SSCP and heteroduplex analysis were performed using slight modifications of the techniques originally described by Orita et al (1994) and White et al (1992) respectively.
2.3.6.1 SSCP Analysis.
2.3.6.1.1 Preparation o f PCR Samples.
4\i\ of each second round PCR product were diluted 1/5 with formamide loading buffer in a MicroSample™ plate. The samples were denatured for 2 mins at 94°C and then quick-chilled on ice ready for immediate electrophoresis.
2.3.6.1.2 Polyacrylamide Gel Electrophoresis.
Glass plates for use with Hybaid vertical electrophoresis tanks were cleaned with detergent and wiped with ethanol. The smaller plate was siliconised by coating with dimethyldichlorosilane solution and HCl washed off with ethanol. Plates were sandwiched together separated by 0.4 mm spacers and clamped along the edges. Acrylamide gels were prepared using MDE™ gel mix according to the manufacturer's instructions. To perform electrophoresis under two conditions (for greater efficiency of mutation detection) gels were made to concentrations of Ix MDE (for 60 ml gels; 30 ml MDE™ gel mix, 3.6 ml lOx TBE, 26.4 ml dHzO 360gl APS and 36gl TEMED) or 0.5x MDE/ 5% glycerol (for 60 ml gels; ISmls MDE™ gel mix, 6mls 50%
glycerol, 35.4 ml dHiO, 360pi APS and 36pi TEMED). The gel mix was poured between the plates using a 50 ml syringe, a sharks tooth comb placed along the top of the gel and allowed to polymerise for Ihr.
4 pi of the denatured samples and a non-denatured control sample were loaded onto the gel and electrophoresed at 4®C, at 6V, for ~20hrs, or until the xylene cynol marker had reached the bottom of the gel.
2.3.6.1.3 Autoradiography.
The small plate was prised away from the gel and a sheet of 3MM chromatography paper pressed onto the gel. The gel was lifted off, covered with cling film and dried for 1 hr at 80®C. The cling film was removed and the gel exposed to X-ograph Blue X-ray film at -70®C overnight.
2,3,6,2 Heteroduplex Analysis.
2.3.6.2.1 Preparation and Electrophoresis o f Samples.
4 pi of the second round (non-radioactive) PCR products were diluted 1/2 with 20% glycerol loading buffer. 4 pi of this dilution was directly loaded onto MDE™ acrylamide gels, prepared and electrophoresed as for SSCP analysis. For each gel a known heteroduplex sample was also run to act as a control.
2.3.6.2.2 Silver Staining.
The small plate was prised away from the gel, and the gel lifted onto 3MM
chromatography paper, as described above. The gel was trimmed and then placed face down in a large tray containing 500ml of 10% ethanol. The chromatography paper was removed and discarded. The gel was fixed in the ethanol for 5 mins, after which the ethanol was aspirated away and 500ml of 1% Nitric acid was added to oxidise the gel for 3 mins. The acid was again aspirated away, and the gel was rinsed in mileiQ dHiO for a few seconds. To impregnate the gel with silver, 500ml of 0.012M AgNOs was applied and shaken sporadically for 20 mins. After removal of the silver solution and a second rinse with mileiQ dHiO, a solution of 0.28M NazClg, 0.019% formaldehyde in mileiQ dHiO was applied to the gel and shaken gently until the DNA bands became visible. When the developing reaction had reached
completion (the colour of the DNA bands had reached the desired intensity) the solution was removed and 500ml of 10% acetic acid was added for 2 min after which the gel was given a final rinse. During the staining procedure the gel expands. To shrink the gel back to the desired size 500ml of 50% ethanol was placed onto the gel and left for ~30 min (the time here is not critical). A fresh piece of 3MM
chromatography paper was placed onto the gel, to which it sticks. The gel was then covered in cling-film and dried for IV2 hrs at 80®C.
2,3,7 Sequencing,
For the majority of this project sequencing was performed manually. However the department recently obtained an ABI Prism 377 DNA Sequencer (Perkin Elmer), and the sequencing projects currently in progress are being performed using automated techniques. Furthermore, a trial sequence run was also performed using an
Automated Laser Fluorescent ALF™ DNA sequencer (Pharmacia).
2.3.7.1 Manual Sequencing.
2.3.7.1.1 Preparation o f Double-Stranded Template.
Plasmid DNA for sequencing was prepared by the methods previously described. An estimated l-5pg DNA were resuspended in 50pl dH20 and denatured in an equal volume of a 0.4M NaOH, 0.4mM EDTA solution for 30 min at 37®C. The DNA was precipitated by adding 0.3M sodium acetate and 2.5 volumes of ethanol and leaving on dry ice for 15 min. The recovered DNA pellet was resuspended in 7pl dHzO
2.3.7.1.2 Sequencing Reactions.
Sequencing was performed using a Sequenase™ Version 2.0 DNA Sequencing Kit. Annealing reactions were set up by adding 2pi Reaction Buffer and 1 pi primer (0.5-1.0 pmol) to 7pi denatured or M l3 single-stranded DNA. The reactions were heated to 65®C in a beaker in a water bath for 2 mins. The beaker was removed and the reactions allowed to cool to 37°C at RT. Meanwhile, 2.5 pi of each Termination Mix (dideoxy-nucleotides) were aliquotted into wells in a MicroSample™ plate. A bulk reaction mix was made for several reactions and 5.5pi, comprising the following, added to each annealed primer-template; 2pl diluted Labelling Mix (1/5 dilution in water), Ipl DTT, 0.5pl [a^^S]-dATP and 2pl diluted Sequenase (1/8 dilution in Enzyme Dilution Buffer). The microsample plate was floated in a 37®C water bath. After 5 min at RT, 3.5 pi of each reaction was added to each Termination Mix with careful mixing. After 5 min, 4pi Stop Solution were sequentially added to termination reactions.
2.3.7.1.3 Polyacrylamide Gel Electrophoresis.
Sequencing plates were cleaned with detergent and wiped with ethanol. The smaller plate was siliconised by coating with dimethyldichlorosilane solution and HCl washed off with ethanol. Plates were sandwiched together separated by 0.4 mm spacers and clamped along the edges. Acrylamide gels were generally 6% and were made according to the manufacturer's instructions. For 60 ml gels, 40 ml diluent, 14.4ml concentrate, 6 ml lOx TBE, SOOpl APS and 30pl TEMED were mixed and poured between the plates using a 50 ml syringe. Sequences were denatured for electrophoresis by placing the MicroSample™ plate on a boiling block for 2 min. 3 pi of each sequencing reaction were loaded onto the gel and samples electrophoresed at 70 V for 2-3 h or 65 V for 6-7 h depending on the required length of gel run.
2.3.7.1.4 Autoradiography.
The small plate was prised away from the gel and a sheet of 3MM
chromatography paper pressed onto the gel. The gel was lifted off, covered with cling film and dried for 45 mins at 80®C. The gel was exposed to Kodak BioMax film at RT. Exposure was overnight for Ml 3 sequences and usually 2-3 overnights for double-stranded sequencing.
2.3,7.2 Automated Sequencing.
Double stranded plasmid DNA sequencing was performed using the ABI
PRISM™ Dye Terminator Cycle Sequencing ready reaction kit, with AmpliTaq^ DNA polymerase, FS (Perkin Elmer). 200-5OOng of double stranded DNA, resuspended in 6pi of mileiQ dH20, was placed in an Omigene PCR tube with 3.2pmol primer and 4pl of the dye terminator reaction mix. Cycle sequencing was performed in a Geneamp 2400™ (Perkin Elmer), using a heated lid programme. For any primer the procedure was as follows; 20 cycles of, 94®C dénaturation, 20 secs; 53®C annealing, 20 secs; 60®C elongation, 2 mins.
30pl of an ethanol/3M sodium acetate solution was added to each sample and incubated at 4®C to precipitate the DNA. After centrifugation and washing in 70®C, the recovered DNA pellet was resuspended in 4pl of ABI loading buffer.
The samples were subjected to electrophoresis through a 10% acrylamide gel (according to the manufacturer’s instructions). Sequence samples were then analysed using the computer software provided with the ABI Prism 377.
Automated sequencing was also performed on the Automated Laser Fluorescent ALF™ DNA sequencer (Pharmacia). To sequence double-stranded plasmid DNA templates, 5-lOpg of DNA were denatured by incubation at RT with 2M NaOH for 10 mins. The DNA was precipitated as described for manual sequencing, and resuspended in lOpl of dHiO. Sequencing was performed using the Autoread™ Sequencing kit according to the manufacturer’s instructions. A primer labelled with fluorescein at its 5’ terminus was annealed to the template and then using standard dideoxy sequencing methods the primer was extended by T7 DNA polymerase in four separate dideoxy reactions. The samples were electrophoresed according to the manufacturer’s instruction, and sequence was analysed with the software provided for the ALF™.
2,3,7,3 Direct Sequencing o f PCR Products,
DNA PCR products were purified using the Magic”^^ PCR DNA Purification Systems in accordance with the manufacturer’s instructions (Promega). The purified DNA was resuspended in dHzO to a concentration of ~100ng/pl. To anneal the PCR products to the required primer, 50ng of primer, Ix Sequenase buffer (Amersham), 1.6pl DMSO and lOOng of the DNA were added to an Eppendorf tube to a final volume of 7pl. The contents were mixed, denatured for 3 mins and then snap-cooled on dry ice. Meanwhile the four termination mixes were prepared as follows:
dideoxy A mix, 0.5mM dCTP, dGTP, dTTP, O.OSmM dATP, O.OSmM ddATP; dideoxy C mix, 0.5mM dCTP, dGTP, dTTP, O.OSmM dATP, O.OSmM ddCTP; dideoxy T mix, O.SmM dCTP, dGTP, dTTP, O.OSmM dATP, O.OSmM ddTTP; dideoxy G mix, O.SmM dCTP, dGTP, dTTP, O.OSmM dATP, O.OSmM ddGTP. 2|il of each termination mix was aliquotted to a Micro Sample™ plate and kept on ice until the next step.
primer/DNA solution. This was vigorously mixed, collected by pulse
centrifugation and then 2 pi was added to each microsample well containing the termination mix. The reaction was incubated for 5 mins at 37®C. After this period, 2pl of Chase solution (0.25mM dNTPs, 10% DMSO) was added to each reaction and incubated for a further 5 mins at 37®C. The sequencing was stopped by addition of 3 pi of stop solution (Amersham). The samples were denatured and subjected to electrophoresis, as for standard manual sequencing techniques.
2.3.7.4 Sequence Analysis,
All computer programs for sequence analysis and database searching can be accessed at the Human Genome Mapping Project (HGMP), Hinxton Hall,
Hinxton, Cambridge. The Basic Local Alignment Search Tool (BLAST; Altschul
et al, 1990) was used to compare sequences with GenBank (Benson et al, 1993), EMBLE (Emmert et at 1994), TREMBLE (Bairoch & Apweiler 1996) and the SWISS-PROT protein database (Emmert et al 1994). The Gene Recognition and Analysis Internet Link (GRAIL; Uberbacher and Mural, 1991) was used to predict coding sequence. The GCG programs, MAP and Bestfit, can also be accessed at the HGMP, as can the Fugu rubripes genome database. YAC and STS data was accessed from the Whitehead Institute database, also via HGMP.