CONCEPCIONES DE LA INFANCIA COMO SUJETO DE DERECHOS
4.4. A manera de conclusión: las concepciones de la infancia como sujeto de derechos
Chemicals and solutions
All company details can be found in appendix 1
All chemicals were obtained from Fischer Scientific except for those listed below.
Acrylamide (19:1) National diagnostics
Agarose Sigma-Aldrich
Ammonium persulphate Biorad
BSA for Denhardts ICN
BSA for Jeffreys PCR buffer(1 Omg/ml) Amersham Pharmacia
Bromophenol blue Merck BDH Chemicals
Ethidium bromide tablets Sigma-Aldrich
Sonicated Herring Sperm DMA (1 Omg/ml) Promega
2-Mercaptoethanol Amersham Pharmacia
dATP, dCTP, dGTP, dTTP for Jeffreys' PCR buffer (each nucleotide bought
separately all at lOOmM) Amersham Pharmacia
DNA polymerisation mix (dATP, dCTP.dGTP, dTTP) (20mM)
Amersham Pharmacia
Xylene cyanole Sigma-Aldrich,
Glycerol Merck BDH Chemicals
Ficoll type 400 Amersham Pharmacia
Radiochemicals
®^PdCTP (0.37 MBq/^l) ICN Biomedicals
Enzymes
All restriction enzymes used were from New England Biolabs
Taq polymerase in storage buffer A (5U/|il) Pfu poiymerase(3U/|il)
Dynazyme EXT (1U/|il) Exonuclease I (lOU/^il)
Shrimp Alkaline Phosphatase (2U/|il)
Promega Promega Finnzyme Amersham Pharmacia Amersham Pharmacia Miscellaneous
Ready Reaction Big dye terminator cycle
Powered silica Glassmilk
Sodium Iodide (pHG-7.4) Raoul Marker (30|xg/|il) Kb ladder (1|ig/|il) Hybond N+ membrane Nick Columns
Multi/ Megaprime labeling kit Puregene DNA extraction kit Super HRE X-Ray Film Spermidine
sequencing kit
PE Applied Biosystems Sigma
Gift from C.Herd Gift from C Herd Quantum Appligene Gibco BRL Amersham Pharmacia Amersham Pharmacia Amersham Pharmacia Gentra Systems Fuji film Promega
Buffers and solutions
All buffers were made up with reverse osmosis (Millipore, Watford) water and stored at room temperature unless otherwise stated.
Glassmilk The glassmilk was made by resuspending finely
powdered silica in Nanopure water. The
suspended silica mix was left to settle for 2 minutes, the supernatant was removed and was left to settle for a further 1 hour. After an hour the supernatant was removed and discarded (1-g sedimentation cut). The slurry was simmered in an equal volume of nitric acid for about 20 minutes.
The mix was allowed to cool and then washed thoroughly with Nanopure water and resuspended in an equal volume of water.
20 X SSC (Saline Sodium Citrate):
3M Sodium Chloride (NaCI), 0.3M sodium citrate (Na3H507.2H20). Adjusted to pH7.0 with saturated citric acid.
5 X TBE (Tris, Boric acid, EDTA):
0.44M Tris, 0.44M Boric acid, 0.01 M EDTA. pH PH8.2-8.4
100 X Denhardts: 2%(w/v) Ficoll type 400, 2% (w/v) PVP, 2% (w/v)
BSA. Approximately pH7. The solution was filter sterilised, aliquoted and stored at -20°C.
Pre-hybrisisation solution: 6xSSC, 0.5% SDS, 5xDenhardts. Prewarmed to 65°C
First wash solution: 2xSSC, 0.1 % SDS, pre-warmed to 65°C
Second wash solution: 0.2 x SSC, 0.1 % SDS, pre-warmed to 65°C
DNA loading buffer: 15% Ficoll type 400. Bromophenol blue and xylene
cyanol to colour
Depurination solution: 0.25M MCI
Dénaturation solution: 1.5M NaCI, 0.5M NaOH
Neutralisation solution: 1.5M Tris, 0.5M NaCI, ImM EDTA. Adjusted to
Buffer IV supplied with AB Taq:
50mM Tris-HCI (pH 8.8 at 25°C), 200mM
(NH4)2S0 4, 0.1% (v/v) Tween20, 15mM MgCb
autoclaved and supplied sterile, stored at -20°C. AB Gene
Buffer 2 supplied with AlwN1:
20mM Tris-HCI, lOmM Mg Acetate, 50mM KAcetate, 1 mM DTT, stored at -20°C. New England Biolabs
Buffer 4 supplied with Hinfl:
lOmM Tris-HCI, lOmM MgCb, ImM DTT, stored at -20°C.
New England Biolabs
NEETwash: lOOmM Sodium Chloride, 50% Ethanol, ImM
EDTA, lOmM Tris
Jeffrey’s buffer (recipe from J. H. Stead): 11.1 x buffer (Jeffreys et al., 1990)
0.49M Tris-HCI (pH8.8), 0.12M (NH4)2S0 4, 0.05M
MgCl2, 44pM EDTA (pH8.0), Ilm M all dNTPs,
1.26pg/pl BSA and 0.7M 2-mercaptoethanol. Aliquoted and stored at -20°C
Slagboom buffer: lOOmM NaCI, lOmM Tris-HCI (pH 8.0), lOmM
EDTA (pH 8.0), 0.5% SDS, 0.2mg/ml proteinase K
Magic Mix: Phenol chloroform containing solution of
undisclosed composition. Kindly supplied by Prof Ian Craig and Bernard Freeman.
Equipment
VacuGeneXL, vacuum blotting system Amersham Pharmacia
Thin walled tubes Perkin Elmer
Molecular biology methods
DNA extraction from whole blood
Blood was collected in ETDA and stored at -70°C until extraction. The Puregene kit was used and the manufacturer's instructions followed for extracting DNA from 3ml of whole blood. If there was less than 3ml of blood the reagents were scaled down as shown in table 2.1.
Reagent
Volume of whole blood
3ml 2ml 1ml 0.5ml
Red blood cell
lysis solution
9ml 6ml 3ml 1.5ml
Cell lysis solution 3ml 2ml 1ml 0.5ml
RNase 15pJ 10pl 5|il 2.5pl Protein precipitation solution 1ml 0.67ml 0.33ml 0.15ml 100% Isopropanol 3ml 2ml 1ml 0.5ml 70% ethanol 3ml 2ml 1ml 0.5ml DNA hydration solution
250^1 lOOpI lOO^il lOOfil
Table 2.1 Scaled reagents for the Puregene kit, which was used for the isolation of genomic DNA from whole blood.
DNA extraction from buccal cells
The buccal cells were collected by rubbing ten cotton bud swabs around the inside of the cheeks and along the gum line. The swabs were then submerged in Slagboom buffer and were kept in the dark at room temperature until extraction. The tube with the cotton bud swabs in was inverted into a 50ml Falcon tube and centrifuged at 647xg at room temperature. The swabs were drained and discarded. 0.3ml Magic Mix was added to the residual liquid and the solution mixed by inversion for one minute. The tubes were then
centrifuged at 8,500xg at room temperature for 25 minutes. The supernatant was removed and to this another 0.3ml Magic mix was added. The solution was mixed for a further minute before centrifugation at 8,500xg at room temperature for 25 minutes. The supernatant was removed. This process extracted the DNA from the buccal cells. To the supernatant 1.8ml 100% isopropanol was added and mixed by inversion. The solution was centrifuged
at 8,500xg at room temperature for 25 minutes. The supernatant was
removed and discarded and the resulting pellet washed with 70% ethanol. The mix was centrifuged at 8,500xg at room temperature for 10 minutes. The supernatant was removed and discarded. The pellet was air dried for 15 minutes and was then re-suspended in 0.4ml DNA hydration buffer.
Agarose gel electrophoresis
All agarose gels were made up with IxTBE and the running buffer was IxTBE. Usually 1-2% agarose gels were used. For minigels 50ml agarose gel was used to which 1 pi (5mg/ml) ethidium bromide was added before pouring. The gels were electrophoresed in 50ml running buffer to which 1 pi (5mg/ml) ethidium bromide had been added. The gels were visualised with a UV transilluminator (Ultra Violet Products (UVP), Cambridge)
Big Dye Sequencing
Each sequencing reaction contained 4pl Ready Reaction mix (Applied Biosystems) and 4pmole oligonucleotide primer, and was made up to 20pl
with autoclaved Nanopure water. The Ready Reaction mix contained
AmpliTaq DNA polymerase FS, pyrophosphatase, deoxynucleotides,
magnesium chloride Tris buffer at pH9 and the fluorescently labelled dye terminators.
The sequencing products were precipitated using the ethanol method,
as described in the ABI (Applied Biosystems Inc.) manual. If oil was used on
top of the sequencing reaction it was all carefully removed and 64pl 95% ethanol and IGpl autoclaved Nanopure water was added to each reaction. The reactions were briefly vortexed before being allowed to stand at room
reactions were centrifuged for 20 minutes at 13,250xg at room temperature. The supernatant was removed and 250^1 70% ethanol added to each pellet. The reactions were centrifuged again for a further 10 minutes at 13,250xg at room temperature. The supernatant was again aspirated. The pellets were dried by heating to 90°C for 1 minute.
Preparation of template for sequencing
Sequencing of point mutation at position 3506 in exon 2
Each PCR reaction contained lOpt buffer IV (ABGene), 0.2mM dNTP
and 2.5U ABTaq DNA polymerase. Primers Exon2A and ExonIS were both
used at a concentration of IpM. 10% glycerol (v/v) was used to prevent secondary structure formation and aid amplification (Lu, 1993). The reaction was made up to lOOfxl with autoclaved Nanopure water and was amplified using the Genius PCR machine. 400ng genomic DNA was amplified and the reaction conditions were; a dénaturation step at 94°C for 15 seconds, an annealing step at 55°C for 30 seconds and an elongation step at 70°C for 30 seconds, and 30 cycles were performed. The PCR product was checked on a 2% agarose minigel, and the PCR product size was 677bp.
To clean up the PCR product for sequencing two methods were used: either enzymatic removal of primers dNTPs etc or extraction of DNA from an agarose gel slice. Both systems are described below.
Enzvmatic treatment
5pl of PCR product (approximately O.Spg) was treated with 10U exonuclease I and 2U shrimp alkaline phosphatase. The mix was incubated at 37°C for 15 minutes followed by a step at 80°C for 15 minutes to denature both the enzymes. 5|xl of this treated PCR product was used as input for the sequencing reaction.
Extraction of PCR product from agarose
The gel was visualised using a Dark Reader transilluminator to prevent UV damage to PCR product; the amplified band was cut out of the gel using the taking as little agarose as possible. The gel slice was weighed and 0.5 volumes of 10% acetic acid (v/v) was added i.e. the gel slice weighed 0.1 g so 50|xl of acetic acid was added. To this, 4.5 volumes of sodium iodide were
added. The mix was then incubated at 60°C until the gel slice had dissolved and 5^il glassmilk was then added. The mix was then left for 10 minutes at room temperature inverting occasionally to ensure the glassmilk stayed in suspension. This mix was then centrifuged briefly to pellet the glassmilk and the supernatant was removed and discarded. 300|il chilled NEET wash was added and the pellet was resuspended by vortexing briefly. The mix was then briefly centrifuged to pellet out the glassmilk and the supernatant was again removed and discarded. This was repeated a further two times and on the final occasion the mix was centrifuged for a minute at 13, 250xg at room temperature. The supernatant was removed and discarded and the pellet was
resuspended in 15pl autoclaved Nanopure water. The mix was then
centrifuged at 13, 250xg for 1 minute at room temperature. The supernatant was removed and used as input for the sequencing reaction.
Primers 2953 and 2954 were used to prime the sequencing reaction. The cycling conditions for the sequencing reaction were 96°C for 30 seconds, 54°C for 15 seconds, 60°C for 4 minutes for 25 cycles.
Sequencing of clones PUM24P and PUMGRep
0.5|ig of purified clone DNA was used as input for the sequencing reaction. For PUMGRep the oligonucleotide primer Exon2S was used, and for PUM24P two reactions were set up one using the pBluescipt vector primer M l3 forward and one using Ml 3 reverse to prime the reaction. Both reactions used the Perkin Elmer thermocycler 480 and a drop of oil was put on top to prevent evaporation during thermocycling. The cycling conditions for the PUM24P reactions were: 96°C for 30 seconds, 55°C for 15 seconds, 60°C for 4 minutes for 25 cycles.
The cycling conditions for the PUMGRep reaction were: 96°C for 30 seconds, 60°C for 6 minutes and 30 seconds for 30 cycles.
ABI Sequencing gels
All sequencing products were run separated by electrophoresis on either an ABI 373 or an ABI 377. The gel for the ABI 373 was prepared by mixing 40g urea, 12ml polyacrylamide (19:1) and 30ml Nanopure water. Once
the urea had dissolved 1g Amberlite resin was added and mixed briefly to deionise the mixture. 8ml lOxTBE and the gel mixture were filtered into the same vessel. This process removed the Amberlite resin. 400pl 10% (w/v) Ammonium persulphate and 45pl TEMED were added to the gel mixture to catalyse polymerisation. The gel for the ABI 377 was made by mixing 18g urea, 5.2ml 40% (v/v) acrylamide, 27.5ml Nanopure water and 0.5g Aberlite resin. The Amberlite resin was removed and 250pl 10% (w/v) ammonium persulphate and 35pl TEMED were added to the gel mixture to polymerise the gel.
To each of the samples 5pl loading buffer (5 volumes formamide, 1 volume 25mM EDTA (pH8.0) with 50pg/pl blue dextran) was added. The samples were denatured for 2 minutes and placed on ice to prevent reannealing while loading was taking place. The gel was run at 50W for 7 hours at a surface temperature of 50°C.
The gels were all analysed using the ABI sequence analysis software (Perkin Elmer).
General method for the Polymerase chain reaction
PCR was performed in either IOp.1 or 25pl reactions using Ipl or 2.5pl 10x buffer IV (ABGene) respectively. Each reaction contained 0.2mM of each dNTP, 0.5pM of each primer, 5% (v/v) glycerol, 0.05U ABTaq DNA polymerase and was amplified using the Genius thermocycler.
PCR products were subjected to electrophoresis on a 2% agarose gel unless otherwise stated. Kb ladder (0.5pg) was used as a size standard.
PCR of the product used to detect the point mutation at position 3506 in exon 2 and of CA microsatellite
Primers used in this reaction were 1946 AGS and Cy5 Exon2AS to amplify around the G to A transition at position 3506 in exon 2 and CAA and Cy5 CAS to amplify the CA microsatellite in intron 6. The reaction volume was made up to 25pl with autoclaved Nanopure water and thin walled tubes were used. Approximately 400ng genomic DNA was subjected to an initial
dénaturation at 95°C for 5 minutes and then cycled at 96°C for 40 seconds, 62°C for 40 seconds, 72°C for 1 minute for 32 cycles.
The PCR product size of the G/A transition in exon 2 was 260bp, and the PCR product size of the CA microsatellite in intron 6 was 176-180bp.
AlwNI digest to type point mutation in exon 2 at position 3506
Approximately 250ng PCR product was digested in a 15pl reaction
containing 1.5pl buffer 4 (New England Biolabs) and 5U AlwNI restrcition
enzyme. Reactions were incubated at 37°C for 3 hours. Products were electrophoresed thorough a 2.5% agarose minigel. Adenine at position 3506 (Gen Bank Reference:MS 1170) gave a band of 240bp, guanine at position 3506 gave a band of 260bp. The PCR product of the CA microsatellite remained uncut.
Analvsis of the CA microsatellite in intron 6 of the MUC1 gene using the A.L.F.
Express machine
PCR products were electrophoresed on a 6% acrylamide gel. The gel was made up by mixing 40ml 6% polyacrylamide (19:1) and 10ml 5xTBE. 400|il 10% (w/v) Ammonium persulphate was added to polymerise the gel.
The electrode buffer was IxTBE. 2pl A.L.F. loading dye (Amersham
Pharmacia), 50ng fluorescent lOObp marker (prepared by L. Vinall), 50ng fluorescent 300bp marker (prepared by L. Vinall) and approximately 50ng of
PCR product were loaded into each well. The gel was subjected to
electrophoresis at 150V, 38mA, 25V for approximately 2 hours or until the 300bp marker had run past the laser.
The gel was analysed using the Fragment Manager program (Amersham Pharmacia)
Analvsis of the G to A transversion at position 4679 in exon 4 of the MUC1
gene
The NCBI single nucleotide polymorphism (SNP) database
fwww.ncbi.nim.nih.Qov/SNP/) was searched for polymorphisms in the MUC1 gene.
intron 5 and one in exon 4 at position (Gen Bank reference: M61170). The
latter polymorphism was a guanine to adenine transversion
(www.ncbi.nlm.nih.Qov/SNP/snp retrieve.cQi?subsnp id=2419730) and caused a valine to methionine substitution and was thus selected to investigate further.
Primers Exon4S and ExonSAS were used to amplify the polymorphic
region and the final reaction volume was 25pi. The PCR product size was
297 base pairs.
NIalll restriction digest to tvoe the SNP at position 4679
The conditions used were the same as for the AlwNI digest described
previously except 5U NIalll were used. The guanine to adenine change
creates the NIalll restriction site, and also within the PCR product there was
an additional unvarying NIalll site. The latter site gave two fragments of 149
and 148 base pairs and acted as an internal control for the efficiency of the digest reaction. If there was an adenine at position 4679 it was expected that three bands of 149, 105 and 43 base pairs would be seen.
Long PCR across the tandem repeat region of the MUC1 gene
For PCRs across the TR region each reaction contained O.BSpI l l . l x Jeffreys’ buffer, 5% glycerol, 2.7mM Tris (not pHed) and 0.25U Dynazyme EXT. The primers were at a concentration 0.25pM and the reaction volume was made up to 7pl with autoclaved Nanopure water. 100-150pg genomic DNA was used as input for the PCR and the reactions were all amplified using a Genius PCR machine with the heated lid on.
For amplification across the TR region for general purposes primers Exon2S and MUC1E2AS were used. Reactions were subjected to an initial dénaturation of 96°C for 1 minute 30 seconds for 1 cycle. Following this PCRs were cycled at 96°C for 40 seconds, 60°C for 30 seconds, 68°C for 3 minutes for 22 cycles.
Allele specific PCR across tandem repeat region
The reagents were the same as for the general purpose long PCR but the forward primers were ExonA2S or Exon g2S (instead of Exon2S). The
reaction conditions were the same as for the general long PCR but the annealing step was at 66°C for 30 seconds.
PCR across tandem repeat for generating allele sizes
The entire 7^1 PCR product was electrophoresed through a 22cm 1% agarose gel at 2.5V/cm for 24 hours. Two lanes containing O.Spg Raoul marker were included on the gel, these were used a size standards. The gels were stained with 500ml running buffer containing 50pl (50mg/ml) ethidium bromide. Gels were then vacuumed blotted on to Hybond-N+ membrane
using a constant pressure of 2 mbars. The DNA was depurinated for 30
minutes, denatured for 30 minutes, and neutralised for 30 minutes. Transfer to the membrane was with 20xSSC for 2 hours. Filters were then baked at 80°C for 2 hours. The filters were then probed and the bands sized as for the
Hinfl digests.
PCR across tandem repeat to generate gel slices
The entire 7pi PCR product was electrophoresed through a 14cm 1% agarose gel at 2.1V/cm for 20 hours. 2pg Kb ladder was used as a size
marker. The gels were stained for 30 minutes after electrophoresis by
submerging in 500ml of running buffer (IxTBE) containing 50pl (5mg/ml)
ethidium bromide. The gels were visualised using a Dark Reader
transilluminator to prevent UV damage to the PCR products. Using sizing
obtained from Hinfl digests it was estimated where the bands would be. Gel
slices were then cut out and broken up with a pipette tip in an eppendorf tube. The gel slice fragments were then resuspended in 50pl 0.05pM sonicated herring sperm DNA. These were then frozen at -70°C and thawed at room temperature 3 times to disrupt the agarose and release the DNA. Gel slices were then centrifuged at 11, 290xg for 2 minutes at room temperature.
PCR for testing gel slices
The general PCR conditions were followed and primers Exon2S and Exon2AS were used. 1 pi of the gel slice supernatant was used as input for the
PCR reaction. The reaction volume was made up to 10pl with autoclaved Nanopure water. The cycling conditions were:
96°C for 40 seconds, 60°C for 30 seconds and 70°C for 1 minute for 27 cycles.
The PCR product size was 205 base pairs.
MVR-PCR of the PDTR to PESR nucleotide changes
MVR-PCR were performed in 7pi using 0.7pl (-40pg) of single allele DNA from the gel slices or O.SpI (~100ng) genomic DNA with 0.57pl of
Jeffrey's 11.1x buffer, 5%(v/v) glycerol, 2.7mM Tris (not pHed), 0.25U Taq