High-resolution mapping of satellite DNA using biotin-labeled DNA probes

We have developed a novel method for high resolution mapping of specific DNA sequences after in situ hybridization. DNA probes, labeled with biotin-nucleotides in conventional nick-translation reactions, are hybridized to cytological preparations and detected with affinity-purified rabbit antibiotin antibodies followed by antibodies to rabbit IgG that are conjugated to fluorescent or enzymatic reagents. Using peroxidase labeled anti-rabbit IgG, we are able to detect and localize specific sequences at both the light and electron microscopic levels. Initial studies were done with repeated DNA sequences previously mapped by light microscope autoradiography to assess the fidelity and resolution of this method. An analysis using biotin-labeled mouse satellite DNA is presented here.

[1]  M. Ferguson-Smith,et al.  Chromosomal localization of a single copy gene by in situ hybridization ‐ human β globin genes on the short arm of chromosome 11 , 1981, Annals of human genetics.

[2]  B. Erlanger,et al.  Localization of 5-methylcytosine in human metaphase chromosomes by immunoelectron microscopy , 1976, Cell.

[3]  S. Hsu,et al.  Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. , 1981, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[4]  W. Knapp,et al.  Immunofluorescence and related staining techniques , 1978 .

[5]  J. Littlefield Three Degrees of Guanylic Acid — Inosinic Acid Pyrophosphorylase Deficiency in Mouse Fibroblasts , 1964, Nature.

[6]  P Berg,et al.  Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. , 1977, Journal of molecular biology.

[7]  K. Hofmann,et al.  Iminobiotin affinity columns and their application to retrieval of streptavidin. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Birnstiel,et al.  RNA-DNA Hybrids at the Cytological Level , 1969, Nature.

[9]  Comings De The rationale for an ordered arrangement of chromatin in the interphase nucleus. , 1968 .

[10]  K. Jones Chromosomal and Nuclear Location of Mouse Satellite DNA in Individual Cells , 1970, Nature.

[11]  J. Sedat,et al.  A direct approach to the structure of eukaryotic chromosomes. , 1978, Cold Spring Harbor symposia on quantitative biology.

[12]  J. Sedat,et al.  SOFT X‐RAY LITHOGRAPHIC STUDIES OF INTERPHASE CHROMOSOMES , 1980, Annals of the New York Academy of Sciences.

[13]  D. Ward,et al.  Immunological method for mapping genes on Drosophila polytene chromosomes. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D C Ward,et al.  Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Gall,et al.  Formation and detection of RNA-DNA hybrid molecules in cytological preparations. , 1969, Proceedings of the National Academy of Sciences of the United States of America.

[16]  L. Manuelidis,et al.  Amount of satellite DNA in four experimentally induced tumors of the central nervous system. Quantitative changes in a glioblastoma producing C-type particles. , 1976, Journal of the National Cancer Institute.

[17]  E. Manuelidis,et al.  Localization of mouse satellite DNA on chromosomes of experimentally induced glioblastomas; non‐centromeric lable in one glioblastoma producing C‐type particles , 1976, International journal of cancer.

[18]  D. Comings The rationale for an ordered arrangement of chromatin in the interphase nucleus. , 1968, American journal of human genetics.

[19]  D. Gerhard,et al.  Localization of a unique gene by direct hybridization in situ. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[20]  B. Hamkalo,et al.  In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold , 1982, The Journal of cell biology.

[21]  T. Ashley Specific end-to-end attachment of chromosomes in Ornithogalum virens. , 1979, Journal of cell science.

[22]  L. Manuelidis A simplified method for preparation of mouse satellite DNA. , 1977, Analytical biochemistry.