LISTA DE MATERIALES PARA LA LINEA TRIFÁSICA Poste de hormigón

DNA was extracted from blood, biopsy and short term cell cultures (between passage 4 and passage 6), using commercially available extraction kits (Qiagen, Crawley, Sussex)

Procedure for isolation of genomic DNA from whole blood

Each sample of frozen blood was removed from -70°C storage and left at room temperature to thaw completely. Subsequently, 3 mis of blood was removed from each sample and placed in a 50 ml polypropylene screw cap tube (Falcon, Marathon Lab Supplies). One volume of ice-cold buffer C l and 3 volumes of ice-cold dH2Ü was added and mixed by

inverting several times until the suspension became translucent. This was then incubated for 10 minutes on ice. The lysed blood was centrifuged for 15 minutes at 1300 x g and the supernatant discarded. One ml of ice-cold buffer C l and 3 ml of ice-cold dH20 were added and the pelleted nuclei resuspended by vortexing. Centrifugation was carried out again for 15 minutes at 1300 x g and the supernatant discarded. After adding 5 mis of buffer G2, the nuclei were resuspended completely by vortexing for 20 seconds and 95 |xl of proteinase K (20 mg/ml) (ICN Biochemicals) was added. The solution was then incubated at 50°C for 60 minutes or until the lysate became clear. A Qiagen Genomic-tip was then placed over a 50 ml polypropylene screw cap tube using a tip holder and the tip equilibrated with 4 mis of buffer QBT. The tip was subsequently allowed to empty by gravity flow. Following incubation, the sample was vortexed for 10 seconds and applied to the Genomic-tip. The DNA binds to the resin of the tip and the residual QBT buffer passed through the tip into the screw cap tube. The genomic-tip was washed twice with 7.5 mis of buffer QC before being transferred to a clean screw cap tube. The DNA was then eluted with 5 mis of buffer QF. Three and a half mis of isopropanol (Sigma Aldrich) was added and mixed with the sample and the DNA spooled with a glass rod. The DNA was then transferred to a 1.5 ml eppendorf tube (Alpha Laboratory Supplies, Hampshire) containing 1 ml TE (pH 8.0) buffer and resuspended. Each DNA sample was sheared with a 1 ml syringe prior to use and any remaining blood not used for DNA extraction was returned to storage at -70^ C.

Procedure for isolation of genomic DNA from tissue

Frozen biopsy samples were removed from LN2 storage, placed in 25 ml universal tubes

and allowed to thaw in a 37°C water bath. Approximately 25 mg of tissue was cut from each sample, under sterile conditions. This was chopped into very small pieces using crossed carbon steel scalpel blades (size 10) and then pipetted into a 1.5 ml eppendorf tube with 180 |il of buffer ATL. Twenty jil of Proteinase K solution (20 mg/ml) was then added and mixed by vortexing for 10 seconds. The solution was then incubated at 55°C for 60 minutes or until the tissue was completely lysed. Two hundred \i\ of buffer AL was added, the sample was mixed by vortexing for 10 seconds and then incubated for a further 10 minutes at 70°C. Following this, 210 |il of ethanol was added and the sample mixed by vortexing again for 10 seconds. A QIAmp spin column (Qiagen tissue kit) was placed in a 2ml collection tube (Qiagen tissue kit) and the sample applied. The hd of the spin column was then closed and the column centrifuged at 6000 x g for 1 minute. The collection tube containing the filtrate was then discarded, the column placed in a clean tube and the DNA was washed with 500 |il of buffer AW. The column was then centrifuged again at 6000 x g for 1 minute. The collection tube containing the filtrate was again discarded and the wash procedure was repeated. The column was placed in a clean collection tube and the DNA was eluted with the addition of 200 |xl of 10 mM Tris-HCl (preheated to 70^C) followed by the centrifugation of the column at 6000 x g for 1 minute. This was repeated twice using the same collection tube. After elution, the DNA samples were stored at -20°C until use.

Procedure for isolation of genomic DNA from cultured cells

One ml of trypsin EDTA was added to each 150 cm^ flask, containing approximately 3 x 10^ cells when the latter were confluent, and incubated at 37°C until all of the cells had detached from the flask. The cell suspension was then transferred to a 50 ml polypropylene

screw cap tube. The empty flask was then washed with 2 ml Phosphate Buffered Saline (PBS) which was then added to the tube.

The cells were recovered by centrifugation at 1500 x g for 10 minutes after which they were resuspended in 4 ml PBS and centrifuged at 1500 x g for a further 10 minutes. The cells were then resuspended in PBS at a concentration of 10^ cells /ml. One volume of ice- cold buffer C l and 3 volumes of ice-cold dH20 were added and mixed by inverting the tube several times until the suspension was translucent. The suspension was then incubated on ice for 10 minutes.

The remainder of the DNA isolation procedure is identical to that used for the isolation of DNA from whole blood from the lysing procedure through to the shearing of extracted DNA.

F L U O R O M E T R IC Q U A N TIFICA TIO N O F DNA

Following DNA extraction, the quantity of DNA in each sample was measured using a DyNAQuant 200 fluorometer (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire) and a DQ 130 capillary cuvette kit (Amersham Pharmacia Biotech). The former is a filter fluorescence photometer designed specifically for the accurate quantitation of low DNA concentrations using Hoechst 33258 dye (Sigma, St. Louis, USA for Amersham Pharmacia Biotech) The DQ 130 kit includes a capillary cuvette and capillary tubes which hold 3 to 9 pi of solution.

As the DNA fluorescence assay is based on a relative measurement of emitted light, a calibration reference value must be estabhshed with a known DNA sample before the concentration of DNA in an unknown sample can be determined. A capillary tube was

filled with the standard of 100 ng/|il calf thymus DNA (Sigma Aldrich) in capillary assay solution inserted into the cuvette and the reference value was entered on the fluorometer.

In order to measure the quantity of DNA in the actual samples, a capillary tube was filled with the sample solution diluted in capillary assay solution and placed in the cuvette. The quantity of DNA measured is given in ng/pl.