PURPOSE: Chromosome microdissection has become a very powerful approach to generate chromosome band-specific library and painting probes for physical mapping or cytogenetic analysis. We have constructed here band-specific painting probes for human chromosomes by microdissection and polymerase chain reaction(PCR). METHODS: We pretreated the microdissected fragments with Topoisomerase I(Topo I) which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of fluorescent in situ hybridization(FISH) probe. RESULTS: It was possible to construct region-specific painting probes for 5q14-q15, 6q23, 16p11.2 and 22q12. The painting probes were successful in determining the corresponding chromosome bands in SNU-484 cell line, IMR-32 cell line. CONCLUSION: These painting probes can be used to detect the related marker chromosomes in congenital malformations and other solid tumors, and the band specific probe pools may be used effectively to analyse the genes.
Cell Line ; Chromosomes, Human ; Cytogenetic Analysis ; DNA ; DNA, Superhelical ; Humans ; Microdissection ; Paint* ; Paintings* ; Polymerase Chain Reaction ; Relaxation

Chromosome microdissection has become a very powerful approach to generate chromosome band-specific library and painting probes for physical mapping or cytogenetic analysis. Here we have constructed the band-specific painting probe for human chromosome 18p11.1-p11.2 by microdissec-tion and polymerase chain reaction (PCR). We pretreated the microdissected fragments with Topoisomerase I (Topo I) which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of fluorescent in situ hybridization (FISH) probe from a single microdissected chromosome. With this method, it was possible to construct region-specific painting probe for 18p11.1-p11.2. The probe has been used successfully to determine the trisomy 18. Thus this painting probe can be applied to detect the related marker chromosomes in various solid tumors, and the band specific probe pools may be used to analyse the genes.
Chromosomes, Human ; Cytogenetic Analysis ; DNA ; DNA Topoisomerases, Type I ; DNA, Superhelical ; Humans ; In Situ Hybridization, Fluorescence ; Microdissection ; Paint* ; Paintings* ; Polymerase Chain Reaction ; Relaxation ; Trisomy

pUDK-HGF, the recombinant plasmid DNA encoding human hepatocyte growth factor (HGF), can treat ischaemic disease. A great quantity of pharmaceutical pUDK-HGF is needed. A pilot-scale production process of pUDK-HGF was established based on a new chromatographic media (plasmidselect), including fermentation, cell harvesting, alkaline lysis, ultrafiltration, RNA removing and buffer exchanging on Sephacryl S-1000, capturing supercoiled plasmid DNA with plasmidselect, and removing the salt with Sepharose 6BFF. The process does not use RNase enzyme and toxic solvents.
DNA, Recombinant ; biosynthesis ; DNA, Superhelical ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Genetic Vectors ; genetics ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Pilot Projects ; Plasmids ; isolation & purification

Painting probe for HSRs in IMR-32 Neuroblastoma, a pediatric malignant neoplasm of neural crest origin, has a wide range of clinical virulence. The mechanisms contributing to the development of neuroblastomas are largely unclear, but non-random chromosomal changes identified over the past years suggest the involvement of genetic alterations. Amplification of the human N-myc proto-oncogene is frequently seen either in extrachromosomal double minutes or in homogeneously staining regions(HSRs) of aggressively growing neuroblastomas. N-myc maps to chromosome 2 band 24, but HSR have never been observed at this band, suggesting transposition of N-myc during amplification. We have constructed and analyzed the region-specific painting probe for HSR in neuroblastoma IMR-32 to determine the derivative chromosomes. Microdissection was performed on HSR using an inverted microscope with the help of microglass needles and an micromanipulator. We pretreated the microdissected fragments with Topoisomerase I which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of Fluorescent in situ hybridization probe from a single microdissected chromosome. With this method, it was possible to construct the region-specific painting probe for HSR. The probe hybridized specifically to the HSRs of IMR-32, and to 2p24, 2p13 of normal chromosome. Our results suggest there was coamplification of N-myc together with DNA of the chromosome 2p24 and 2p13. Moreover, the fluorescent signals for the amplified chromosomal regions in IMR-32 cells were also easily recognized at a Thus this painting probe can be applied to detect the similar amplification of N-myc in neuroblastoma tissue, and the probe pool for HSR may be used to identify the cancer-relevant genes.
Cell Line* ; Chromosomes, Human, Pair 2 ; DNA ; DNA Topoisomerases, Type I ; DNA, Superhelical ; Genes, myc ; Humans* ; In Situ Hybridization, Fluorescence ; Microdissection ; Needles ; Neural Crest ; Neuroblastoma* ; Paint* ; Paintings* ; Polymerase Chain Reaction ; Relaxation ; Virulence