La Evaluación

The method used to determine the nucleotide sequence of a given piece of DNA was based on the one described by Murphy & Kanvanagh (1988) and required highly purified plasmid DNA in large quantities. An aliquot (20 pi) of DNA isolated as described in Section

2.2.5.3.2, or highly purified DNA (10 pg/20pl in TEqi buffer) was denatured by addition

of 1 pi of 5 M NaOH, for 5-10 min at room temperature. In the meantime the mini- gelfiltration columns for the spindialysis were prepared as follows. The base of a small Eppendorf tube (0.8 ml) was punctured using just the tip of a syringe needle (23-Gauge x 11/4) and the base of a large Eppendorf tube (1.5 ml) was punctured using the same needle but pushing it fully through. The lids of both the tubes were cut off and the small tube was placed inside the large tube which was placed in a 5 ml plastic test tube which fitted the Rodwell H-103N centrifuge. Glass beads (Ballotini no. 1), which had been acid-washed and

were stored at room temperature in TEq \ buffer, were transferred to the small tube to a

depth of 2 mm after which the tube was filled to the rim with Sepharose CL-6B which had

been prepared as a 66% (v/v) slurry in TEqj buffer, autoclaved and stored at 4°C, using a

sterile Pasteur pipette. The 5 ml plastic test tube containing the large and small Eppendorf tubes was placed in the Rodwell H-103N centrifuge and spun at 3400 g for 3 min at room temperature. The denatured DNA was then loaded on the column, the large Eppendorf tube replaced by a fresh one without a hole and the whole apparatus centrifuged again exactly as described above. Between 16-20 pi of denatured DNA was recovered, and either used directly for determination of the nucleotide sequence or frozen and stored at -20°C.

An aliquot of the denatured DNA (8 pi) was transferred to a fresh small Eppendorf tube, and 1 pi of TM consisting of 100 mM TrisCl, 100 mM MgCl2, pH 8.0 and 1.5 pi of primer at a concentration of 10 pg/ml in distilled water, were added. If more than two DNA samples were used, a TM-mix consisting of TM and primer in a ratio of 1:15 was freshly prepared and an equal volume of 2.5 pi of the TM-mix was added to each DNA sample. Annealing of the primer was established by incubation at 37°C for 15-20 min. In the meantime an aliquot

(2 pi) of each of the dideoxy-mixes, A, T, C and G, which had been prepared as outlined in Table 2 and stored at -20°C, was pipetted into a large Eppendorf tube and kept in ice-water. The Klenow-mix was prepared as follows (for 10 DNA samples): 9 pi of TM, 62 pi of distilled water, and 15 pi of a 8,3 mM [a-35s]dATP solution (specific activity ± 44.4 TBq/ mmol = 1200 Ci/mmol) to which 5 p i Klenow fragment of DNA polymerase I (5 U/pl) was added were mixed together and kept in ice-water. An aliquot of the Klenow-mix (8 pi) was added to each of the annealing reaction mixtures which had been allowed to cool to 0°C in ice-water. An aliquot of the Klenow-mix/annealing reaction (4.5 pi) was placed on the inside of the wall of each Eppendorf tube containing one of the four dideoxy mixes. The reaction was started by a 2 s spin in an Eppendorf microfuge at room temperature, and continued by incubation at 42°C for 10 min.

After 8 min, an aliquot (2 pi) of NTP-mix of 0.5 mM dNTPs (A, C, G and T)

prepared and stored as described by Maniatis et al. (1982), was placed on the inside of wall

of the Eppendorf tube. After 10 min the NTP-mix was spun to the bottom of the tube by means of an Eppendorf microfuge for 2 s at room temperature, and the incubation continued for an additional 5 min at 42°C. An aliquot (4 pi) of formamide mix consisting of 10 ml deionized formamide (formamide stirred with BDH Amberlite' monobed resin MB-3 until the pH was 7.5, vacuum-filtered through Whatman no. 1 filterpaper in a Buchner funnel to remove the resin and aliquoted into 500 pi portions which were stored at -20°C), 0.01 g bromophenolblue, 0.01 g xylenecyanol FF and 2 ml 0.5 M EDTA pH 8.0, was added, and the samples either boiled for 2 min or incubated in a heating-block at 85°C for 15 min; the

latter procedure was used to concentrate the samples. An aliquot of the sample (5 pi)

was loaded onto a polyacrylamide sequencing gel which was run and carried through the procedure described in Section 2.2.12.1.3. After drying the gel, a sensitive monitor was used to determine the distribution o f the radio activity across the gel. If the top of the gel registered 300-500 cps, an overnight exposure to X-ray film produced a strong signal.

Table2. Composition of dideoxynucleotide mixes* Solution* ** A C G T 0.5 mM d-CTP 135 pi 10 pi 200 pi 200 pi 0.5 mM d-GTP 135 pi 200 pi 10 pi 200 pi 0.5 mM d-TTP 135 pi 200 pi 200 pi 10 pi 10 mM dd-ATP 2 pi - - 10 mM dd-CTP - 8 pi - - 10 mM dd-GTP - - 16 pi - 10 mM dd-TTP - - - 53 pi TEqj buffer 397 pi 392 pi 384 pi 347 pi

* Mixes were aliquoted (22 pi) and stored at -20°C.

** The solutions were prepared in TEqi buffer and stored at -20°C.