Sanger Sequencing


The dideoxy chain termination DNA sequencing procedure has the advantage of being fast, simple to perform, and very accurate.  The procedure is based on the enzymatic elongation of oligonucleotides that are complementary to the single-stranded DNA template. Labeling of the synthesis products is achieved either by incorporation of radioactively or fluorescently labeled nucleotides or by extending primers labeled respectively. Chain extension competes with the infrequent but specific termination by incorporation of a dideoxyribo nucleotide. The products of four nucleotide-specific reactions can be separated on a polyacrylamide gel. In the case of a radioactive labeling system, an autoradiogram of such a gel provides the sequence information; fluorescently labeled fragments are used in automated sequencers where a laser beam serves as the detector; biotinylated fragments can be detected after transfer to a nylon membrane through chemoluminescence.  The synthetic oligonucleotide primers required for the synthesis of the labeled strands can easily be synthesized in automatic synthesizers.

In principle, there are two different ways of obtaining DNA sequence information by the dideoxynucleotide method. The first strategy involves the creation of a set of subclones to bring different areas of the DNA fragment to be sequenced in the proximity of the primer binding site of the vector. This allows for sequencing of the respective fragment with one oligonucleotide and can be achieved using different subcloning strategies. One possibility would be random subcloning after physically breaking up the original fragment (e.g., by shearing). This methodology requires a high number of recombinant clones, many time-consuming and costly sequencing reactions, and a computer for putting the data together. Alternatively, defined subclones can be constructed after restriction mapping the area of DNA to be sequenced, characterized, and finally sequenced. This approach is more straightforward, but also takes a large amount of work and time. As a third possibility, a set of nested deletions can be introduced into the original DNA fragment, originating close to the primer binding site. These nested deletions extend various lengths along the target DNA, thus bringing the area next to the deletion into sequencing range. Again, this procedure requires additional recombinant DNA work on the original clone and the preparation of many DNA templates.

The second approach is based on starting sequencing with primers that are complementary to a known sequence. This can either be the vector or any other sequence adjacent to the DNA to be analyzed. Sequencing proceeds by successive synthesis of new primers at the edges of the newly obtained sequence in such a way that their 3′ ends are pointing off into the unknown target DNA (“primer walking method”). This method has the advantage of not requiring the subcloning procedures. The sequencing work can be done on the original clone. The design of the second primer depends on the result of the first sequencing reaction. For one individual plasmid, only one sequencing reaction can be performed in one direction at a time. Therefore, it is often advisable to produce a small set of subclones and sequence these from both ends simultaneously using the “primer walking method.”



Various primers for a number of commonly used cloning vectors are commercially available. For sequencing, oligonucleotides should be deprotected. High-performance liquid chromatography (HPLC) purification is not necessary, as the coupling efficiency of the synthesis process lies between 98 and 99.5%. Oligonucleotides are generally delivered in a lyophilized state with information about the amount and instructions for the preparation of a solution of a certain molarity (see Notes 1 and 2). The concentration of a primer in the sequencing reaction should be approx 1 μM (corresponding to 5.6 μg/mL for a 17-mer). However, even much higher concentrations have been used successfully.

Template Preparation (Double-Stranded Plasmid DNA)

  1. Lysozyme solution: 50 mM glucose, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0). This solution should be filter-sterilized and stored at 4°C. Prior to use, crystalline lysozyme is added (5 mg/mL) and completely dissolved (see Note 3).
  2. Sodium dodecyl sulfate (SDS) solution: SDS (1%, [w/v]) in NaOH (0.2M). This solution should not be stored longer than 1 wks. Store at room temperature.
  3. Sodium acetate solution: 3 M sodium acetate, pH 5.2. This solution is prepared by dissolving 246.1 g of water-free CH3COONa in 800 mL of H2O, adjusting the pH to 5.2 with glacial acetic acid, and then bringing the volume to 1 L. Store at room temperature after sterilization by autoclaving.
  4. Phenol-chloroform solution: Melt crystalline phenol at approx 65°C. Only distilled phenol should be used. Equilibrate the liquid with 0.5 M Tris-base, let phases separate, and remove aqueous phase. Add 50 mM Tris-HCl, pH 8.0, and repeat the last three steps until the pH of the phenol solution is approx 8.0. One volume of chloroform solution is then mixed with one volume of the equilibrated phenol solution to obtain the final reagent. Store in a brown glass bottle (if possible, under a nitrogen atmosphere) at 4°C up to 1 mo.
  5. Ethanol: 96 and 70% (v/v).
  6. TE buffer: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 7.5.
  7. RNase solution: 10 mM Tris-HCl, pH 7.5, 15 mM NaCl, RNase A (10 mg/mL). Dissolve enzyme completely and keep the solution at 100°C for 15 min to inactivate possible DNase contamination. Let cool to room temperature, divide into small aliquots, and store at –20°C.
  8. Chloroform solution: Mix chloroform and isoamyl alcohol in a ratio of 24:1.

Sequencing Reactions

Primer Annealing

  1. Template: single- or double-stranded DNA, 0.25 μg/μL.
  2. NaOH solution: 2 M NaOH.
  3. Sodium acetate solution: 3 M sodium acetate, pH 4.5.
  4. Ethanol: 96 and 70% (v/v).
  5. Primer solution: 0.8 μM in TE buffer or H2O.
  6. Annealing buffer: 1 M Tris-HCl, pH 7.6, 100 mM MgCl2, 160 mM dithiothreitol (DTT).

Labeling Reaction

  1. Sequenase Version 2.0 T7 DNA Polymerase, suitable for sequencing: Be careful to keep the enzyme at –20°C constantly. The polymerase should be removed from the freezer only for the purpose of taking an aliquot, and this should be done quickly. The aliquot is then diluted with dilution buffer and used for sequencing. The dilution can be kept on ice until the sequencing reactions are set up.
  2. Dilution buffer: 20 mM Tris-HCl, pH 7.5, 5 mM DTT, bovine serum albumin (BSA) (50 μg/mL).
  3. Radioactive nucleotide: [a-32P]dATP or [a-32S]dATP, 370 MBq (10 mCi)/mL and 370–463 MBq (10–12.5 mCi)/mL, respectively. Both nucleotides are incorporated equally well into the growing oligodeoxyribonucleotide chain by T7 DNA polymerase. Using 35S, sharper bands are obtained in the autoradiogram and, owing to the avoidance of scattering, longer sequences can be deciphered in one run. The shelf life of this isotope is longer, and the radiation dose of the personnel is reduced. Advantages of 32P are that shorter exposure times are required and that the sequencing gel does not need to be dried before autoradiography.
  4. Labeling mix: 1.375 mM dCTP, 1.375 mM dGTP, 1.375 mM dTTP, 333.5 mM NaCl. This mix can be stored in aliquots at –20°C.

Termination Reaction

  1. Termination mixes:

Table Composition of Termination Mixes

Component    A-Mix μM    C-Mix μM    G-Mix μM    T-Mix μM

dATP            93.5           840          840         840

dCTP            840            93.5         840         840

dGTP            840            840          93.5        840

dTTP             840           840          840         93.5

ddATP            14             –              –            –

ddCTP            –              17             –            –

ddGTP            –               –            14            –

ddTTP             –               –             –           17

Tris- Hcl pH       7.6             40           40          40

NaCl              50              50           50          50


  1. Stop solution: 97.5% (v/v) deionized formamide, analytical grade, 10 mM EDTA, pH 7.5, 0.3% (w/v) xylene cyanol FF, 0.3% (w/v) bromophenol blue. This solution should be stored at –20°C (under a nitrogen atmosphere, if possible). T7 DNA polymerase is also available in a kit, together with most of the required solutions.


Primer Design

As a rule for primer design, the following formula has been reported: primer length = 18 + 1 extra nucleotide for each 2% off of 50% G + C.  This is because oligonucleotides with too high an A + T content might not prime satisfactorily with special template DNA strands. Most important is the 3′ end of the primer: the last six nucleotides should contain 50% G + C. However, working with an A + T-rich organism (Clostridium acetobutylicum), use 17-mer primers with G + C contents between two and nine nucleotides.

New primer and known sequence should overlap well to avoid not being able to read the starting nucleotides of the new part and to have an internal control of each sequence start. As a rule of thumb, the 3′ end of the primer should be located approx 30–40 nt from the end of the already known sequence.

Before synthesizing a primer, its sequence should be compared carefully to the whole known sequence of template DNA and vector. This will identify regions where undesired hybridizations could take place. Again, of special importance in this respect are the last ten 3′-terminal bases. An 80% homology in this region might cause major background problems, particularly in G + C-rich templates. If undesired homology can be found, a new primer should be designed. Furthermore, a primer should not contain inverted repeats or sequence repetitions.

Template Preparation

Various kits distributed by several molecular biology companies are available for the purification of plasmid DNA. In most cases, the DNA is purified by passage over anion-exchange resin, resulting in a highly purified product. This procedure is the isolation of plasmid DNA detailed in the following list:

  1. Centrifuge 5 mL of bacterial culture grown overnight at 6000g for 5 min at 4°C (see Note 6).
  2. Discard supernatant and suspend the sediment in 150 mL of lysozyme solution.
  3. Transfer the preparation to a sterilized microcentrifuge tube and incubate for at least 5 min at room temperature.
  4. Add 300 mL of SDS solution and mix gently by hand for a few seconds.
  5. Immediately add 225 mL of sodium acetate solution.
  6. Mix gently by hand for a few seconds and incubate for 15 min at 0°C.
  7. Centrifuge for 5 min at 4°C in a microcentrifuge at maximal speed. Use either a model with a cooling system or place the centrifuge in the cold room.
  8. Transfer supernatant to a new microcentrifuge tube using a microliter pipet with sterilized tip. Be very careful to take only the supernatant.
  9. Add 600 mL of isopropanol to precipitate the DNA and vortex a few seconds.
  10. Incubate for 5 min at room temperature.
  11. Centrifuge for 5 min at room temperature in a microcentrifuge at maximal speed.
  12. Remove supernatant completely and rinse sediment with 1 mL of cold ethanol (70%).
  13. Let the sediment air-dry (3–5 min) and then suspend it in 100 mL of TE buffer.
  14. Add 1 mL of RNase solution and incubate for 15 min at room temperature.
  15. Add 260 mL of H2O and 40 mL of sodium acetate solution.
  16. Add 1 vol of phenol-chloroform solution.
  17. Vortex-mix for a few seconds and centrifuge for 5 min at room temperature in a microcentrifuge at maximal speed.
  18. Transfer the upper phase to a new microcentrifuge tube and add 1 vol of chloroform solution.
  19. Mix gently by inverting the closed tubes several times.
  20. Centrifuge a few seconds for phase separation and transfer upper phase to a new microcentrifuge tube.
  21. Add 2.5 vol of ethanol (96%) to precipitate the DNA, vortex-mix gently, and leave at room temperature for 15 min.
  22. Centrifuge for 10 min at room temperature in a microcentrifuge at maximal speed.
  23. Wash the sediment with 1 mL of ethanol (70%) and dry it.
  24. Suspend the DNA in water. This procedure usually yields 10–20 mg of plasmid DNA from a 5-mL culture.

Sequencing Reactions

Primer Annealing

Single-stranded phage DNA can be used directly as a template, double-stranded plasmid DNA must be denatured first.

  1. Pipet 8 mL of double-stranded plasmid DNA (containing 2 mg of nucleic acid) into a sterilized microcentrifuge tube (see Note 7).
  2. Add 2 mL of NaOH and vortex-mix the tube briefly.
  3. Centrifuge the tube for a few seconds in a microcentrifuge at maximal speed to concentrate the complete solution at the bottom of the tube.
  4. Incubate the tube for 10 min at room temperature.
  5. Add 7 mL of sterilized H2O and 3 mL of sodium acetate solution.
  6. Add 60 mL of cold (–20°C) ethanol (96%), mix well, and incubate the cup for 20 min at –70°C.
  7. Centrifuge for 10 min at room temperature in a microcentrifuge at maximal speed and discard supernatant.
  8. Add 300 mL of cold ethanol (70%) and mix briefly.
  9. Centrifuge for 10 min at room temperature in a microcentrifuge at maximal speed.
  10. Remove the supernatant carefully and dry the sediment under vacuum (3–5 min).
  11. Suspend the pellet in 10 mL of sterilized H2O.
  12. Add 2 mL of annealing buffer and 2 mL of primer solution.
  13. Incubate for 20 min at 37°C.
  14. Keep the cup at room temperature for at least 10 min. If the sequencing reaction is not carried out subsequently, the solution should be stored at –20°C until needed.

For single-stranded DNA the following procedure can be used:

  1. Pipet 10 mL of the template (containing 2 mg of DNA) into a sterilized microcentrifuge tube.
  2. Add 2 mL of primer solution and 2 mL of annealing buffer.
  3. Vortex-mix the tube briefly.
  4. Centrifuge the tube for a few seconds in a microcentrifuge at maximal speed to concentrate the complete solution at the bottom of the tube.
  5. Incubate the cup for 10 min at 60°C.
  6. Keep the cup at room temperature for at least 10 min. If the sequencing reaction is not carried out subsequently, the solution should be stored at –20°C until needed.

Labeling Reaction

Because T7 DNA polymerase exhibits a high processivity and a high rate of polymerization, the proper sequencing reaction is divided into two parts. In the first step (labeling reaction), low concentrations of the four deoxyribonucleotides and a relatively low temperature result in the formation of short oligodeoxyribonucleotide chains (approx 20–30 bases). Because a radioactive nucleotide is present in the reaction mixture, all fragments become uniformly labeled, which is important for equal band intensities in the autoradiography step. In the second stage (termination reaction), the four standard reaction mixtures are set up, each of which contains high concentrations of the four deoxyribonucleotides and a single dideoxyribonucleotide. Higher temperature and nonlimiting dNTP concentration then allow a high rate of polymerization that is terminated only by incorporation of a ddNTP.

  1. Dilute T7 DNA polymerase with dilution buffer to a concentration of 1.5 U/mL.
  2. Add 0.8–1 mL of radioactive dATP (3.7*105 Bq) to the microcentrifuge tube containing annealed primer/template.
  3. Add 3 mL of labeling mix.
  4. Add 2 mL of diluted T7 DNA polymerase.
  5. Mix by pipetting several times up and down in the tube with a microliter pipet and a sterilized tip.
  6. Incubate for exactly 5 min at room temperature and then proceed directly with the termination reaction.

Termination Reaction

  1. Set up appropriate vials for the termination reaction (microcentrifuge tubes or a microtiter plate) and mark them carefully with “A,” “C,” “G,” and “T.”
  2. Pipet 2.5 mL of the respective termination mix into the corresponding cup or well. Steps 1 and 2 should be done in advance (e.g., during the incubation period of the annealing reaction).
  3. Warm mixtures to 37°C in a water bath (takes approx 1 min).
  4. Pipet 4.5 mL of the labeling mixture into each tube or well. Use a fresh tip for each transfer.
  5. Incubate the reaction mixture for exactly 5 min at 37°C.
  6. Add 5 mL of stop solution to each tube or well.
  7. Denature an aliquot (approx 3 mL) by heating for 3 min at 80°C.
  8. Put vials on ice.
  9. Load 1.8–2 mL of each mixture onto a sequencing gel. The remaining material of the termination reactions can be stored at –20°C and used for further sequencing runs if necessary. Be sure to denature the aliquots before loading onto a gel.
  10. To read as many bases as possible, two samples from each reaction mixture should be run on the same gel, with a period of electrophoresis (approx 2–3 h, until the first colored marker band reaches the end of the gel) between the two loadings.

Starting with prepared template DNA and primers, one round of sequencing including the autoradiography can be performed in 24 h (calculating overnight as exposure time).


  1. Oligonucleotide solutions are sensitive toward nucleases. We recommend storing oligonucleotides in the lyophilized state and dissolving them directly before use. The solution should be treated with care, always kept on the ice, and stored at –20°C.
  2. If the concentration of an oligonucleotide is not given, then both the molar and the mass concentration can be calculated from the absorption of the solution at 260 nm:

c [pmol/mL] = A260 nm * 100/N

c [mg/mL] = A260 nm * 0.033

where N equals the number of nucleotides of the oligonucleotide. This is an estimation based on an average composition of bases with an average molecular weight of 333/nucleotide.

  1. In many cases, the plasmid preparation works equally well if the first solution is prepared without lysozyme.
  2. To avoid band compressions by G–G or G–C band pairing nucleotide analogs such as 7-deaza-dGTP, 7-deaza-dATP, or dITP can be used.
  3. We strongly recommend checking the template sequence carefully after every elongation. Ambiguous regions can thus be resequenced at once, which might help to avoid later unnecessary primer synthesis. Direct computer analysis will also enable finding matching stretches if sequencing was started from both ends of the template. If only the end of a newly obtained sequence is read and used for new primer design, this could finally lead to the unnecessary sequencing of large parts of the vector.
  4. Best sequencing results are obtained by isolating the template DNA from logarithmically growing cells. Therefore, cells should be harvested after growth at 37°C until OD600nm reaches 1.5 or after overnight growth at 30°C.

7. If multiple sequencing reactions with the same double-stranded plasmid are planned, it is recommended that a large amount of plasmid be used in the denaturation reaction. This will save time for future sequencing reactions. The denatured DNA should be dissolved in sterilized H2O and stored in 10-mL aliquots with the correct amount of DNA for one sequencing reaction at –20°