CLONING OF MINISATELLITE-CONTAINING SEQUENCES FROM 2-DIMENSIONAL DNA-FINGERPRINTING GELS REVEALS THE IDENTITY OF GENOMIC ALTERATIONS IN LOW-GRADE GLIOMAS OF DIFFERENT PATIENTS

Citation
K. Marczinek et al., CLONING OF MINISATELLITE-CONTAINING SEQUENCES FROM 2-DIMENSIONAL DNA-FINGERPRINTING GELS REVEALS THE IDENTITY OF GENOMIC ALTERATIONS IN LOW-GRADE GLIOMAS OF DIFFERENT PATIENTS, Electrophoresis, 18(9), 1997, pp. 1586-1591
Citations number
21
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemical Research Methods
Journal title
ISSN journal
0173-0835
Volume
18
Issue
9
Year of publication
1997
Pages
1586 - 1591
Database
ISI
SICI code
0173-0835(1997)18:9<1586:COMSF2>2.0.ZU;2-J
Abstract
Two-dimensional (2-D) DNA fingerprinting was used to investigate genom ic changes in human low-grade gliomas of different subtypes. DNA varia tions were identified in the 2-D hybridization patterns as spot losses or gains. Computer-aided matching of spot patterns from different pat ients revealed a clustering of spot changes at particular areas in the gel. Representative spots of each cluster were cloned using a spot cl oning protocol which includes the preparation of a duplicate and a mas ter gel. The DNA fragments of the 2-D gels were transferred to DEAE an d nylon membrane, respectively. After hybridization of the master blot with a minisatellite core probe, the position of a particular spot wa s determined with reference to the lambda DNA fragments used as extern al 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 o f the 2-D blots. One of these probes detected a spot loss in 7 of 28 l ow-grade gliomas of different subtypes analyzed. Another probe reveale d a characteristic intensity shift in 8 of 9 pilocytic astrocytomas be tween two neighboring spots. The target sequence of this highly specif ic effect was assigned to chromosome 11q14 by in situ hybridization of a P1 clone harboring the affected genomic region. Thus, we successful ly established a spot cloning procedure for the generation of locus-sp ecific probes that may be instrumental in the discovery of the ciritic al early events of glioma pathogenesis.