DNA Sequencing

All sequencing reactions were carried out in 0.5ml plastic centrifuge tubes (Eppendorf). 4pl of pUC 18 B7-2 (Ipg DNA) was added to 3pl of distilled water, 5pl of plasmid denaturing reagent (lOmM Tris-HCI, pH 7.5, ImM EDTA, 50% Glycerol, 50% Ethylene Glycol) and 1^1 of forward primer (2.0pmol/pl 5'- GTTTTCCCAGTCACGACGTTGTA-3’), mixed thoroughly and heated to 95°C for 5 minutes to denature the DNA. The mixture was chilled in an ice water bath and 2^1 of plasmid reaction buffer (400mM Tris-HCI, pH 7.5, lOOmM MgCl2, 250mM NaCI) was added. The mixture of template, buffer and primer

was then incubated at 37°C for 10 minutes to allow annealing of the primer to the template and then the mixture was chilled in ice. To label the DNA strands 1|il DTT(O.IM), 2pl labelling mix (1.5|iM 2'-Deoxyguanosine 5'-triphosphate (dGTP), 1.5|liM 2'-Deoxycytidine 5’-triphosphate (dCTP), 1.5pM 2’- Deoxythymidine 5'-triphosphate (dTTP), 0.5pl (5pCi) [^^S] 2’-Deoxyadenosine 5'-triphosphate (dATP) and 2|liI sequenase polymerase (13 units/pl in 20mM

KPO4, pH 7.4, 1mM DTT, 0.1 mM EDTA, 50% Glycerol diluted 1:8 with ice cold

Enzyme Dilution Buffer) was added to the ice cold annealed DNA mixture. This was mixed and incubated at room temperature for 8 minutes.

Prior to termination of the generation of the DNA strands 4 0.5ml microcentrifuge tubes were labelled G, A, T and C respectively. Into the tube labelled G was added 2.5^il of the ddGTP Termination mix (80|iM 7-deaza- GTP, 80|iM dATP, 80pM dCTP, 80pM dTTP, 8pM ddGTP, 50mM NaCI) and

similarly into the A,T and 0 tubes was added 2.5^\ of the ddATP (80pM 7- deaza-GTP, 80pM dATP, 80^M dCTP, 8 0 |liM dTTP, 8|aM ddATP, 50mM NaCI),

ddTTP (80pM 7-deaza-GTP, 80|iM dATP, 80|aM dCTP, 80^iM dTTP, 8pM

ddTTP, 50mM NaCI) and ddCTP (80|iM 7-deaza-GTP, 80pM dATP, 80pM dCTP, 80|iM dTTP, 8 pM ddCTP, 50mM NaCI) termination mixes respectively.

These termination tubes were then prewarmed to 37°C and on completion of the labelling reaction 3.5pl was added to each of the termination tubes, mixed and reincubated at 37°C for 5 minutes. 4pl of stop solution (95% formamide, 20mM EDTA, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol FF) was added to each of the termination reactions, mixed thoroughly and stored on ice until ready to load onto the sequencing gel.

2.2.1.1 Preparation of Acrylamide Gel and Running of Sequencing Reactions

The sequencing gel (8% acrylamide) was made by dissolving 75g urea

in 67.5mls of distilled water. 6 mls of 20x glycerol tolerant gel buffer (216g Tris

base (Tris[hydroxymethyl]aminomethane), 72g Taurine (2-Aminoethanesulfonic acid), 4g NaEDTA.2 H2 0 , made up to lltr volume with distilled H2O) and 30mls

persulphate was added and prior to pouring of the gel was added SO^il of TEMED (N,N,N’,N’ tetramethylethylenediamine). Prior to this the glass plates, graduated spacers and comb were thoroughly cleaned and then rinsed with distilled H2O. The inner surface of the smaller glass plate was coated with a

film of sigmacote® (Sigma) and left to dry. The glass plates were then taped together with insulating tape to ensure a watertight seal, with the sigmacoted surface of the smaller plate facing inwards and the graduated spacers at each margin of the glass plates. The acrylamide solution was the poured between the glass plates immediately after addition of the TEMED (Sigma), taking care to remove any air bubbles and the flat side of the shark’s tooth comb was inserted into the top of the plates when the solution had reached the top of the plates. The comb was the clamped in position using bulldog binder clips. The gel was then left until it had set and then the insulating tape was removed and the gel loaded onto a Model S2 Sequencing Gel Electrophoresis Apparatus (Bethesda Research Laboratories, Life Technologies Incorporated) with the smaller glass plate facing inwards. The top reservoir and the bottom reservoir of the gel tank was then filled with 1x Glycerol Tolerant Gel Buffer so that the lip of the smaller glass plate was covered. The comb was then reversed to provide discrete wells for loading of the sequencing reactions and each well was flushed with the gel buffer prior to loading of the sequencing reactions. 4pl of each of the 4 sequencing reactions was loaded into each well, after being heated to 75°C for 2 minutes and run at 1400 volts for 3 hours. A careful note of the order of loading of the sequencing reactions was taken. The gel was removed from between the glass plates by blotting it onto Whatman 3MM paper, which was then laid on a further two pieces of the 3MM paper before

being covered in Saran Wrap (Dow Chemical Company). The 3MM paper and the Saran Wrap was then trimmed so that they were only slightly larger than the gel. The gel was then dried under vacuum for three hours in a Biorad Model 483 gel dryer. Once the gel was dry the gel was developed using Kodak Scientific Imaging Film (35x43cms) placed over the dry gel in a exposure cassette for 24 hours at room temperature.

2.2.1.2 Generation of Nested Deletions

4pg of pUC 18/B7-2 was digested with the restriction enzymes Sal I and

Sph I (section 2.2.3.1). The mixture was then incubated at 37°C for 3 hours to ensure full digestion of the DNA with both restriction enzymes. Prior to digestion with Exonuclease III (Exo III), the DNA was cleaned with a Qiagen clean up column as described in section 2.2.3.4., except that the DNA was eluted from the column with 45pl H2O. The cleaned digested DNA was topped

up to 45pl with distilled H2O if required, transferred to a 0.5ml microcentrifuge

tube (Eppendorf) and 5pl of Exo III lOx buffer (660mM Tris-HCI, pH 8.0, 6 .6 mM

MgCl2) was added. The mixture was then warmed to 34°C and 7.5pl of SI mix

(172pl distilled H2O, 27pl 81 7.4x Buffer (0.3M potassium acetate pH 4.6, 2.5M

NaCI, lOmM ZnSO^, 50% glycerol), 60 units 81 nuclease) was added separately to 12 wells of a 96 well round bottomed plate (Nunclon) and then placed on ice. 500 units of Exo III was added to the digested pUCB7-2, incubated at 34°C and after 30 seconds a 2.5pl aliquot was removed and put in well 1 of the 81 mix ensuring the sample was well mixed. This process was repeated at 15 second intervals until 12 samples had been aliquoted. The samples were taken off ice and incubated at room temperature for 30 minutes.

After this time 1pl of S1 stop buffer (0.3M Tris base, 0.05M EDTA) was added to each of the samples, which were then heated at 70°C for 10 minutes to inactivate the enzymes. To determine the extent of digestion 2pi aliquots were removed to run on a 1% agarose gel. To prepare the gel, 1g of agarose (Gibco) was weighed and added to 125mls of 0.5x TBE (5x 54g Tris base, 27.5g Boric acid, 20mls 0.5M EDTA (pH 8.0). The mixture was heated in a microwave oven until the agarose had completely dissolved. 5pl of ethidium bromide (lOmg/ml) was added to the melted agarose and the mixture poured into the gel plate, sealed with watertight tape, with a 30 well comb in place. The agarose was allowed to cool until completely solidified and the tape and comb were carefully removed. The solidified gel in its tray was inserted into the gel tank and 0.5x TBE was poured in until the gel was submerged. Each of the aliquots was added to 2\i\ of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water) and 6pl of distilled H2O, before being

loaded into a well in the gel. The DNA was then run at 5 volts/cm until the DNA had run far enough into the gel, as indicated by the dyes in the loading buffer. The digested DNA was visualised using a UV light box and the gel photographed (Polaroid 667). The remainder of the samples were then transferred to a 37°C incubator and 1pl of Klenow mix (30pl Klenow buffer(20mM Tris-HCI, pH 8.0, lOOmM M gCy, 5 units Klenow DNA polymerase) was added to each sample, incubated for 3 minutes at 37°C, before the addition of Ipl of dNTP mix (0.125mM each of dATP,dCTP,dGTP and dTTP) to each well. The DNA was then incubated for a further 5 minutes at 37°C. At room temperature 40pl of ligase mix (790pl deionised H2O, lOOpI

50% PEG, 10|il lOOmM DTT, 5 units T4 DNA polymerase) was added to each well, mixed and incubated at room temperature for 1 hour. 1 0pl of each ligation

product were then transformed into E. coli (strain TG-1) competent cells.