Method A. Sequencing Reactions for Double-Stranded Plasmid DNA
1. Denature double-stranded DNA as follows:
a. To denature plasmid DNA, the alkaline denaturation method is recommended. Transfer an appropriate volume of purified plasmid DNA (approximate 1 mg/ml) to a microcentrifuge tube and add one volume of freshly prepared alkaline solution containing 0.4 M NaOH and 0.4 mM EDTA (pH 8.0). Incubate at 37°C for 35 min.
Tip: The amount of DNA to be denatured should be in excess of the amount of DNA to be sequenced. For example, if one uses 3 to 5 fig DNA for one primer sequencing reaction, the amount of DNA for two primer sequencing reactions for both strands at opposite directions will be 6 to 7 fig. If the yield of DNA following denaturation, neutralization and precipitation is 80%, the amount of DNA used for denaturation should be 7.2 to 8.4 fig. We routinely double the amount of DNA for denaturation and measure the DNA concentration after it is precipitated. This ensures a sufficient amount of DNA for sequencing.
b. After denaturation is complete, add 0.1 volume of 3 M sodium acetate buffer (pH 5.2) to the denatured sample to neutralize the mixture.
c. Add 2 to 5 volumes of 100% ethanol to the mixture and precipitate DNA at -70°C for 15 min.
d. Centrifuge at 12,000 x g for 10 min and decant the supernatant. Wash any remaining salt from the DNA pellet with 1 ml of chilled 70% ethanol and spin down at 12,000 x g for 5 min. Dry the DNA under vacuum for 15 min.
e. Dissolve the DNA in 10 fl dd.H2O and place the tube on ice. Quickly measure the concentration of DNA using 1 to 2 f1 of the sample at A260 and A280 nm. Immediately proceed to the primer annealing reaction.
2. Carry out template-primer annealing as follows:
a. Transfer 1 to 2 mg or 1 pmol of freshly denatured plasmid DNA to a microcentrifuge tube on ice and add dd.H2O to a total volume of 7 ml. Add 2 ml of reaction buffer and 1 ml of 5'-biotinylated sequencing primer (2 to 3 pmol). This gives approximately 1:1 (template:primer) molar sto-ichiometry with a total volume of 10 ml.
DNA + dd.H2O, 7 ml 5X Reaction buffer, 2 ml 5'-Biotinylated primer, 1 ml Total = 10 ml b. Place the tube in a plastic rack or its equivalent and heat at 65°C for 2 to 3 min. Quickly transfer the rack together with the tube to a beaker or tray containing an appropriate volume of 60 to 63 °C water and then let cool slowly to <30°C or room temperature over 20 to 30 min. If one uses a heating block, the tubes can be heated at 65°C for 2 min followed by slow cooling to room temperature by turning off the heat so that the heating block will slowly cool down. When the temperature drops to <30°C, the annealing is complete. The real annealing temperature is 50 to 52°C. Some laboratories prefer to anneal at 50 to 55°C for 15 to 30 min followed by slow cooling.
a. While the annealing mixture is cooling, remove the necessary materials from a commercial sequencing kit that is usually stored at -20°C. Thaw the materials on ice.
b. For each template-primer mixture, label four microcentrifuge tubes A, G, T and C, representing ddATP, ddGTP, ddTTP and ddCTP, respectively.
c. Transfer 2.5 ml of the termination mixture of ddATP, ddGTP, ddTTP and ddCTP to the labeled tubes A, G, T, and C, respectively. Cap the tubes and keep at room temperature until use.
d. Dilute the labeling mixture fivefold as a working concentration and store on ice prior to use. For example, 2 ml of labeling mixture is diluted to total 10 ml with dd.H2O.
Tips: (1) Mix the mixture in each stock tube well by pipetting it up and down prior to removal of an appropriate amount of mixture from each tube. (2) Two sets of labeling mixtures and termination mixtures are in the sequencing kit (USB). For regular noncompression sequencing, the one labeled dGTP should be used. However, if compression appears due to G-C-rich sequences, the one labeled dITP is strongly recommended. dITP replaces the nucleotide analog dGTP. We have found that it significantly eliminates the compressed bands.
e. Dilute Sequenase version 2.0 T7 DNA polymerase (1:8 dilution) as follows:
Ice-cold enzyme dilution buffer, 6.5 ml
Pyrophosphatase, 0.5 ml
DNA polymerase stored at -20°C, 1 ml
Tips: The enzyme should not be diluted in glycerol enzyme dilution buffer if one uses TBE in the gel and in the running buffer. The diluted enzyme should be stored on ice and be used within 1 h.
4. Perform a labeling reaction when the template-primer annealing is complete.
a. Briefly spin down the annealed mixture and store on ice until use.
b. Add the following to the tube containing 10 ml of annealed mixture in the order shown:
For use of a biotinylated primer: DTT (0.1 M), 1 ml dd.H2O, 2.5 ml
Diluted Sequenase DNA polymerase, 2 ml For use of a biotinylated nucleotide DTT (0.1 M), 1 ml Diluted labeling mixture, 2.5 ml Diluted Sequenase DNA polymerase, 2 ml Tip: Add 1 ml of Mn buffer to enhance the bands close to the primer.
c. Gently mix well to avoid any air bubbles. For use of a biotinylated nu-cleotide in a labeling reaction, incubate at room temperature for 3 to 5 min. Proceed to the next step.
5. Carry out the termination reactions as follows:
a. Carefully and quickly transfer 3.5 ml of the mixture to each of the termination tubes (A, G, T, C) prewarmed at 37°C for 1 min. Mix and continue to incubate the reaction at 37°C in a heating block for 4 to 5 min.
b. Add 4 ml of stopping solution to each tube, mix and cap the tubes. Store at -20°C until use.
Tip: The samples should be electrophoresed within 4 days even when stored at -20° C. Denature the sample at 75 to 80° C for 2 to 3 min prior to loading into a sequencing gel.
5X Reaction Buffer
100 mM Dithiothreitol
5X Labeling Solution for dGTP 7.5 mM dGTP 7.5 mM dTTP 7.5 mM dCTP or dATP Biotinylated dATP or dCTP
5X Labeling Mixture for dITP 7.5 mM dITP 7.5 mM dTTP 7.5 mM dCTP or dATP Biotinylated dATP or dCTP
ddATP Termination Mixture for dGTP 80 mM dATP 80 mM dGTP 80 mM dCTP 80 mM dTTP 8 mM ddATP
ddGTP Termination Mixture for dGTP 80 mM dATP 80 mM dGTP 80 mM dCTP 80 mM dTTP 8 mM ddGTP
ddCTP Termination Mixture for dGTP 80 mM dATP 80 mM dGTP 80 mM dCTP 80 mM dTTP 8 mM ddCTP
ddTTP Termination Mixture for dGTP 80 mM dATP 80 mM dGTP 80 mM dCTP 80 mM dTTP 8 mM ddTTP
ddATP Termination Mixture for dITP 80 mM dATP 80 mM dITP 80 mM dCTP 80 mM dTTP 8 mM ddATP
ddGTP Termination Mixture for dITP 80 mM dATP 160 mM dITP 80 mM dCTP 80 mM dTTP 8 mM ddGTP
ddCTP Termination Mixture for dITP
80 mM dATP 80 mM dITP 80 mM dCTP 80 mM dTTP 8 mM ddCTP
ddTTP Termination Mixture for dITP 80 mM dATP 80 mM dITP 80 mM dCTP 80 mM dTTP 8 mM ddTTP
Sequence Extending Mixture for dGTP 180 mM dATP 180 mM dGTP 180 mM dCTP 180 mM dTTP
Sequence Extending Mixture for dITP 180 mM dATP 360 mM dITP 180 mM dCTP 180 mM dTTP
Mn Buffer (only for dGTP) 150 mM Sodium isocitrate 100 mM MnCl2
Enzyme Dilution Buffer
Glycerol Enzyme Dilution Buffer 20 mM Tris-HCl, pH 7.5 2 mM DTT 0.1 mM EDTA 50% Glycerol
Sequenase Version 2.0 T7 DNA Polymerase 13 units/ml in 20 mM KPO4, pH 7.4 1 mM DTT
Pyrophosphatase 5 units/ml in 10 mM Tris-HCl, pH 7.5 0.1 mM EDTA, pH 7.5 50% Glycerol
20 mM EDTA, pH 8.0 95% (v/v) Formamide 0.05% Bromophenol blue
0.05% Xylene cyanol FF
Method B. Sequencing Reactions for Single-Stranded DNA
The procedures in Method B are very similar to those described in Method A except for the following:
• No double-stranded DNA denaturation is mandated. Instead, the appropriate primer and template DNA can be directly annealed in an annealing reaction.
• The temperature for primer-template annealing can be 50 to 55°C in a heating block for 15 to 25 min followed by slow cooling by turning off the heat.
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