Fluorescent in situ hybridization (FISH) is a powerful technology for studying the chromosome organization and aberrations as well as for searching the homology between chromosomal regions in mammals. Currently, FISH is used as a simple, rapid, and reliable technique for analyzing chromosomal rearrangements and assigning chromosomal breakpoints in modern diagnosing of chromosomal pathology. In addition to cloned DNA fragments, the DNA probes produced by sequence-independent polymerase chain reaction are widely used in FISH assays. As a rule, the DNA probes generated from a genomic or chromosomal DNA by whole genome amplification are enriched for repetitive elements and, consequently, efficient FISH analysis requires that repetitive DNA hybridization is suppressed. The linker-adapter polymerase chain reaction (LA-PCR) using the genomic DNA hydrolyzed with HaeIII and RsaI restriction endonucleases allows the repetitive DNA fraction in DNA probe to be decreased and gene-rich DNA to be predominantly amplified. The protocol described here was proposed for production of the DNA probes for enhanced analysis of the C-negative regions in human chromosomes.
C-negative regions Linker-adapter polymerase chain reaction (LA-PCR) FISH Supernumerary marker chromosomes
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The study was supported by the Siberian Branch of the Russian Academy of Sciences (grants nos.VI.53.2.1, 6.1, and 75), and state contract no. 8289.
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