Skip to main content
SpringerLink
Log in

Oligonucleotide PRINS DNA Synthesis on Extended Chromatin Preparations

  • Protocol
PRINS and In Situ PCR Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 71))

Abstract

One of the inherent problems with conventional fluorescent in situ hybridization (FISH) on metaphase chromosomes has been the difficulty in resolving closely associated markers. Any targets separated by less than about 1 Mb (1 × 106 bp) tend to appear as a single locus on metaphase chromosomes. The first approach to improving this resolution was hybridization to interphase nuclei (1,2), in which, because the chromatin is decondensed, the markers are further apart and, therefore, more easily resolved. However, the chromatin in interphase nuclei is not identifiable as discrete strands, so it is both difficult to see whether two markers are definitely linked and impossible to tell whether, if they are physically linked, the chromatin between them follows a straight course or a convoluted one. Thus, it is necessary to measure the distance between a large number of signal pairs to establish modal and average figures for the distance between them. By this means, it has been possible to establish physical distances between markers as little as 50–100 kb (5 × 104–105 kbp) apart.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Trask, B. J. (1991) Fluorescence in situ hybridization: applications in cytogenetics and gene mapping. Trends Genet. 7, 149–154.

    PubMed  CAS  Google Scholar 

  2. Van den Engh, G., Sachs, R., and Trask, B. (1992) Estimating distance from DNA sequence location in cell nuclei by a random walk model. Science 257, 1410–1412.

    Article  PubMed  Google Scholar 

  3. Heng, H. H. Q., Squire, J., and Tsui, L.-C. (1992) High resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc. Natl. Acad. Sci. USA 89, 9509–9513.

    Article  PubMed  CAS  Google Scholar 

  4. Wiegant, J., Kalle, W., Mullenders, L., Brookes, S., Hoovers, J. M. N., Dauwerse, J. G., van Ommen, G. J. B., and Raap, A. K. (1992) High-resolution in situ hybridization using DNA halo preparations. Hum. Mol. Genet. 1, 587–591.

    Article  PubMed  CAS  Google Scholar 

  5. Parra, I. and Windle, B. (1993) High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature Genet. 5, 17–21.

    Article  PubMed  CAS  Google Scholar 

  6. Heiskanen, M., Karhu, R., Hellsten, E., Peltonen, L., Kallionemi, O.P., and Palotie, A. (1994) High resolution mapping using fluorescence in situ hybridizaion to extended DNA fibers prepared from agarose-embedded cells. BioTechniques 17, 928–933.

    PubMed  CAS  Google Scholar 

  7. Senger, G., Jones, T. A., Fidlerová, H., Sanséau, P., Trowsdale, J., Dutt, M., and Sheer, D. (1944) Released chromatin: linearized DNA for high resolution fluorescence in situ hybridization. Hum. Mol. Genet. 3, 1275–1280.

    Article  Google Scholar 

  8. Koch, J. E., Kølvraa, S., Petersen, K. B., Gregersen, N., and Bolund, I. (1989) Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ. Chromosoma 98, 259–265.

    Article  PubMed  CAS  Google Scholar 

  9. Gosden, J., Hanratty, D., Starling, J., Fantes, J., Mitchell, A., and Porteous, D. (1991) Oligonucleotide primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding and investigation of sequence organization. Cytogenet. Cell Genet. 57, 100–104.

    Article  PubMed  CAS  Google Scholar 

  10. Gosden, J. and Lawson, D. (1994) Rapid chromosome identification by oligonucleotide primed in situ DNA synthesis (PRINS). Hum. Mol. Genet. 3, 931–946.

    Article  PubMed  CAS  Google Scholar 

  11. Gosden, J. and Lawson, D. (1995) Instant PRINS: a rapid method for chromosome identification by detecting repeated sequences in situ. Cytogenet. Cell. Genet. 68, 57–60.

    Article  PubMed  CAS  Google Scholar 

  12. Shibasaki, Y. and Gosden, J. R. Manuscript in preparation.

    Google Scholar 

  13. Lichter, P. and Ried, T. (1994) Molecular analysis of chromosome aberrations, in Methods in Molecular Biology, vol. 29: Chromosome Analysis Protocols (Gosden, J. R., ed.), Humana, Totowa, NJ, pp. 449–478.

    Chapter  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Medical Research Council, Human Genetics Unit, Western General Hospital Edinburgh, Scotland

    Yoshiro Shibasaki & John R. Gosden

Authors
  1. Yoshiro Shibasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John R. Gosden
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Western General Hospital, Edinburgh, UK

    John R. Gosden

Rights and permissions

Reprints and permissions

Copyright information

© 1997 Humana Press Inc, Totowa, NJ

About this protocol

Cite this protocol

Shibasaki, Y., Gosden, J.R. (1997). Oligonucleotide PRINS DNA Synthesis on Extended Chromatin Preparations. In: Gosden, J.R. (eds) PRINS and In Situ PCR Protocols. Methods in Molecular Biology™, vol 71. Humana Press. https://doi.org/10.1385/0-89603-395-3:45

Download citation

  • DOI: https://doi.org/10.1385/0-89603-395-3:45

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-395-5

  • Online ISBN: 978-1-59259-557-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation