MÉTODOS QUÍMICOS

Dideoxy-sequencing was performed by a variety of techniques, depending on the DNA under analysis, the isotope available, and the number of samples to be sequenced. PCR products were sequenced directly by cycle sequencing, using either end-labelled primers or (where appropriate) by performing a labelling step to incorporate Clones were sequenced either by cycle sequencing using ^^P- or fluorescently-labelled primers, or by incorporating a labelling step, or by the conventional method with the

incorporation of or fluorescent ATP. In all cases, the primer is annealed to a single­ stranded DNA template and then extended in the presence of the four dNTPs and one dideoxynucleotide (ddNTP). Incorporation of a dideoxynucleotide prevents further DNA synthesis, and so a fraction of the extending products will terminate at each site where the ddNTP can be incorporated. Four separate reactions, each with a different ddNTP, give the complete sequence information. Acrylamide gel electrophoresis size fractionates the products, and inclusion of a labelled nucleotide (or primer) allows the products to be visualised (by fluorescence or autoradiography) and the sequence to be read.

2.11.1 Conventional sequencing with

Conventional manual sequencing was performed with the Sequenase Version 2.0 DNA Sequencing Kit (Amersham). DNA (3-5 pg) was denatured by mixing with 0.1 volumes 2 M NaOH/2 mM EDTA and incubating at 37°C for 30 minutes. The mixture was then neutralised by adding 0.1 volumes 3 M sodium acetate, pH 4.8, and the DNA

precipitated with 2 volumes ethanol on dry ice for 15 minutes. The DNA was pelleted by centrifugation at 13,000 rpm for 10 minutes, then washed with 70% ethanol and redissolved in 7 pi ddHiO. The primer was annealed to the DNA by mixing 2 pi reaction buffer and 1 pi primer with the 7 pi denatured DNA, with warming to 65°C for 2 minutes followed by slow cooling (over 15-30 minutes) to < 35°C. The labelling reaction was set up on ice by adding 1 pi 0.1 M dithiothreitol (DTT), 2 pi labelling mix (diluted 1:4 with ddH20), 0.5 pi [a-^^S]-dATP (10 pCi/pl, 1000 Ci/mmol; ICN) and 2 pi Sequenase polymerase (diluted 1:8 in enzyme dilution buffer). This was incubated at room temperature for 2-5 minutes, then 3.5 pi of the labelling reaction was aliquoted into each of four termination tubes (G, A, T and C) containing 2.5 pi termination mix preheated to 37°C. The termination reactions were incubated at 37°C for 5 minutes then stopped by the addition of 4 pi bromophenol blue stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol EE). Products were electrophoresed through 6% denaturing acrylamide gels (section 2.12.1).

2.11.2 Conventional sequencing with fluorescently-labelled primers

Reactions were performed with the AutoRead sequencing kit (Pharmacia) with a fluorescently labelled Universal or Reverse primer (Pharmacia), which are suitable for sequencing from most common cloning vectors, including pBluescript, pGEMT and pTAg. This protocol requires clean DNA, so plasmid DNA was prepared using the Qiagen isolation procedure (section 2.9.2).

The template DNA (5-10 pg) was denatured by mixing 32 pi DNA with 8 pi 2 M NaOH and incubating at room temperature for 10 minutes. DNA precipitation was achieved by adding 7 pi 3 M sodium acetate (pH 4.8), 4 pi ddH20 and 120 pi ethanol and standing in dry ice for 15 minutes. Following centrifugation to pellet the DNA, it was washed with 70% ethanol then resuspended in 10 pi ddH20. The primer was annealed by mixing 10 pi denatured DNA, 2 pi fluorescent primer and 2 pi annealing buffer with

incubation at 65°C for 5 minutes followed by incubation at 37°C for 10 minutes then at room temperature for 10 minutes. The annealed template-primer was mixed with 1 pi extension buffer, 3 pi DMSO and 2 pi T7 DNA polymerase (diluted 1:1 with enzyme dilution buffer) then 4.5 pi aliquots were mixed with 2.5 pi ddNTP mix preheated to 37°C. These termination reactions were incubated at 37°C for 5 minutes then stopped by adding 5 pi dextran blue stop solution (formamide containing 5 mg/ml Dextran Blue 2000). Samples were electrophoresed through 6% acrylamide gels using the automated laser fluorescence sequencer (section 2.12.3).

2.11.3 Solid-phase sequencing with fluorescent dATP

Solid-phase sequencing was used to sequence PCR fragments with the automated laser fluorescence sequencer. PCR reactions were carried out with a biotinylated primer, allowing the products to be captured on magnetic beads and denatured. The single stranded product is then used as a template for the sequencing reaction, using

fluorescent dATP. Since f-dATP is light-sensitive, the reactions were kept wrapped in foil as much as possible after the fluorescent nucleotide had been added.

PCR reactions were carried out with a biotinylated primer, then the products were captured by mixing 40 pi PCR reaction with 40 pi streptavidin-coated magnetic Dynabeads (Dynal) resuspended in binding buffer (10 mM Tris HCl pH7.5, 1 mM EDTA pHS.O, 2 M NaCl and 0.1% Tween-20) according to the manufacturer’s

instructions. After incubation at room temperature for 20 minutes, the unbound material was removed by washing the beads with 40 pi binding buffer and then 40 pi TE. The DNA was denatured for 15 minutes in 10 pi freshly made 0.1 M NaOH then washed with 40 pi 0.1 M NaOH, 40 pi binding buffer and then 40 pi TE. The single stranded product, still attached to the beads, was then resuspended in 12 pi ddH2 0 and used as

the template for the sequencing reaction.

The primer (2 pi, 200 pmol) was annealed to the 12 pi single stranded DNA in the presence of 2 pi annealing buffer. The mixture was heated to 65°C for 10 minutes, then 37°C for 10 minutes, then at room temperature for 10 minutes. Once the annealing step was complete, 1 pi fluorescein-15-dATP (f-dATP; Pharmacia) and 0.5 pi T7 DNA Polymerase were added to each template tube and then immediately placed at 37°C for

10 minutes to allow the labelling step to take place. After this time, 1 pi extension buffer was added and the mixture immediately aliquoted in volumes of 3.5 pi into each of four prewarmed termination tubes containing 1 pi DMSO and 3 pi dideoxy

termination mix (ddATP, ddCTP, ddGTP or ddTTP). The termination reaction was incubated at 37°C for 5 minutes then stopped by the addition of 5 pi dextran blue stop solution. Products were stored at 4°C and then electrophoresed on the automated laser fluorescence sequencer as described (section 2.12.3).

2.11.4 Cycle sequencing with ^^P-end-Iabelled primers

2.11.4.1 Radioactive end-labelling of primers

Primers were end-labelled with [y-^^P]-ATP using T4 polynucleotide kinase which catalyses the replacement of an hydroxyl group at the 5’ end of the primer with a labelled phosphate group. Each primer was labelled in a total volume of 25 pi

containing 10 pmoles primer, 1 x forward reaction buffer, 10 Units T4 polynucleotide kinase (Gibco BRL) and 0.5 pi [y-^^P]-ATP (adenosine 5’ triphosphate; 7000 Ci/mmol, 150 pCi/pl; ICN). The reaction was incubated at 37°C for 30 minutes, then stopped by heating to 65°C for 10 minutes. Labelled primers were stored for up to one week at - 20°C.

2.11.4.2 Cycle sequencing

Cycle sequencing reactions were performed with the ThermoSequenase cycle

sequencing kit (Amersham). The reaction mix contained 12 pi template DNA (50 ng-1 pg), 2 pi reaction buffer, 2 pi ThermoSequenase DNA polymerase and 1 pi (1 pmol) end-labelled primer (see section 2.11.4.1). After mixing, 4 pi of the reaction was aliquoted into four tubes each containing 4 pi ddNTP termination mix. Each aliquot was overlaid with a drop of mineral oil then placed in the ThermaCycler for the cycling reactions, consisting of an initial dénaturation step of 94°C for 3 minutes, then 60 cycles of 55°C for 30 seconds (annealing), 72°C for 2 minutes (extension) and 94°C for 30 seconds (dénaturation). Products were mixed with 4 pi bromophenol blue stop solution and electrophoresed through 8% denaturing acrylamide gels (see section 2.12.1).

2.11.5 Cycle sequencing with

incorporation: 3-dNTP labelling

method

In this protocol, the primer is labelled by extending it in the presence of only three of the four dNTPs, one of which is radioactively labelled. Since the fourth dNTP is missing, DNA synthesis stops when it reaches that nucleotide, so the primer is extended by only a few bases. This protocol therefore requires prior knowledge of the fragment sequence just 3’ to the primer site, to determine which of the four dNTPs should be omitted, and

has the limitation that the sequence must be such that at least one labelled nucleotide can be incorporated in the labelling step. Therefore not all fragments are suitable for this method of cycle sequencing.

For example, if the base to be omitted was T, then the labelling mix contained 1 pi (0.5 pmol) primer, 2 pi reaction buffer, 10 pi (-0.1 pg) DNA, 1 pi dGTP cycle mix, 1 pi dCTP cycle mix, 0.5 pi [a-^^S]-dATP (10 pCi/pl, 1000 Ci/mmol; ICN) and 2 pi ThermoSequenase. The mix was overlayed with mineral oil and cycled with the program: 95°C 15 s, 60°C 30 s, for 50 cycles. The labelling mix was then split into 3.5 pi aliquots and each mixed with 4 pi ddNTP termination mix, then subjected to the cycling program: 95°C 30 s, 72°C 120 s, for 50 cycles. The reaction was stopped with the addition of 4 pi bromophenol blue stop solution and the products electrophoresed through 6% acrylamide gels (section 2.12.1).

2.11.6 Cycle sequencing reactions with fluorescent primers

Reactions were performed with the ThermoSequenase fluorescent labelled primer cycle sequencing kit (Amersham). Plasmid DNA was prepared using the Wizard isolation procedure (section 2.9.1). For each template, four tubes were set up, each containing 5 pi (0.1-1 pg) template DNA, 1 pi fluorescent primer (Pharmacia) and 2 pi A, C, G or T sequencing reagent. The reaction mix was overlaid with a drop of mineral oil then placed in a ThermalCycler for the cycling program consisting of an initial dénaturation step of 95°C for 5 min, followed by 25 cycles of 95°C for 30 sec (dénaturation) then 60°C for 30 sec (annealing and extension).

Reaction products were separated from unincorporated primer and reaction buffer prior to electrophoresis. The mineral oil was removed by trickling the mixture down a slope

of Nesco film, then the DNA was precipitated by the addition of 1/10 volume 3 M sodium acetate and 2 volumes 100% ethanol. After incubation at room temperature for 5 minutes, the DNA was pelleted by centrifugation for 10 minutes at 13,000 rpm at 4°C, washed gently with 80% ethanol then resuspended in 5 pi dextran blue stop solution. The reaction products were analysed by electrophoresis on the automated laser fluorescence sequencer (section 2.12.3).

2.12 ACRYLAMIDE GEL ELECTROPHORESIS