Cloning of minisatellite-containing sequences from two-dimensional DNA fingerprinting gels reveals the identity of genomic alterations in low-grade gliomas of different patients.
Two-dimensional (2-D) 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 11q14 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 critical early events of glioma pathogenesis.
Marczinek
K
K
Institute of Medical Genetics, Charité Medical School, Berlin, Germany.
Sugiyama
A
A
Hampe
J
J
Thiel
G
G
Lehmann
K
K
Neumann
R
R
de Leeuw
W J
WJ
Nürnberg
P
P
D002874:Chromosome Mapping; D002880:Chromosomes, Human, Pair 11; D003001:Cloning, Molecular; D016172:DNA Fingerprinting; D015180:Electrophoresis, Gel, Two-Dimensional; D005910:Glioma / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D018598:Minisatellite Repeats; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction