Marco Conceptual

10 ml of am niotic fluid w as centrifuged at 2000 rp m for 10 m inutes. The fluid w as d e c a n te d alm o st c o m p letely a n d the cell p e lle t re s u sp e n d e d in the rem aining 0.5 ml of am niotic fluid and transferred to a flat sided 10 ml leighton tube. 2 mis of FIO complete m edium (appendix 7.1) was ad d ed and the culture

w as incubated at 37°C for 5 days. After this the culture w as exam ined for cell g ro w th w ith a 100 x in v e rte d m icroscope every 48 h o u rs an d the m ed iu m changed every 72 hours. W hen there w as an actively d iv id in g m onolayer or sev eral active colonies the FIO m ed iu m w as replaced w ith 1 m l of C hangs complete m edium and incubation proceeded at 37°C and 5% CO2 for 24 hours. Colchicine was a d d e d to a final concentration of 0.1 m g /m l an d incubation continued for 3 hours at 37°C and 5% C O 2- The contents of the Leighton tube were transferred to a conical bottom ed centrifuge tube and the leighton tube was w ashed out into the centrifuge tube w ith 1 m l of 1 x H anks solution. 1 ml of trypsin/E D T A was ad d ed to the Leighton tube and this w as view ed u n d e r an inverted microscope until the cells h ad lifted off the flat side of the tube. These were transferred to the concial bottom ed centrifuge tube and centrifugation was carried out at 1000 rp m for 5 m inutes. The supernatant w as discarded and the cell p ellet resu sp en d ed in 5 m is of 0.075 M KCl for 15 m inutes at 37°C. The harvest then proceeded as for blood cultures.

3.3.3 G banding of m etaphase spreads.

Slides were baked overnight in a 60°C oven and then imm ersed for 10 minutes in 1 : 1 lOX Hanks Balanced salt solution w ithout sodium bicarbonate : IX H anks Balanced salt solution w ith 0.3g/l sodium bicarbonate. After rinsing in p H 6.8 b u ffer the slides w ere im m ersed in 2.8% b a cto try p sin in p H 6.8 b u ffer for b etw een 20 and 60 seconds. The slides w ere w ashed in 0.9% saline and then stained in Leishmans, freshly diluted 1 in 5 w ith pH 6.8 buffer, for 2-4 m inutes.

A fter w ashing in tap w ater a coverslip w as applied and the banding assessed using a Zeiss Axioskop microscope.

3.3.4 DNA extraction

3.3.4(i) DNA extraction from blood

Using a 15ml centrifuge tube 5mls of low salt buffer, TKMl, (appendix 7.3) and 125^1 of NP-40 to lyse the cells w as a d d ed to 5mls of w hole blood an d m ixed thoroughly by inversion. 800After centrifugation at 2200rpm for 10 m inutes at room tem perature the supernatant was discarded and a fu rth u r 5mls of TKMl w as a d d e d to the n u clear pellet. A second 10 m inute sp in at 2200rpm w as follow ed by the addition of 800^1 of high salt buffer, TKM2, (appendix 7.3) and 50/zl of 10% SDS. A fter m ixing w ell w ith a p lastic p ip ette the so lu tio n w as tran sferred to a large e p p en d o rf tube an d incubated at 55°C for 10 m inutes. 300jul of 6M NaCl was added and the whole suspension w as m ixed well before centrifugation at 1200rpm for 5 m inutes. The precipitated protein pellet at the bottom of the tube w as discarded and 2 volum es of ice-cold ethanol was added to the soupem atant w hich contained the DNA. The tube w as inverted several times as the DNA precipitated and the DNA was then rem oved and w ashed in 1ml of 70% ice-cold ethanol. Following centrifugation at 1200rpm for 5 m inutes the pellet was lyophilised and resuspended in TE buffer and stored at 4°C. The c o n cen tratio n of D N A ex tracted by this p ro c e d u re w as m ea su re d u sin g a flurometer.

3.3.4(ii) DNA extraction from tissue

100/zl of tissue buffer (appendix 7.3) was ad d ed to the cells in a small eppendorf

tube. A fter a d d in g 2^1 of 10% SDS an d 2^1 of lO m g /m l p ro te in a se K the

suspension was mixed well and left at 37°C overnight. 50/zl of phenol was added together w ith 50/zl of isoamylalcohol : chloroform (1:24) and mixed by inversion before microcentrifuging for two m inutes at high speed. The aqueous layer was transferred into a clean eppendorf and 50/zl of phenol was ad d ed w ith 50fil of isoam ylalcohol : chloroform (1:24) as before. Following centrifugation at high speed for tw o m inutes the aqueous layer w as transferred into a clean eppendorf and lOOfil of isoamylalcohol: chloroform was added. The solution was mixed by inversion and then m icrocentrifuged at high speed for two minutes. The aqeuos layer was again transferred and the isoamylalcohol: chloroform extraction was repeated. To the aqueous layer w as a d d e d l / 10 volum e of 4M NaCl and two volum es of absolute eth an o l before leaving a t -20°C for 1 ho u r. Follow ing centrifugation at top speed for 10 minutes the supernatant was discarded and the DNA was lyophilised and resuspended in 20//1 TE buffer and stored at 4°C.

3.3.4 (iii) DN A extraction from amniotic fluid

Between 2mls an d 5 mis of am niotic fluid w as centrifuged at 2500rpm for 10 m inutes and the supernatant was discarded. The cell pellet was resuspended in SOOjul of 50m M S o d iu m H y d ro x id e a n d w as b o ile d fo r 20 m in u te s. The specim ens w ere neutralised w ith lOOjul IM Tris (pH7.5) an d 5^1 w as used in a 50]ul DOP-PCR reaction.