2-1 : Isolation of cosmid DNA
The cosmid was streak out directly from the -70° C gycerol stock onto an LB plate with 25pg/ml ampicillin, in order to obtain single colonies. The plate was incubated overnight at 37°C. A 50ml Falcon tube, containing 5ml LB broth with 25pg/ml ampicillin was innoculated with a single colony and incubated overnight in shaking incubator at 37°C.
1.5ml of culture was transferred to an Eppendorf tube and centrifuged for 15 min 13000 rpm. The supernatant was poured off into a large beaker containing Tegadyne and the pellet resuspended in 200pl GTE. 5pl RNAase A stock [lOmg/ml] was added. 400pl 0.2 M NaOH/1% SDS (freshly made) was added. The tube was mixed by inversion and placed on ice for 30 min. 300pl 3M KAc was added , the tube inverted to mix and placed on ice for 30 min. The tube was centrifuged for 10 min at 13000 rpm in a microflige. The supernatant was decanted into an Eppendorf tube containing 1 ml cold ethanol, briefly vortexed and left to stand for 1 min. The tube was centrifuged 10 min at 13K rpm in microflige. The ethanol was carefully removed using the vacuum pump fitted with a drawn out glass pasteur pipette. 200pl cold 70% ethanol was added, briefly vortexed, centrifuged 10 min at 13K rpm in microfuge and the ethanol removed carefully using the vacuum pump fitted with a drawn out glass pasteur pipette. The pellet was air dried. 50pl 1 x TE was added and the tube left to resuspend overnight at 4° C in ftddge.
The DNA concentration was estimated by measuring the optical density of a 1:100 dilution at 260 nm in a spectrophotometer. Based on the constant that a DNA solution of
1 .Opg/pl will give a reading of 0.5 OD260 unit, the concentration was worked out by
multiplying the OD260 value by a factor of 2 to give the concentration of the stock in
pg/pl.
2-2: ProCipitate Purification of cosmid DNA.
Cosmid DNA destined to be used as template for direct sequencing was prepared in the same way as in the previous protocol, adding 100 pi of ProCipitate along with KAc at step 7. The ProCipitate was thoroughly mixed before addition.
2-3: Restriction digests
Restriction digestion of DNA was carried out for a variety of purposes such as the assessment of DNA quantity and quality, obtaining fingerprints in order to determine the extent of overlap between different clones and to generate Southern blots.
Commercial enzymes were used (New England Biolabs, Boehringer Mannheim) and the reactions carried out for at least one hour at 37°C using 20 units of enzyme and the buffers supplied. Digests were separated on agarose gels.
2-4: Agarose gel electrophoresis
DNA fragments were separated and sized by gel electrophoresis. Agarose gels were made up in Ix TBE buffer and ethidium bromide added to a final concentration of 500 ng/ml. The strength of agarose gel varied according to the size of DNA fragments. Large fragments, such as cosmid digests and large PCR products were run on 0.8% (w/v) agarose gels. DNA fragments smaller than 2 kb were separated on 1.5 % (w/v) agarose gels. The size of gel used depended on the application. Typically, a 20 cm gel would have been used for cosmid digests and 10 cm minigels used for most PCR checking gels. Commercially prepared molecular weight markers were loaded on agarose gels to aid the sizing and quantification of fragments.
2-5: Alkali Transfer Southern Blot
2 |ig cosmid DNA was mixed together in an Eppendorf tube with 1 |il of restriction enzyme, 1 jil of the restriction enzyme buffer and 6 jil of Filter sterilised, autoclaved
MilliQ water. The reaction was incubated at 37°C for 1 hour and then run out on 0.8% agarose gel. The gel was photographed, placing a ruler along the left hand edge to provide a scale The gel was placed in a tray, covered with 0.25 M HCl, shaken for 10 minutes and rinsed in water. The gel was covered with denaturing buffer, shaken for 30 minutes and the excess buffer poured off. The gel was equilibrated by covering with alkali transfer buffer. The blot was set up in the following manner: a platform was set up inside a tray at least 5 cm longer and wider than the gel. A wick the same width as the gel and 10 cm longer was cut out of chromatography paper (Whatman 3MM). Three further pieces of chromatography paper the same size as the gel were also cut out, along with a piece of nylon membrane (Genescreen +) also the same size as the gel. The wick was placed on the platform, with the ends touching the bottom of the tray and wet with alkali transfer buffer. Air bubbles were eliminated with a suitable roller and the gel placed on top. The membrane was placed on top of the gel, the bubbles chased out and the three pieces of chromatography paper placed on top of the membrane. A stack of absorbent paper around 5 cm thick was placed on top of the blot and a weight around 750 g on top of the absorbant paper. The bottom tray was topped up with alkali transfer buffer and the blot left overnight. The chromatography paper and agarose gel were discarded and the membrane rinsed in 2x SSC. The membrane was placed in a shaking bath of neutralising buffer for 30 minutes then air dried for 30 minutes. The DNA was cross-linked to the membrane in the UV Cross linker using the optimal cross link setting. The membrane was wrapped in cling film.
2-6: DNA Filter Hybridisation
The filters were placed in 20x20 cm polyethylene ‘pizza storage container’, covered with 100 ml Hybridisation Buffer and the boxes sealed with their lids. The filters were pre hybridised by incubating in shaking hybridisation oven for a minimum of two hours. For homologous probes (from the same species as the library), the temperature was set at 65°C. In the case of a heterologous probe (from a different species to the library), the temperature was set at 50°C. Probes were labelled using the rediprime kit (Amersham) according to the manufacturers instructions. The probe was added to Hybridisation mix, taking great care to minimise exposure to radiation of self and others and adhering strictly to local rules. The container was returned to hybridisation oven and left overnight. Hybridisation buffer was discarded via the designated sink. Wash buffer was added, swirled around the box to rinse and discarded. More wash buffer was added, the container sealed and left on shaking platform 10 mins. Wash buffer was discarded and activity monitored at 1 cm from the surface of the filters with portable Geiger counter fitted with p-particle probe. If the activity was uniformly above 100 counts per seconds (cps), wash was repeated. If after the second wash the activity remained high, wash was repeated at 50 °C in hybridisation oven. Filters were wrapped individually in cling film, placed in exposure cassettes with filter number at top left had comer and taken to dark room. A sheet of X-ray film was placed in each cassette and these were stored in -80°C freezer overnight. Cassettes were left to come to room temperature for 20 mins and films fed into a film processor. Films were checked for correct exposure (background grid of the array visible and positive signals clear and discrete). If another exposure was
required (longer or shorter), another sheet of film was placed in each cassette and they were returned to -80°C. Autoradiographs were examined on a light box and positive clones identified by referring to the interpretation sheet. Filters were stripped by
incubation in a solution of 0.4 M NaOH (freshly made) at 45 °C with shaking for 30 mins followed by equilibration in Neutralising Buffer at room temperature for a minimum of
15 minutes. Filters were wrapped in cling film and stored in designated fridge.
2-7: Preparation of random cosmid DNA fragments for ligation into M13 or pUC vector
A 2pg aliquot of DNA was placed into an 1.5ml tube and the solution made up to 200pl with sterile Milli-Q water. The DNA was subjected to 20 cycles of shearing at speed setting 3 using a Hydroshear, which should generate ffangments in the range of l-2kb. The sheared DNA was incubated for two hours at room temperature with 5pl 2mM dNTPS, 5pl lOx T4 DNA polymerase buffer, 0.5pl T4 DNA polymerase in an eppendorf tube in order to end-fill the ragged ends.
An equal volume of 20% PEG solution was added. The tube was vortexed and incubated at room temperature for 5-10 minutes. The tube was then spun for 10 minutes at 13000 rpm. The supernatant was removed, the pellet rinsed with 70% ethanol, dried and resuspended in 20pl of sterile Milli-Q water. 5pl was run out with DNA marker VI on a
2-8: Ligation and transformation of sonicated, end-repaired and size-selected cosmid fragments into the PUC plasmid vector.
Vector was prepared by digesting 1 Opg of pUC 18 plasmid with 1 p 1 of Smal fori hour at 37° C and the enzyme heat inactivated by incubating the plasmid at 65° C for 20 mins. The Jinearised pUC18 plasmid was dephosphorylated, in order to prevent the plasmid from religating to itself in cloning ligations, with Ip l of shrimp alkaline phosphatase (SAP) at 37° C for 30 mins. The SAP was inactivated by incubating at 80° C for 15 mins. The DNA was diluted to 50ng/pl.
The sheared DNA fragments were ligated into the prepared vector in the following reaction: 0.5pl Smal cut pUC18, Ipl sheared DNA, Ipl lOx ligation buffer, Ipl T4 DNA ligase and 6.5pl sterile Milli-Q water were mixed together in a 0.5 ml tube and incubated overnight at 15° C.
A negative control was set up, in which the DNA is replaced by water and a positive control, in which the (|)X174/HaeIII molecular weight marker, a blunt-ended DNA of similar size distribution was used.
The recombinant plasmids were used to transform ultracompetent E. coli XL-2 cells (Stratagene). 2pl of B-Mercaptoethanol was added to a tube of XL-2 Blue E. coli cells. The ligation mix was added to 15pl of ultracompetent cells and left on ice for 10 mins. The cells were heat shocked at 42° C for 47 seconds. 500pl of SOC (pre-warmed to 37° C) was added and incubated with shaking at 37° C for 1 hour. 75pl of IPTG/X-Gal was added and mixed (25pl of 200mM fPTG and SOpl of 8% X-Gal). 500pl of
cells/SOC/IPTG/X-Gal mix was poured onto the required number of TYE/ampicillin plates, which were left to dry and incubated overnight at 37° C.
The transformants (white colonies) were picked into 96 well plates containing 160pl of 2xTY and ampicillin (25pg/ml). The cells were grown overnight at 37° C. After
incubation, SOpl of 50% glycerol was added. The cells were then stored at -20° C.
2-9: DNA preparation from plasmids
Deep well blocks (2ml) were filled 1ml aliquots of 2xTY/ampicillin [25pg/ml]. They were inoculated from the glycerol stocks using a 96 pin hedgehog and incubated for 22 hours in a shaking incubator, 320 rpm, at 37° C. The blocks were spun in a centrifuge (Jouan GR422) for 2 mins at 4K rpm to pellet the cells. The supernatant was poured off and the block inverted on tissue to allow the residual supernatant to drain away. SOpl of GTE/RNAse was added and the blocks gently shaken until all the cells had been
thoroughly but gently. The blocks were incubated on the bench for an additional 2-5 mins (no longer than 5 mins). 80^1 of 3.6M KO Ac was added and mixed thoroughly but gently. An FB capture plate (Millipore) was placed in the bottom of a vacuum manifold (CP labs) and and MultiScreen-NA filter plate (Millipore) placed on top. 1 SOpI of lysate was transferred well-to-well from the deep well plate to the NA plate using a
multichannel pipettor. The vacuum pump was turned on and the vacuum adjusted to below 8 inches Hg to ensure uniform filtration. The suction was applied for a further 3 min, drawing the lysate through the NA plate into the FB plate. The FB plate was removed and 150pl of Bind Solution added to each well. The clear lysate was mixed thoroughly by rapid pipetting. A waste plate was placed in the bottom of the manifold, the FB plate placed on top and the vacuum applied at full strength for 1 min to bind the DNA to the FB plate. 200pl of 80% ethanol was added to each well and vacuum applied at full strength for 1 min. This was repeated, the FB plate removed, blotted dry and vacuum dried for 5 mins. The DNA was eluted by adding 70pl of dH20 to each well and spinning at 4K rpm for 2-5 mins.
2-10: Sequencing pUC plasmid DNA preparations
2-10-1: Dye terminator sequencing using Big Dyes:
3pl of template DNA was transferred to each well of a thermofast plate along with 4pl Big Dye mix, 0.32pl appropriate sequencing primer [lOpM] and 3pl dH20 per reaction.
The placed were put through the following temperature cycles on an MJ Tetrad: 25 cycles of: 96°C for 10 secs, 50°C for 5 secs and 60°C for 4 mins and then held at 15°C .
2.5pl of 2M EDTA was added to each well of a polypropylene 96 well plate (Costar). The reaction product was recovered by adding 25pl 100% ethanol to each sample, mixed by pipetting and transferred to the pooling plate.
The plates were spun at 4000 rpm for 30 mins at 4°C on Jouan GR422 centrifuge. The alcohol was flicked out, 50pl 70% ethanol added and the plate spun for 15 min at 4K rpm, at 4°C.
The alcohol was flicked out, the plate tapped out on tissue papers and dried 5 min in vacuum drier (Jouan ???). 4pl loading buffer (dilute formamide) was added, the plate sealed with cycle seal, vortexed, heated to 95°C for 2 min using the MJ Tetrad and stored on ice prior to loading.
2-10-2: Dye primer sequencing using energy transfer (ET) chemistry (Amersham):
4 thermofast plates were labelled A, C, G and T and 2pl DNA added to each well. 8pl Amersham ET Dye Primer Sequencing kit with Thermosequanase A mix was added to each well of A plate, on ice. 8pl Amersham ET Dye Primer Sequencing kit with Thermosequanase C mix was added to each well of C plate, on ice. 8pl Amersham ET Dye Primer Sequencing kit with Thermosequanase G mix was added to each well of G plate, on ice. 8pl Amersham ET Dye Primer Sequencing kit with Thermosequanase T mix was added to each well of T plate, on ice. The plates were sealed using the heat sealer, spun briefly and cycled on a MJ Tetrad.( 20 cycles of [92°C for 15 secs, 50°C for 15 secs and 70°C for 60 secs]; 15°C hold.) lOpl 3M NaAc was added to each well of a polypropylene 96 well plate (Costar). Finished reactions were pooled with a 12 channel 5-50 pipettor by adding lOOpl cold ethanol to each sample on top row of A plate, mixing by pipetting up and down six times, transferring to top row of C plate, mixing, transferring to G plate and so on, and finally transferring to pooling plate. The process was repeated for all rows. The pooling plate was spun for 30 mins at 4000rpm using Jouan CR422 centrifuge. lOOpl 70% alcohol was added, the plate spun 10 min the alcohol flicked out and the plate tapped out on tissue. The plate was vacuum dried for 20 min. 4pl of loading buffer (dilute formamide) was added to each well, the plate sealed with cycle seal, vortexed, heated to 95°C for 2 min using the MJ Tetrad (Instant heating cycle) and stored on ice prior to loading.
2-10-3: Use of automated sequencers
Sequencing reactions were read on polyacrylamide (PAGE) slab gel automated sequencers (ABI 373S and 377) or capillary automated sequencers (ABI 3700 or Molecular Dynamics Megabace) according to the manufacturers instructions. Polyacrylamide gels were prepared by mixing 20 ml 5% PAGE PLUS (Southern Biotech), 150 pi 10% (w/v) Ammonium Persulphate (APS) and 15 pi TEMED. The gel was poured between the glass plates clipped together, separated by their spacers. The casting comb was inserted as per the manufacturer’s instruction and left to polymerise for 90 minutes. The set gel was carefully cleaned, mounted on the machine and pre-run as per the manufacturer’s instruction. 2 pi of each sequencing reaction was loaded according to the manufacturer’s instruction. Capillary sequencers were operated according to the manufacturer’s instruction.
2-11: Solutions:
A garose loading dye (lO x): 20% (w /v ) F icoll 4 0 0 , 0.4% (w /v ) brom ophenol blue, 0.1 M E D T A . A lk ali Transfer Buffer: 0.25M N aO H , 1.5M N aC l.
B ind Solu tion for plasm id preps: 6.1M potassium iodide.
Church Gilbert buffer: 1 m M E D T A , 0.5 M N aH P0 4 (pH 7.2), 7% (w /v ) S D S . Filter sterilised. Church Gilbert w ash buffer: 1 m M E D T A , 4 0 m M N aH P0 4 (p H 7.2), 1% (w /v ) SD S. F ilter sterilised. D enaturing Buffer: 1.5M N aO H , 0 .5M N aC l.
Form am ide loading dye:
GTE: 50 m M g lu co se, 25m M Tris(pH 7 .5), 10 mM EDTA . N eu tralisin g Buffer: IM N aC l, 0.5M Tris pH 7.2
PEG (20% ): 20 g p olyeth ylen e g ly co l (PEG ) 8 0 00, 66pl IM M gC l2, 20 m l 3 M K ac. M ade up to 100 ml w ith d dH 20. N o t autoclaved.
S eq u en cin g gel mix: 2 0 m ls Page plus g el m ix, 15pl TEM ED, 150p l 10% A m m onium Persulphate (A P S l.O g in 10m l S D W ).
SSC (20x): 3M N aC l, 0.3 M sodium citrate (pH 7).
TBE buffer: 0 .0 9 m M T ris, 0 .0 9 m M borate, 0 .0 0 2 nm E D T A pH 8
X -G al: 8% (w /v ) 5-brom o-4-chloro-3-indolyl-P -D -galactosidase in dim ethylform am ide. Stored at -20°C , wrapped in foil to sh ield from light.
FGF2 to SPAF
C hapter Three
Analysis of a highly conserved genetic region in man, mouse and Fugu: FGF2 to SPAF (human 4q25-27)
3-1 Introduction
The aim of this part o f the project is to compare the sequence o f a Fugu genomic region to the equivalent region in man and mouse. This permits the evaluation of currently available bioinformatics tools available and establishes the foundations for further comparative sequencing studies.
The criteria for selecting such a region were that it had to be: • gene rich
• containing well conserved genes • showing conserved gene order • sequenced in man and mouse.
It was important for the region to be gene rich in order to have plenty o f data for comparative analyses. The presence of highly conserved genes would provide a greater number of species for comparison and conserved linkage groups may show conservation of intergenic features such as regulatory elements. The region around human FGF2, on chromosome 4q25-27, shows a high degree o f similarity to the corresponding mouse (strain Svl29) region, containing a number o f genes such as SPAF and NUDT4 (originally named GFG) (Lyons, 2000). Genomic sequencing of the region in the mouse strain Svl29 highlighted the presence o f a ‘caltractin related gene’ (Paul Lyons, personal communication).
FGF2 to SPAF
The human sequence for the region is being obtained as part o f the Human Genome