Sequenase Version 2 Protocol

Table 2.8. Solutions for Sequenase reaction.

5 X sequenase reaction buffer

labelling mix

enzyme dilution buffer

ddATP termination mix

ddCTP termination mix ddTTP termination mix ddGTP termination mix 200mM Tris-HCl pH 7.5,100mMMgCl2, 250mM NaCl (USB) 7.5pM dGTP, 7.5pM dCTP, 7.5pM dTTP (USB) lOmM Tris-HCl pH 7.5, 5mM DTT, 0.5mg/ml bovine serum albumin (USB) 80pMdGTP, 80pM dATP, 80pM dCTP, 80pMdTTP, 8pM ddATP, 50mM NaCl 80|iM dGTP, 80pM dATP, 80pM dCTP, 80pM dTTP, 8pM ddCTP, 50mM NaCl 80pM dATP, 80pM dTTP, 80pM dCTP, 80pM dGTP, 8pM dTTP, 50mM NaCl 80pM dATP, 80pM dTTP, 80^iM dCTP, 80^iM dGTP, 8pM dGTP, 50mM NaCl

For exon 17, the 7-deaza-dGTP analogue which forms weaker bonds with dCTP was used in place of dGTP. This improved the quality of the sequencing for this exon. 7-deaza-dGTP sequencing solutions (USB) contained 7-deaza-dGTP in place of dGTP and, otherwise, were the same as those described in table 2.8.

2.5.6.1 Annealing reaction.

Unless otherwise stated, the "Sequenase" reaction proceded as follows: The DNA template for sequencing was prepared as described in sections 2.5.2, 2.5.3, 2.5.4 and 2.5.5. Annealing of the sequencing primer to the DNA template was achieved by adding Ipl of primer to 7|il of DNA and 2pl of 5 X sequenase reaction buffer. The mixture was heated to 65“C in a beaker of water for 2 minutes and left in the beaker as the water cooled to <37°C.

2 5.6.2 Labelling reaction.

The labelling reaction is performed to allow the incorporation of [a-^^S]dATP into the DNA molecule as it is being extended from the sequencing primer by T7 DNA polymerase. The labelling mix containing the dNTPs was diluted 1:5 when sequencing was carried out according to the procedures described in sections 2.5.2 and 2.5.3. For asymmetric sequencing (see 2.5.4), or the biotin-streptavidin method (section 2.5.5), the labelling mix was diluted 1:15. A lower concentration of dNTPs in the sequencing reaction is preferable to allow sequence close to the primer to be read. When the DNA template-primer from 2.5.6.1 was cooled to <37°C, Ifil of O.IM DTT, 2.0|il of diluted labelling mix and 0.5|il of [a-^^S]dATP (1,000 Ci/mmol) was added. Sequenase Version 2.0 (USB) DNA polymerase (on ice) was diluted 1:8 in enzyme dilution buffer and 2.0|il was added to the reaction which was then incubated at room temperature for 5 minutes. After the labelling step, the labelled DNA molecules are extended rapidly with Sequenase in the presence of a high concentration of dNTPs and terminate when a ddNTP, also present in the reaction, is incorporated.

2.S.6.3 Termination reaction.

During the labelling reaction, 2.5p,l of one of the ddNTP termination mixes was added to each of 4 tubes (ddATP, ddGTP, ddTTP, ddCTP). These tubes were prewarmed at 37°C for 1 minute. Following the labelling reaction, 3.5|il of the solution was added to each of the 4 ddNTP tubes. The termination reaction proceeded at 37°C for 4 min, following which, 4.0|il of Sequenase stop mix was added (unless stated otherwise in the relevant section). The tubes were then placed on ice or frozen.

2.5.7 Sequencing gels.

Either 4% or 6% polyacrylamide (0.3-0.6mm) thick wedge gels were prepared for analysing the products of sequencing reactions depending on the length of the sequence which was to be resolved on the sequencing gel. The following provides a guide:

Table 2.9 Conditions for sequencing gels.

length from 3' end of primer polyacrylamide time (hours) concentration

10-200 bp 6% 2

65-300 bp 6% 4

up to 320 bp 4% 3

Glass gel plates were prepared as described in section 2.4.2.5. For 6% gels, the sequencing gel (20cm X 30cm X 0.5cm) solution consisted of 27ml of 40% acrylamide (containing 1:20 bis acrylamide:acrylamide), 75.6g of urea, 18ml of 10 X TBE pH 8.3 and water to 180ml. For 4% gels, 18ml of 40% acrylamide was used. The urea was dissolved by gently heating the mixture. Wedge gels (0.2mm-1.0mm thick) were poured as described in section 2.4.2.5, except that wedge-shaped spacers (BRL) were used. Because urea can quickly settle into the wells forming crystals, which retard the radioactively labelled molecules, the wells were flushed out with 1 X TBE using a

needle and syringe before loading the samples onto the gel. If this simple precaution was not taken streaks would appear on the autoradiograph.

The DNA samples were denatured at 95°C for 3 minutes before loading on the sequencing gel. Because it takes a while to load the gel it was important to keep the waiting samples at >85“C until their turn. Gels were then electrophoresed at room temperature in 1 X TBE buffer in a BRL SlOO sequencing tank. The time of electrophoresis depended on the length of the sequence which was to be read (see table 2.9). It was also possible to do both a 2 hour and a 4 hour run on the same gel by staggering the loading times. Following electrophoresis, the glass gel plates were prised apart and the gel attached to the non-siliconised plate was fixed in a 10% methanol/ 10% acetic acid bath for 45 minutes to remove the urea which would otherwise make the gel brittle. Following fixation, two layers of 3mm chromatography paper were carefully laid on top of the gel so that it adhered to them. The gels were dried in a vacuum dryer and exposed to XAR-5 (Kodak) film overnight. If the signal was faint, however, it was necessary to re-expose them for longer.

2.6 SOUTHERN BLOTTING.

Table 2.10 Solutions for Southern Blotting.

S.O.C.

Hybridisation buffer

lOX oligolabelling buffer

20 X SSC

LB

Denaturing solution

Neutralising solution

2 % bactotryptone, 0.5% yeast extract, lOmM NaCl, 2.5mM KCl, lOmM MgCl2, lOmM MgS04, 20mM glucose

4XSSC, 50|ig/ml sonicated salmon sperm DNA, lOX Denhardts, 0.1%SDS

0.25M Tris pH 8 ,0.025M MgCl2, lOO^iM dATP, lOO^iM dTTP, lOOjiMdOTP,

0.05M B-Mercaptoethanol,

IM Hepes pH6, 30 OD260 U/ml random hexanucleotides

3M sodium chloride,

0.3M sodium citrate pH 7.0

1% bacto-tryptone, 0.5% bacto-yeast extract, 0.17M sodium chloride, pH 7.0 0.5M sodium hydroxide, 1.5M sodium chloride

0.5M Trizma base, 0.3M sodium citrate, 3Msodium choloride, pH 5.5.

2.6.1 Transformation.

The RB1 cDNA (4.7R) which was in the Bluescribe vector (kind gift of Dr. T Dryja) was used to transform DH5a competent E.coli host cells for the preparation of probes for hybridisation analysis. Transformed cells were selected in the presence of the colorimetric indicator, 5-bromo-4-chloro-3-indolyl B-D-galactopyranoside (X-gal) and

inducer, isopropyl B-D-thio-galactopyranoside (IPTG). E. coli competent cells (Gibco BRL) were thawed on ice and 1-lOng of DNA was added to lOOpl of competent cells, together with Img of X-gal (dissolved in N,N, dimethylformamide) and 0.5mg IPTG. The cells were incubated on ice for 30 minutes then heat shocked by placing in a 42“C water bath for 45 seconds. The heat-shocked cells were returned to ice for 2 minutes. 0.9 ml of S.O.C. was added to each tube which was then shaken at 37“C for 1 hour. The mixture was plated onto LB plates (LB containing 15g/l bacto-agar and 50|ig/ml of amplicillin) and incubated overnight at 37“C. Single white colonies were selected and cultured in LB containing ampicillin overnight at 37°C and glycerol stocks were prepared (Sambrook et al, 1989).