Methods Sample preparation

a Serum samples were pre-treated with 0.1 M NaOH for 1 h at 37°C, and thereafter neutralized with 0.1M HCl, prior to PCR.

b DNA was prepared from PMBC essentially as described by Landgren et al. (1994). After centrifugation at 4000 x g for 5 min the cell pellet was lysed in buffer containing 10mM Tris HCl (pH 8.4), 1 mM EDTA, 0.5% Tween 20, 0.5% IGEPAL (Sigma) and 400 •g/ml proteinase K for 1 h at 55°C, followed by heat inactivation at 95°C for 10min. DNA was isolated by ethanol precipitation in the presence of salt at -20°C, washed with ethanol and acetone, and subsequently solubilized in DNAse free water. c Amniotic fluid was centrifuged, 5 min 5000xg, and the cell deposit separated from supernatant. Cell pellet was digested by treatment with proteinase K in lysis buffer prior to DNA precipitation as described for isolation of DNA from PBMC. The supernatant fluid was subjected to alkali treatment as outlined for serum samples.

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d Broncheoalveolar lavage (BAL) specimens were centrifuged as above, the cell pellets separated from supernatant, and each treated as described for amniotic fluid.

e CSF was subjected to alkali denaturation, followed by neutralization as described for serum samples.

f Vitreous body was treated with proteinase K in lysis buffer and the DNA precipitated as described previously, whilst vitreous humour was treated with alkali, and subsequently neutralized as described.

g Biopsy tissues were finely chopped with a scalpel prior to digestion with proteinase K (400^g/ml) in a lysis buffer volume of 1ml. DNA was prepared as described previously. Where necessary gross blood contamination was removed by gentle washing of the specimen with buffered saline prior to chopping. In particular, lysates of brain tissue were extracted with phenol and chloroform prior to DNA preparation.

h Placental tissue was prepared as described above for biopsies.

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