DNA?Sequencing

The sequencing reactions described below work perfectly well if you are short of cash to buy sequencing kits.It is based on the Dideoxy sequencing method of Sanger et al.,1977.However,due to the number solutions that need to be made,I recommend purch asing a sequencing kit,we use either the T7 Sequencing Kit (Pharmacia,100 reactions) or the Sequenase 2.0 Sequencing Kit (USB via Amersham in the U.K.,100 reactions).

Reactions are performed in sterile 1.5ml microcentrifuge tubes.Primers are synthesised on an Applied Biosystems 381A DNA synthesiser.

Approximately 3ug denatured,high quality dsDNA (i.e. prepared as described in 'Plasmid Isolation using PEG') are typically used for standard sequencing reactions.

DNA Sequencing Reactions

You will need:
      Freshly made 2M NaOH
      3M sodium acetate, pH 4.5
      Sterile, distilled water
      Absolute ethanol
      70% ethanol
      7 x DNA annealing buffer (280mM Tris.Cl,pH 7.5,100mM MgCl2,350mM NaCl)
      Termination mixes (40mM Tris.Cl,pH 7.5,50mM NaCl,10mM MgCl2,150mM dTTP,150mM dATP,150mM dCTP,150mM c7 deaza-dGTP and 15mM of the respective ddNTP)
      5 x DNA labelling mix (10mM dGTP,10mM dCTP,10mM dTTP,200mM Tris.Cl,pH 7.5,250mM NaCl)
      300mM DTT
      [a-35S]-dATP (~ 1000Ci/mmol,Amersham or DuPont)
      T7 DNA polymerase (Pharmacia)
      Stop solution (95% deionized formamide,20mM EDTA,pH 7.5,0.1% each of bromophenol blue and xylene cyanol FF)

1)Denature dsDNA by the addition of 8ul DNA (approximately 3ug) to a sterile microcentrifuge tube containing 2ul freshly made 2M NaOH vortexed briefly and incubate at room temperature for 10 minutes.

2)Neutralise DNA by the addition of 3ul 3M sodium acetate,pH 4.5 and 7ul sterile, distilled H2 O and precipitate by the addition of 60ul ethanol.Recover DNA by centrifugation,at maximum speed, for 10 minutes in a microfuge.Rinse DNA briefly in 70% ethanol, air dry and re-dissolve in 10ul sterile, distilled H2O.

3)To a microcentrifuge tube containing 10ml denatured template DNA,add 4.44ng primer (2ul of a 2.22ng/ul stock) and 2ml 7 x annealing buffer.Heat the mixture to 65°C for 2 minutes and allow to cool slowly,over a period of about 30 minutes,to roo m temperature.

4)While the annealing reaction is taking place, take 4 sterile microcentrifuge tubes per sample and label G,A,T,C,respectively.Place into each tube 2.5ul,respectively,of the corresponding termination mix.Pre-warm tubes to 37°C.

5)After completion of the annealing reaction,the labelling reaction is initiated by the addition to the annealed template/primer of 2ul 1 x labelling mix,1ul 300mM DTT,1ul [a-35S]-dATP (~ 1000Ci/mmol) and 3 units T7 DNA polymerase (2ul of a 1.5 units/ ul solution).The solution is pipetted briefly to mix the components and incubated at 4°C for 2-5 minutes.

6) Termination is achieved by transferring 4.5ul of the labelling reaction into each of the 4 tubes labelled G, A, T, C, respectively and incubating at 37°C for 2-5 minutes.

7)After termination,5ul stop solution should be added to each tube,mixed by pipetting and the samples stored at -20°C for later use.

DNA sequencing gels

Gels used for DNA sequence analysis are of the wedge type.These produce a voltage gradient which decreases as DNA migrates down the gel,thus retarding the rate of migration of smaller fragments and allowing more readable sequence information to be obtai ned from one gel.DNA sequencing gels are cast between the 38 x 50cm and 38 x 47.5cm glass plates of the Bio-Rad SequiGen?sequencing system.

You will need:
      A standard detergent
      2% dichlorodimethyl silane in hexane
      Absolute ethanol
      6% sequencing acrylamide (5.7% acrylamide,0.3% bisacrylamide,48% urea,1x TBE)
      25% AMPS (freshly made)
      TEMED

N.B:Wear gloves while handling solutions of unpolymerised acrylamide.Unpolymerised acrylamide is a neurotoxin.

1)Clean the glass plates extensively with detergent and water,tap water,distilled water and finally ethanol.Wipe dry with a clean paper towel.

2)Siliconize the smaller of the two plates using the 4% solution of dichlorodimethylsilane in hexane.The solution should be spread evenly over the plate and allowed to dry before being repeated.Once dry,the plate should be washed with 100% ethanol and again wiped dry using a clean paper towel.

3)Gel plates are then assembled as described in the manufacturers instructions using two 0.25 -1mm wedge spacers.Polyacrylamide sequencing mix for use in the gels was stored at 4°C in a dark bottle.

4)35ml of the acrylamide mix is used to first plug the bottom of the gel.Chill the acrylamide on ice and add 150ul 25% AMPS and 150ul TEMED.Mix by swirling and then poured briskly into the gel mould.The quantities of AMPS and TEMED may have to be esti mated empirically to cause setting in approx.5 minutes.

5)Once the plug has set,85ml of acrylamide is then used to form the main gel itself.To the acrylamide (chilled on ice beforehand),add 110ul 25% AMPS and 110ul TEMED.The solutions are mixed thoroughly,placed into a 50ml syringe and injected,carefull y,between the glass plates.In order to facilitate ease of pouring,the glass plates were inclined at an angle of approximately 10° to the horizontal in a large developing tray to prevent spills.Again,the quantities of AMPS and TEMED used may need to be varied in order to give polymerisation in approx.30 minutes - this may be especially critical if the ambient temperature is abnormally warm.

N.B: It is critical to chill the acrylamide for the main gel in order to prevent polymerisation while the gel is being poured.You may also need to adjust the AMPS/TEMED quantities used.You should aim to have the plug set in ~5 mins and the main g el after ~30 mins.

6)Immediately after the gel is poured,a flat 0.25mm spacer (or reversed shark tooth comb) should be placed into the acrylamide on the gel top such that it intrudes into the gel by approximately 10mm.This allows the formation of a flat gel surface essen tial to the effective use of the shark tooth combs during electrophoresis.Clamp large bulldog clips across the top of the gel plates during gel polymerisation to ensure a leak-free fit of the combs. Allowed to polymerise for 1 hour at room temperature an d then use directly or store overnight at 4°C,tightly wrapped in clingfilm to prevent dehydration of the gel.

7)Remove the gel former and pre-electrophorese the gel at 1800V to heat the gel and running buffer to the required operating temperature (55°C) prior to the loading of the samples.Running buffer is 1 x TBE.

8)Insert sharks tooth combs such that the tips protrude approximately 0.5mm into the gel surface.

9)Thoroughly wash the wells immediately prior to the loading of the samples with running buffer to remove any urea which leaches from the gel.

10)Sequencing reaction mixtures,containing loading buffer,should be boiled for 2-3 minutes to denature any secondary structure and loaded into the wells (3ul/well),in the order G,A,T,C.

11)Electrophorese at 1800V (preferably 75W constant power) until the xylene cyanol dye front is approximately 5 cm from the bottom of the gel.Monitor the gel temperature to ensure it stays at 60°C or below (preferably 50-55°C).

N.B:Allowing the gel temperature to exceed 60°C for extended periods of time will cause the hydrolysis of urea in the gel.

12)After electrophoresis is complete,combs should be removed and the small siliconized glass plate gently removed from the remaining plate.The large plate,with the gel still attached,is then immersed in a fixative solution containing 10% acetic acid,10% methanol for approximately 15-20 minutes.This process is used to remove urea from the gel.

13)Transfer the gel to a large sheet of Whatman 3MM paper and dry on a vacuum gel drier at 85°C for 75 minutes prior to autoradiography.

This addendum describes the use of formamide in sequencing gels to alleviate problems with base stacking due to G-C compressions.The following method describes the production of 200ml of a 25% formamide,6% acrylamide sequencing gel mix but it can be scaled accordingly.You can also vary the formamide concentration upto 40%.

You will need:
      Ultra-pure urea
      Formamide
      Acrylamide
      Bis-acrylamide
      TEMED
      Ammonium persulphate (AMPS)
      Amberlite MB-1 resin (Sigma)
      10 x TBE buffer

1)Mix together 82g urea,50ml formamide,11.4g acrylamide,0.6g bis-acrylamide and ~60ml H2 O.

2)Warm the mixture to ~40°C,with stirring,to dissolve solids.

N.B.:Do not heat the mixture above 55°C as this leads to hydrolysis of the urea.

3)Once dissolved,add ~5g Amberlite MB-1 resin and stir for 20 minutes.

4)Filter solution (we typically use Whatman 3MM paper) and make up to 180ml with H2 O.

5)Add 20ml fresh 10 x TBE buffer.

6)For a gel of ~100ml,use 200ul TEMED and 200ul fresh 25% AMPS solution to initiate polymerisation.

N.B.:For the above quantities of TEMED and AMPS,you MUST chill the gel mix to ~4°C prior to addition of the catalysts - this prevents gel polymerisation mid-way through pouring the gel! Polymerisation of formamide gels requires longer than normal as does the actual electrophoresis.

7) Electrophorese at 80W,constant power (should be ~45 - 50°C for a 38x50cm gel).