Cloning of minisatellite-containing sequences from two-dimensional DNA fingerprinting gels reveals the identity of genomic alterations in low-grade gliomas of different patients
✍ Scribed by Karola Marczinek; Akio Sugiyama; Jochen Hampe; Gundula Thiel; Karin Lehmann; Regina Neumann; Wilio J. F. de Leeuw; Peter Nürnberg
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 674 KB
- Volume
- 18
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
Cloning of minisatellite-containing sequences from two-dimensional DNA fingerprinting gels reveals the identity of genomic alterations in low-grade gliomas of different patients DNA fingerprinting was used to investigate genomic changes in human low-grade gliomas of different subtypes. DNA variations were identified in the 2-D hybridization patterns as spot losses or gains. Computer-aided matching of spot patterns from different patients revealed a clustering of spot changes at particular areas in the gel. Representative spots of each cluster were cloned using a spot cloning protocol which includes the preparation of a duplicate and a master gel. The DNA fragments of the 2-D gels were transferred to DEAE and nylon membrane, respectively. After hybridization of the master blot with a minisatellite core probe, the position of a particular spot was determined with reference to the lambda DNA fragments used as external markers in both gels. The gel spot DNA was recovered from the DEAE membrane by high salt elution and was polymerase chain reaction (PCR)-amplified after ligation of adaptor oligo cassettes. The PCR products were cloned and used as locus-specific probe for the rehybridization of the 2-D blots. One of these probes detected a spot loss in 7 of 28 low-grade gliomas of different subtypes analyzed. Another probe revealed a characteristic intensity shift in 8 of 9 pilocytic astrocytomas between two neighboring spots. The target sequence of this highly specific effect was assigned to chromosome llq14 by in situ hybridization of a P1 clone harboring the affected genomic region. Thus, we successfully established a spot cloning procedure for the generation of locus-specific probes that may be instrumental in the discovery of the ciritical early events of glioma pathogenesis. Keywords: Two-dimensional DNA fingerprinting / Gliomas / Genomic changes / Spot cloning 2 Materials and methods 2.1 Patient material Gliomas were obtained from unrelated patients cared for at the Charite hospital in Berlin. Histological classifica-