Electrophoretic isolation of extrachromosomal DNA from tumor cells.
Gene amplification allows transformed cells to overexpress specific genes and gain a survival advantage. For this reason, cloning and characterization of amplified genes can improve our understanding of the biology of transformed cells. The techniques of in-gel renaturation and chromosome microdissection can enrich for amplified DNA sequences, but both are labor intensive and have other drawbacks. We have developed an alternative strategy of enriching for amplified DNA sequences that involves two-directional agarose gel electrophoresis of extrachromosomal circular DNA. Extrachromosomal circles can be detected with repetitive DNA probes and can be used to produce DNA probes suitable for fluorescence in situ hybridization for location of genomic origin. The ability to enrich for amplified DNA without specialized equipment or transformed cell metaphases should prove useful in the search for new genes which are important in tumor cell progression.
VanDevanter
D R
DR
Tumor Institute, Swedish Medical Center, Seattle, Washington, USA.
Tseng
J C
JC
Yirdaw
G
G
D001483:Base Sequence; D002738:Chloroquine; D002877:Chromosomes, Human; D017931:DNA Primers; D015342:DNA Probes; D004270:DNA, Circular / Q000302:isolation & purification*; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007624:KB Cells; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D015342:DNA Probes; D004270:DNA, Circular; D004273:DNA, Neoplasm; D002738:Chloroquine