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