SÍNTESIS DE LOS DETERMINANTES POLÍTICOS AMBIENTALES

DOP-PCR am plification of DNA extracted from paraffin em bedded m aterial often resulted in DNA that was too small to be labelled using nick translation or PCR labelling m ethods. The Kreatech universal linkage system offered a non- enzymatic alternative. DNA from normal pooled blood and IN2809 were used as controls to optimise the technique. The DNA samples were sonicated for 3 x 20 seconds before being labelled w ith d-Green. The labelled DNA was initially co­ h y b rid ised w ith Spectrum R ed-labelled norm al reference DNA to norm al m etaphase slides. As "hom o-hybridisations" had produced better results in the DOP-PCR experim ents it w as decided to co-hybridise d-G reen ULS-labelled tu m o u r w ith rhodam ine ULS-labelled norm al DNA. D espite a n u m b er of attem p ts a successful h y b rid isatio n could not be p ro d u ced . A fter m uch discussion w ith the technical d epartm ent at Kreatech a problem w ith the reagents was discovered, which resulted in new packaging being produced for the ULS reagents. The problem arose because plasticisers w ere being leeched from the plastic used to m anufacture the lids of the tubes the reagents w ere

packaged in, and contaminating the labelling reagents. This led to inhibition of the labelling step. Even after new reagents had been provided successful results could not be obtained. IN859 & IN 1265 were also used to evaluate the m ethod, b ut the hybridisation nearly always resulted in "norm al" CGH profiles being obtained (see Figures 3.11 and 3.12).

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Figure 3.11: IN859 sonicated and labelled with d-Green ULS. The red reference DNA was norm al pooled blood DNA sonicated and labelled w ith rhodam ine ULS. This m ethod did not show any of the CNAs seen w ith the nick translation technique.

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Figure 3.12: IN1265 sonicated and labelled with d-Green ULS. The red reference DNA was norm al pooled blood DNA sonicated and labelled w ith rhodam ine ULS. This m ethod did not show any of the CNAs seen w ith the nick translation technique.

After personal communications with Judith Jeuken in Nijmingen, H olland it was felt the reaction could be inhibited by contaminating RNA in the sam ple or by DNA cross links. A new extraction method was used which is based on a salting out procedure and incorporated sodium thiocynate treatm ent to destroy cross links and RNase treatm ent to destroy residual RNA. This technique resulted in DNA that was large enough to be nick translated. DNA that had been extracted by earlier m ethods was treated with RNase before being labelled w ith ULS as before.

U38 was pre-treated w ith RNase before being labelled w ith d-Green ULS and co­ hybridised w ith rhodam ine ULS -labelled normal DNA. The resulting profile can be seen in Figure 3.13a. There appeared to be a lot of CGH artefact and no real chrom osom e alterations. Though the profiles appeared to be quite sm ooth all regions of "alteration" show ed loss and no regions of gain. The regions of loss w ere p resen t at chrom osom al positions w here prev io u s nick tran slatio n

experim ents of other ependym om a had show n gain or amplification, e.g. Iq, 4 and 7q. This patient is also female and the X chromosome is approaching the 0.8 threshold (indicating loss) rather than being on the 1.0 threshold w hich is w hat w ould be expected for a sample from a female patient. A CGH experim ent using U38 DNA labelled by nick translation resulted in a CGH profile w ith no aberrations (see Figure 3.13b)

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Figure 3.13a: U38 labelled w ith d-Green ULS and co-hybridised w ith rhodam ine ULS labelled normal pooled blood DNA. The profiles show w hat appears to be a large am ount of genomic loss from chromosomes 1, 4, 6, 7, 12, 15, 16, 17, 19 and 20. H ow ever these alterations were not seen in DNA that had been labelled by the nick translation technique and were taken to be artefacts of the hybridisation.

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Figure 3.13b: U38 labelled by nick translation and co-hybridised w ith nick translated norm al reference DNA. The profile shows a norm al karyotype. The X chromosome profile is centred along the 1.0 threshold line indicating the patient is female.

A M H 97/590 DNA w as extracted from paraffin em b ed d ed sections using jeuken's m ethod. The DNA was considered too large to label using the ULS procedure and was labelled by nick translation. The resulting profile can be seen in Figure 3.14. No CNAs could be seen in this tum our but as the X chromosome w as consistent w ith the p a tien t's sex (male) this w as taken to be a true representative composite for this tumour.

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Figure 3.14: AMH97 / 590 labelled with SpectrumGreen dUTP by nick translation and co-hybridised with SpectrumRed labelled normal DNA. There are no CNAs but the X chromosome is true to the patient's sex so this composite was taken to be a true representation of the tumour.