The DNA digests were separated as described in section 2.4.7.I. The gel was denatured in denaturing solution (section 2.3.4) for 30 minutes on a shaking platform. The denatured gel was inverted and placed on a sponge in
a photographic tray. The sponge was covered with 3MM Whatman paper pre-soaked in 20XSSC, which acts as a wick.
A piece of charged nylon membrane (Hybond N+) was cut to the exact size of the gel, soaked in 2XSSC and placed on top of the gel. 2 pieces of 3MM Whatman paper pre-soaked in 2XSSC were placed on top of the Hybond N+ and air bubbles were removed by gently rolling a 10ml plastic pipette over the surface. Clingfilm was placed around all four sides of the gel to prevent evaporation and three paper towels were unfolded and laid out flat over the gel. Paper towels were then placed on the gel to a height of approximately 1 Gems to draw the 20XSSC and therefore, the DNA onto the filter.
After 16 hours the filter was removed from the gel and the position of the wells marked with a permanent marker. DNA was fixed to the filter by soaking in 0.4M NaOH for 30 minutes and neutralised by rinsing in 2XSSC until the pH of the SSC was 7.0. The filters were blotted to remove excess liquid and wrapped in clingfilm until required.
2.4.8.1 Oligolabelling of DNA
Plasmid or insert DNA was diluted in TE to a concentration of lOng/pl and a total of 50ng of probe was labelled per reaction. The probe was placed in a 1.5ml tube with a pierced lid to avoid a build up of pressure while heating. The probe was denatured for 5 minutes in a boiling water bath and placed on ice to prevent renaturation. To this was added lOpI of OLB, Ip l BSA (lOmg/ml), 3pl of alpha ^Zp.qcTP (3000Ci/mmoI, Im Ci = lOOpI) and 2.5 units of Klenow fragment of DNA polymerase 1. The reaction was mixed and left to stand at room temperature for 2-4 hours.
After labelling, unincorporated nucleotides were separated from the labelled DNA by filtration through a Sephadex G-50 column in a 1 ml syringe plugged with an autoclaved glass bead. The column was equilibrated with TE. The column was placed in a centrifuge tube and the probe was applied to the column, the elute was discarded. Radio-labelled DNA was eluted by applying an equal volume of TE to the column. The radioactivity of the eluted DNA was determined by counting a 4pl aliquot in a calibrated Bioscan QC2000 bench top beta counter. 1x10® dpm of probe per ml of hybridisation solution was added to pre-hybridising filters.
2.4.8 2 Hybridisation of labelied DNA to filters
The filter was soaked in 2XSSC, rolled up and placed in a hybridisation bottle (Hybaid) containing tOmls of hybridisation solution. The hybridisation bottle was placed in a 65®C rotary oven (Hybaid) and allowed to pre-hybridise for 2 hours to saturate non-specific sites in the DNA and reduce the level of non specific binding. After prehybridisation, the radiolabelled probe was denatured in a boiling water bath, placed on ice, then added to the hybridisation bottle containing the filter to give a final concentration of 10® dpm/ml hybridisation solution.
When competitive sequences were present, 100pg of sheared human placental DNA (lOmg/ml) per ml of hybridisation solution was added to the filter and pre-hybridised for at least 3 hours. In addition, the probe was boiled for 5 minutes and added to 1.8ml of GOOpg of sheared human placental DNA, hybridisation solution and 20XSSC (final concentration 0.5XSSC). This was maintained at 65®C for 90 minutes and then added to the pre-hybridised filter. After hybridisation, the filters were washed in solutions of increasing stringency. The filters were monitored with a beta emission counter until the signal detected was at background level. Generally, the unhybridised probe was removed by washing the filter in 3XSSC/0.1% SDS at room temperature on a shaking platform for 1 hour, followed by washing at 65®C in a shaking water bath in 1XSSC/0.1% SDS, 0.5XSSC/0.1% SDS and 0.1XSSC/0.1% SDS for 30,15 and 10 minutes respectively, depending on the probe. Filters were dried on 3MM Whatman paper, wrapped in clingfilm and exposed to X-ray film in a cassette backed with an intensifier screen. The cassette was placed at -70°C for 12-16 hours, depending on the Intensity of the radioactive signal. The film was developed using a Fuji RGII X-ray film processor.
2.4.8.3 Quantification of radioactivity using a phosphorimager
Phosphorimager screens were erased by exposing them to yellow light for 6 minutes, prior to use. Labelled filters for dosage analysis were placed in a phosphorimager cassette box and exposed to the phosphorimager screen for 24-72 hours. The exposed phosphorimager screen was scanned and the radioactive signal quantified using a phosphorimager (Molecular Dynamics) and the ImageQuant software. Dosage was determined by measuring the ratio of radioactive signals from the test probe and a control probe using the phosphorimager.
2.5 Patients
3731 is the third child of healthy parents. The neonatal period was uneventful but by two years of age global developmental delay was apparent. 3731 has blonde hair, blue eyes with pale fundi, microbrachycephaly, a wide smiling mouth and a prominent chin. Her voluntary movements are jerky and she has never developed any speech. An EEG performed at five years of age showed the slow wave changes characteristic of AS. High resolution chromosome analysis performed by the Northern region genetics advisory service was normal.
13581 presented at clinic at 7 years of age and is the only child of normal parents. Clinical features include developmental delay, especially in the area of speech, a history of seizures and a smiling happy personality. Her mother was 7 weeks pregnant. High resolution chromosome analysis performed by the North East Thames regional cytogenetic service was normal.
B.S has a borderline intellectual deficit whereas D.H. has mild to moderate mental retardation. Neither individual has the characteristics of AS. High resolution chromosome analysis showed a deletion of 15q (Michaelis et al., 1995).
Family R were referred to the molecular genetics unit at IGH when the mother of two siblings with AS was 17 weeks pregnant. Both siblings were mentally retarded, had a speech impairment and a sociable disposition.
Family W were referred to the molecular genetics unit at IGH. The affected male sibling was retarded with a prominent lower jaw and mid facial hypoplasia. He had a history of seizures. The affected female sibling had developmental delay and a protruding tongue. Both siblings had an EEG pattern consistent with a diagnosis of AS.
Patient 6468 was referred to the molecular genetics unit at IGH with global developmental delay. Her clinical features were consistent with a diagnosis of AS. High resolution chromosome analysis performed by the North East Thames regional cytogenetic service was normal.
Patients EA, KC and HR were collected as part of the study by J. Clayton- Smith (1992) and had clinical features consistent with a diagnosis of AS. Patient HR did not have the paroxysms of laughter or seizures that are commonly associated with a diagnosis of AS and high resolution chromosome analysis performed by the Northern region genetics advisory service was normal. High resolution chromosome analysis performed by Dr. Tessa Webb was normal for patient EA. Patient KC was reported as having a deletion of one of the chromosome 15 homologues.