PubMed · 7184272
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W Schnedl. 1982. [Methods in human genetics].. https://pubmed.ncbi.nlm.nih.gov/7184272/
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OBJECTIVE: To establish a rapid and efficient technique of constructing human chromosomal band specific probe pools and their libraries. METHODS: A modified method of combining chromosome microdissection with degenerate oligonucleotide primed PCR(DOP-PCR) was used. 3p23-p26, 3q21-q22 and 4p12- p16 band from human chromosomes were microdissected and amplified as probe pools. The origins of the PCR products were determined by chromosome fluorescence in situ hybridization. The PCR products and pUC19 were digested by Xho I and Sal I respectively, and linke up. The DH5alpha were transformed by the recombinated vectors as the specific band libraries. The inserts were digested by EcoR I and Hind III, then measured by electrophoretic analysis. And the copies of inserts were identified by in situ bacterial colony hybridization with genomic DNA. RESULTS: All the three probe pools showed the special yellow-green signals in their microdissection responsible bands. The sizes of DOP-PCR products ranged from 300bp to 1800bp. 3q21-q22 probe pool generated about 1.2 x 10(4) clones. The average size of inserts was about 420bp by analysis of 30 positive clones. The rate of single-copy and low-repeated sequences was about 81%(178/220), while the rate of middle-repeated and high- repeated sequences was about 19%(42/220). CONCLUSION: The results proved that the modified microdissection combining DOP-PCR technique provided a simple and efficient method to construct the human chromosome band-specific probe pools and might contribute to gene cloning and complete sequencing of human genome.
OBJECTIVE: To develop a simple method for detecting DNA probes directly on R-banded chromosomes. METHODS: After sixty-seven hours culture, human peripheral blood lymphocytes were synchronized for 5 to 6 hours by adding Hoechst 33258 and BUdR, and then arrested by standard cytogenetic procedures. The slides were mounted with 2 x SSC and exposed with a 20W UV light which was about 10cm above the slides for 20 min at 75 degrees C. The biotinylated probes, such as the cosmid and YAC clones on 5p specific region and pBamX7, were hybridized on to the slides. After washing, the slides were treated with avidin-FITC and amplified with additional layer of biotinylated anti-avidin and avidin-FITC, and counterstained with propidium iodide in an antifade solution. Fluorescent signals and R-bands were observed simultaneously under Olympus BX 60 fluorescence microscope equipped with a WIB filter. RESULTS: The chromosomal location of the greenish-yellow signals could be directly identified on the R-banded chromosome background. CONCLUSION: This method can serve as a rapid and precise system for chromosomal localization of DNA markers.
SPOCK, previously identified as testican, is a modular proteoglycan that carries both chondroitin and heparan sulfate glycosaminoglycan side chains. The overall genomic organization has been established. The SPOCK gene spans at least 70 kb and is composed of 11 exons: the first half of the gene is dramatically expanded, but the second half is more compact. In situ hybridization and YAC mapping independently linked the SPOCK gene to 5q31, a region containing an impressive number of genes encoding growth factors, cytokines, and neurotransmitter and hormone receptors. The gene is located between the IL9 and the EGR1 genes, bordering the smallest commonly deleted region of chromosome 5.