to be sequenced must be available.
A single strand of DNA may be copied. Suppose that a strand of 150 bases is being copied. A complete copy of the strand would be a complementary strand 150 bases long. If the strand copying process could be interrupted randomly, some chains would be two bases long, others three bases long,..., others 149 bases long, and still others a full 150 bases long. Electrophoresis on a denaturing polyacrylamide gel allows the separation of strands according to molar mass. A mixture of strands varying in length from 2 to 150 bases may be resolved during urea-polyacrylamide gel electrophoresis. Addition of dideoxyribonucleotides to a chain synthesizing mixture terminates chain growth. Dideoxyribonucleotides have no available hydroxyl groups in the 3'-deoxyribosyl position. They cannot sustain polymerization of DNA, which requires the linking of 3'- and 5'- positions of neighbors through phosphodiester links. Addition of a small amount of dideoxyadenine triphosphate to a chain-synthesiszing-mixture causes a chain mixture to be produced in which all (or nearly all) possible chains terminating with adenine will be produced. This assumes that the dideoxyadenine triphosphate randomly replaces dATP and terminates chain growth. Addition of a small amount of dideoxycytosine triphosphate to a chain-synthesiszing-mixture causes a chain mixture to be produced in which all (or nearly all) possible chains terminating with cytosine will be produced. Suppose four different reaction mixtures are prepared, one each containing a small amount of one of the 4 possible dideoxyribonucleotides. Each will produce a mixture of chains. Mixing all four resulting chain mixtures would give all possible chains. There would be a band in the electrophoretogram for each chain. If each chain were somehow labelled, then these bands could be detected. Radioactive labelling and autoradiography are usually employed. Performing the reactions separately, however, provides an incredibly clever means of determining base sequence. By running 4 channels side-by-side, a sort of stair stepping of the final bands appears. Each step appears in only one channel. If adenine ends a chain, then there is no corresponding chain ended by cytosine or guanidine or thymidine. The four channels provide a means of "reading," directly, the base sequence.
- Scour the plates. Rinse with tap water. Rinse with distilled water. Water beading is intolerable. Repeat until absolutely no water beading is detected. Wipe using a lint-free towel saturated with 95% ethanol. Wipe one side of the plate with a siliconizer. Place two 0.3 mm-spacers on the siliconized side of a glass plate. Set a second glass plate on top of the spacers. The top plate is slightly smaller and has a beveled edge. The gel will be cast between the 2 plates. Set the bevel so that a "Vee" slot forms from this sandwich. Three edges of the sandwich are taped. Proper alignment prevents leaking. The tape must fit very tightly.
- Acrylamide is toxic. Wear a mask, goggles, and gloves when preparing the acrylamide stock solution. Wear gloves to avoid contact with the acrylamide solution. Prepare a gel casting mixture according to a selected protocol. Usual ingredients include buffer, acrylamide, urea solution, ammonium persulfate, and N,N'-methylene-bis-acrylamide as a crosslinker. Filter the solution and keep under vacuum for deaeration. N,N,N',N'Ñ-tetramethylethylenediamine (TEMED) is added to catalyze the polymerization.
- Prop the plate sandwich nearly vertically. Apply two large binder clips to the bottom of each side. Draw the gel mixture into the syringe barrel and inject it into the space between the plates. Remove the plates from the near vertical position and set on a prop in a near horizontal position. (Note that liquid emerges when the plates are lowered.)
- A comb will cause wells to form in the gel. Insert the comb between the plates at the top. Allow the gel to harden.
- Remove the comb. Remove the bottom tape. Insert foam pads in the top if needed. Place the gel sandwich vertically in the electrophoresis apparatus. Fasten bottom clamps. Install and fasten side clamps. Fill the tank with TBE buffer. Apply Radioactivity Hazard tape in anticipation of filling the wells with labelled material. Use a Pasteur pipet to flush out the wells. Complete the tank assembly.
- Connect the power cords to the tank and to the power supply. Turn on the power. "Pre-run" for 2 hours at 1,700 volts to prepare gel, to remove ammonium persulfate, and to heat the buffer. The temperature will rise to approximately 60°C during the electrophoresis.
- Turn off the power. Disconnect the power cords. Remove the tank cover. Treat the radioactive sample with a loading buffer solution that contains two dyes (bromphenol blue and xylene cyanol), glycerol, EDTA, and buffer. Add a sample to each well. Reinstall the tank cover. Reconnect the power cords. Follow a suitable protocol for determining voltage and duration. Turn on the power. Apply power until the blue dye boundary has moved the full length of the gel. Turn off the power. Remove the power cords. Remove the tank cover.
- Loosen the side clamps. Loosen the bottom clamps; the buffer will drain to the bottom of the tank. Remove the side clamps. Remove the plates. Remove the tape from each side. Use a PVC wedge to loosen the short plate. The purpose of the siliconizing is to facilitate this step. Remove the top plate. The gel remains on the bottom plate.
- Remove the plate with the gel. Cover the gel with a large sheet of Whatman 3MM paper. Press gently to smooth out. Lift the gel from the bottom plate onto this paper. Place the paper supporting the gel on the gel dryer. Follow the dryer manufacturer's instructions. Cover the paper with plastic wrap to prevent radioactive material from contaminating the dryer. Place a hard plastic sheet on top. Cover with the dryer lid. Vacuum dry the gel at 60°C for about 90 minutes.
- Once dried, examine the gel by autoradiography. In a dark room, the gel, wrapped in plastic wrap, is placed in contact with a suitably-sized piece of X-ray film, and left in place for a period of one to several days at -70°C in a cassette. Reduce exposure time by using intensifier screens. (An alternative procedure based upon Ü&Mac222;S is becoming more common because it is safer, and the isotope has a more suitable half-life, and the resolution is improved. The label is introduced via deoxyadenosine triphosphate in which an alpha-phosphate oxygen has been replaced by Ü&Mac222;S. Gels used for the alternative procedure are fixed to remove urea and prevent quenching. Intensifier screens are not employed.)
- Learn to distinguish good-quality from poor-quality autoradiographs. See the movie for examples.
- Read the base sequence from the autoradiograph. The gel wells are always filled, from left to right, with the dideoxycompounds for adenine, cytosine, guanine, and thymidine.
- Instruments that use a different strategy for sequencing are becoming available. These often involve labelling the chain terminus with characteristic fluorescent compounds for A, C, G, and T, and detecting the fluorescence of chains as they emerge from a suitable chromatography column.
- This completes a DNA sequencing cycle.