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Dideoxy Sequencing of DNA

  1. S Wilton

Published Online: 24 OCT 2002

DOI: 10.1038/npg.els.0003768

eLS

eLS

How to Cite

Wilton, S. 2002. Dideoxy Sequencing of DNA. eLS. .

Author Information

  1. Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Australia

Publication History

  1. Published Online: 24 OCT 2002
Recipe 1. T4 Polynucleotide kinase (PNK) 10× buffer
IngredientFinal concentrationVolume/amount
Mix gently and store at −20°C.
Tris-HCl (1.0 mol L−1, pH 7.5)500 mmol L−1500 μL
MgCl2 (1.0 mol L−1)100 mmol L−1100 μL
Dithiothreitol (1.0 mol L−1)50 mmol L−150 μL
Spermidine (1.0 mol L−1)1 mmol L−11 μL
Sterile distilled water to final volume 1 mL
Recipe 2. 5× Sequencing buffer
IngredientFinal concentrationVolume/amount
Mix gently and store at room temperature.
Tris-HCl (1.0 mol L−1, pH 9.0)250 mmol L−1250 μL
MgCl2 (1.0 mol L−1)10 mmol L−110 μL
Sterile distilled water to final volume 1 mL
Recipe 3. Sequencing Stop/loading buffer
IngredientFinal concentrationVolume/amount
Mix well, dispense into 1 mL aliquots and store at −20°C. A good stability guide is to check that the buffer is frozen at −20°C. Discard if the buffer does not freeze. Use another de-ionized preparation of formamide.
Formamide98%9.79 mL
NaOH (10 mol L−1)10 mmol L−110 μL
Bromophenol blue (1%)0.01%100 μL
Xylene cyanol (1%)0.01%100 μL
Total 10 mL
Table 1. Extension/termination reactions (values are μmol L−1)
ComponentA stopC StopG StopT Stop
ddATP350   
ddCTP 200  
ddGTP  30 
ddTTP   600
dATP20202020
dCTP20202020
7-deaza dGTP20202020
dTTP20202020
Recipe 4. 10× TBE
IngredientFinal concentrationVolume/amount
  1. a

    Dissolve at room temperature. Store at room temperature. Stable for several months.

Tris basea890 mmol L−1108 g
Boric acid890 mmol L−155 g
Disodium EDTA25 mmol L−19.3 g
Double-distilled water to final volume 1000 mL
Recipe 5. 6% polyacrylamide sequencing gel mix
IngredientFinal concentrationVolume/amount
  1. a

    Gentle heat (not greater than 30°C) may be applied to facilitate dissolving.

  2. b

    Add resin once solids have dissolved. Gently mix for 30 min. Filter the solution through Whatman #54 (or similar) to remove mixed bed resin. NOTE: When using high-quality or premixed acrylamide preparations, the deionization step is not necessary.

  3. c

    De-gas to remove dissolved oxygen, which can inhibit polymerization, just before use. Store at 4°C. Stable for at least one month.

Acrylamide5.7% (w/v)11.4 g
Bis-acrylamide0.3% (w/v)0.6 g
Urea7 mol L−184 g
Double distilled watera 170 mL
Mixed bed resinb 10 g
Double-distilled water to final volume 180 mL
10× TBE20 mL
Final volume 200 mL
Recipe 6. 25% ammonium persulfate
IngredientFinal concentrationVolume/amount
Store at 4°C.
Ammonium persulfate25% (w/v)2.5 g
Double-distilled water to final volume 10 mL
Recipe 7. Gel washing/fixing solutions
IngredientFinal concentrationVolume/amount
Acetic acid10% (w/v)100 mL
Methanol10% (w/v)100 mL
Double-distilled water to final volume 1000 mL
Table 2. Standard thermal cycling conditions
94°C2 min15 s15 s 
55°C 15 s  
72°C 2 min2 min 
4°C   HOLD
  25 cycles10 cycles 
Table 3. Hazards associated with this procedure
Acetic acid CH3COOHCorrosive. Causes severe burns. Glacial acetic acid is flammable. Harmful if swallowed, inhaled or absorbed through skin. Material extremely destructive of tissues of mucous membranes, upper respiratory tract, eyes and skin. Inhalation may be fatal. Used as a fixative. Solutions are irritant. Use a fume hood and wear face protection and gloves when preparing solutions and at all times when using glacial acetic acid.
Acrylamide CH2CHCONH2Neurotoxin. May cause cancer and heritable genetic damage. Toxic through skin contact and if swallowed. Danger of serious damage to health by prolonged exposure. Dispense in fume hood and weigh in closed container or balance. Wear protective clothing and gloves and use face protection.
Ammonium persulfate (NH4)2S2O8Harmful by inhalation and if swallowed. Oxidizing agent.
Bisacrylamide (N,N-methylenebisacrylamide) C7H10N2O2Harmful. Avoid contact. Wear gloves. Do not breathe dust.
Boric acid H3BO3Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Possible Teratogen. Reproductive hazard. In case of contact with eyes, rinse immediately with plenty of water for 15 min and seek medical advice. In case of contact, immediately wash skin with soap and copious amounts of water. If inhaled, remove to fresh air. If not breathing give artificial respiration. If breathing is difficult, give oxygen. If swallowed, wash out mouth with water provided person is conscious.
Bromphenol blue Bromophenol blueAvoid contact and inhalation.
Chloroform (trichloromethane) CHCl3Harmful if swallowed. Irritating to skin. Possible risk of irreversible effects and serious damage to health by prolonged exposure through inhalation and if swallowed. Use in fume hood and wear appropriate gloves. Chloroform should be handled with care, and disposed of by means appropriate for organic solvents.
Dimethyldichlorosilane (Inerton-DMCS) (Inerton DW-DMC) C2H6Cl2SiHighly flammable. Flash point −4°C. Reacts violently with water. Causes severe burns. Irritating to respiratory system. Use in fume hood. May also be supplied as dilute solution in 1,1,1-trichloroethane.
Dithiothreitol (DTT) HSCH2(CHOH)2CH2SHHarmful in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin.
EDTA (diaminoethanetetraacetic acid; ethylenediaminetetraacetic acid)Harmful if swallowed. Irritating to eyes, respiratory system and skin.
ElectrophoresisGreat care must be exercised when using any electrophoresis equipment, especially high-voltage or constant-current supplies. If possible, always use commercially supplied apparatus that has been designed and built to international electrical safety standards: home-made equipment is always suspect in this regard. Always check that all wiring connections are properly made and any interlocks fitted are secure before switching on the power supply. Always switch off the power supply before disconnecting the apparatus. Arrange the work area to reduce the risk of water or reagents splashing on to the power pack, leads, cables or chambers. Preferably use power supplies fitted with electrical earth leakage detection circuitry and automatic cut-off.
Electrophoresis of radioactive materialElectrophoresis of highly radioactive probes is dangerous. Extreme care is needed. Take local advice concerning the level of radiation protection necessary. See also entries for ‘Electrophoresis’ and ‘Radioisotopes’.
Ethanol (ethyl alcohol) C2H5OHHighly flammable. Flash point 12°C. Use in well-ventilated area away from sources of ignition.
Formamide HCONH2Harmful by inhalation, in contact with skin and if swallowed. May cause irritation to skin. May cause birth defects following chronic exposure. Do not breathe vapour. Prepare solutions in fume hood. Wear protective clothing, face protection and gloves.
Hydrochloric acid (HCl)May be fatal if inhaled, swallowed or absorbed through skin. Causes burns. Material extremely destructive of tissues of upper respiratory tract, eyes and skin. Wear protective clothing and gloves and use face protection when using concentrated solutions.
Magnesium chloride MgCl2Irritating to eyes, respiratory system and skin.
Methanol (Methyl alcohol) CH3OHToxic. Flammable. Flash point 10°C. Used as a fixative and a solvent for stains. Toxic by inhalation and if swallowed. Irritating to eyes, respiratory system and skin. Wear suitable protective clothing, gloves and face protection. Use in well-ventilated area away from sources of ignition.
Radioactive materialWear gloves and protective shield. Treat radioactivity according to your laboratory rules
Sodium hydroxide NaOHCauses severe burns. Wear glasses. Eye contact: Rinse immediately with plenty of water for 15 min and seek medical advice. Skin contact: Immediately wash skin with soap and copious amounts of water. Ingestion: If the chemical has been confined to the mouth give large quantities of water as a mouthwash. Ensure the mouthwash is not swallowed. If the chemical has been swallowed, give about 250 mL of water to dilute it in the stomach. In severe cases, obtain medical attention.
Spermidine (N-(3-aminopropyl)-1,4-butanediamine) NH2(CH2)4NH(CH2)3NH2Corrosive. Causes burns. Wear suitable protective clothing, gloves and use face protection.
Tris (tris(hydroxymethyl)aminomethane; 2-amino-2-hydroxymethylpropane-1,3-diol)Irritating to eyes, respiratory system and skin.
Ultraviolet light sourcesAlways wear UV goggles or visor. Do not look directly at the light source in transilluminators for unnecessary periods of time even if goggles are being worn. Allow for reflected UV light. Do not expose skin to UV illumination for unnecessary periods of time. If long periods of viewing are necessary, use a UV face visor. Ensure that the eye protection provides adequate UV absorption for the intensity and frequency of UV light being used. Long-wavelength UV is less dangerous than short-wavelength UV.
Xylene cyanol FFIrritating to eyes, respiratory system and skin. Avoid contact.
Table 4. Primer labelling
Primer lengthng primer to equal 10 pmol
20mer67
24mer80
30mer100