Principles and Applications of PRINS in Cytogenetics

Jørn Koch1

1 Laboratory of Molecular Pathology, Institute of Pathology Aarhus Kommunehospital, Aarhus, Denmark
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.11
DOI:  10.1002/0471142956.cy0811s27
Online Posting Date:  February, 2004
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Abstract

A flexible, low‐cost alternative to FISH, primed in situ labeling (PRINS) has traditionally been used to detect tandemly repeated target sequences in chromosomes and nuclei. The technique is capable of discriminating among closely related DNA sequences in situ and has the advantage of using very small probes which easily penetrate to almost any target. This unit describes basic PRINS and the alternative version, dideoxy‐PRINS, which can increase the sensitivity of the reaction by an order of magnitude. New material on multicolor PRINS and quantitative PRINS has been added. Protocols for detection of single‐copy sequences and for application to the study of in‐vivo activity of DNA‐modifying enzymes are planned.

Keywords: molecular cytogenetics; PRINS; DNA detection in situ; DNA synthesis in situ; sequence discrimination in situ; quantitative DNA detection in situ; combinatorial in situ staining

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Basic Prins on Chromosome Spreads
  • Basic Protocol 2: Multicolor Prins (M‐PRINS) and PRINS‐FISH Reactions
  • Basic Protocol 3: Quantitative PRINS (Q‐PRINS)
  • Basic Protocol 4: Rolling‐Circle PRINS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Basic Prins on Chromosome Spreads

  Materials
  • Slides containing chromosome spreads (unit 8.2)
  • Reaction mixture (see recipe)
  • Stop buffer (see recipe)
  • Wash buffer (see recipe)
  • Blocking solution (see recipe)
  • 2 ng/ml fluorochrome‐labeled anti‐digoxigenin (Fab fragment; Roche) or fluorochrome‐labeled streptavidin (Roche, Vector Laboratories)
  • Antifade mounting medium (Vectashield from Vector Laboratories or p‐phenylenediamine from Sigma) containing either of the following:
    • 0.4 µM DAPI (Sigma; for blue counterstaining of DNA)
    • 0.5 µg/ml propidium iodide (Sigma; for red counterstaining of DNA)
  • Coverslips of appropriate size (e.g., 24 × 60 mm)
  • 94°C and 55° to 65°C heating blocks and insulating lids to cover slide and block or dedicated PRINS/in situ PCR machine (Hybaid, MJ Research, or Techne)
  • Fluorescence microscope with standard excitation and emission filters (e.g., 81000 filter set from Chroma Technology)

Basic Protocol 2: Multicolor Prins (M‐PRINS) and PRINS‐FISH Reactions

  Materials
  • Labeled nucleotides with two different labels (e.g., digoxigenin‐11‐dUTP and fluorescein‐dUTP) to be used in preparation of reaction mixture (see recipe)
  • Ethanol series, ice cold (see recipe)
  • Additional labeled nucleotide with rhodamine‐dUTP (Roche) or biotin‐dUTP (Enzo) label (optional)
  • Additional reagents and equipment for PRINS (see protocol 1)

Basic Protocol 3: Quantitative PRINS (Q‐PRINS)

  Materials
  • Slides containing ethanol‐fixed cells (see, e.g., unit 7.5 for fixation technique)
  • Low‐salt buffer: e.g., PBS or SSC (see appendix 2A for recipes)
  • 0.1% (w/v) pepsin in 0.1 M HCl: freshly prepared and preheated to 37°C (it may take several minutes to dissolve pepsin even at 37°C)
  • Ethanol series (see recipe)
  • RNase A mixture (see recipe)
  • Restriction enzyme mixture (see recipe) containing suitable restriction enzyme (see step )
  • λ‐exonuclease mixture (see recipe)
  • Padlock probe(s) (unit 8.8)
  • Hybridization mixture (see recipe)
  • DNA ligase mixture (see recipe)
  • Padlock removal solution: 2× SSC ( appendix 2A) containing 30% (v/v) deionized formamide
  • Reaction mixture for rolling‐circle PRINS (see recipe)
  • Identifier oligonucleotides (see annotation to step )
  • Antifade mounting medium (Vectashield from Vector Laboratories or p‐phenylenediamine from Sigma) containing either of the following:
    • 0.4 µM DAPI (Sigma; for blue counterstaining of DNA)
    • 0.5 µg/ml propidium iodide (Sigma; for red counterstaining of DNA)
  • Coverslips of appropriate size (e.g., 24 × 60 mm to completely cover a standard slide)
  • 37°C humidified incubator
  • Fluorescence microscope with standard excitation and emission filters (e.g., 81000 filter set from Chroma Technology)
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Figures

Videos

Literature Cited

Literature Cited
   Andersen, C.L., Wandall, A., Kjeldsen, E., Mielke, C., and Koch, J. 2002. Active, but not inactive, human centromeres display topoisomerase II activity in vivo. Chromosome Res. 10:305‐312.
   Bloch, K.D. and Grossman, B. 1995. Digestion of DNA with restriction endonucleases. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and, K. Struhl, eds.) pp. 3.1.1‐3.1.21. John Wiley & Sons, New York.
   Hindkjær, J., Brandt, C.A., Koch, J., Lund, T.B., Kølvraa, S., and Bolund, L. 1995. Simultaneous detection of centromere specific probes and chromosome painting libraries by a combination of PRimed IN Situ labeling and chromosome painting (PRINS‐painting). Chromosome Res. 3:41‐44.
   Kadandale, J.S., Wachtel, S.S., Tunca, Y., Wilroy, R.S. Jr., Martens, P.R., and Therapel, A.T. 2000a. Localization of SRY by primed in situ labeling in XX and XY sex reversal. Am. J. Med. Genet. 95:71‐74.
   Kadandale, J.S., Tunca, Y., and Therapel, A.T. 2000b. Chromosomal localization of single copy genes SRY and SOX3 by primed in situ labeling (PRINS). Microb. Comp. Genomics 5:71‐74.
   Koch, J. 1999. PRINS: PRimed IN Situ labeling and hybridization in one step. In Nonradioactive Labelling and Detection of Biomolecules, 2nd ed. (C. Kessler, ed.) pp. 407‐416. Springer Verlag, Heidelberg.
   Koch, J., Kølvraa, S., Gregersen, N., and Bolund, L. 1989. Oligonucleotide‐priming methods for the chromosome‐specific labelling of alpha satellite DNA in situ. Chromosoma 98:259‐265.
   Koch, J., Hindkjær, J., Kølvraa, S., and Bolund, L. 1995. Construction of a panel of chromosome specific oligonucleotide probes (PRINS‐primers) useful for the identification of individual human chromosomes in situ. Cytogenet. Cell Genet. 71:142‐147.
   Krejci, K. and Koch, J. 1998. Improved detection and comparative sizing of human chromosomal telomeres in situ. Chromosoma 107:198‐203.
   Krejci, K. and Koch, J. 1999. An in situ study of variant telomeric repeats. Genomics 58:202‐206.
   Nilsson, M., Krejci, K., Koch, J., Kwiatkowski, M., Gustavson, P., and Landegren, U. 1997. Padlock probes reveal single‐nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21. Nature Genet. 16:252‐255.
   Pellestor, F., Girardet, A., Andréo, B., and Charlieu, J.P. 1994. A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS). Hum. Genet. 94:346‐348.
   Pellestor, F., Girardet, A., Lefort, G., Andréo, B., and Charlieu, J.P. 1995. Rapid in situ detection of chromosome 21 by PRINS technique. Am. J. Med. Genet. 56:1‐8.
   Pellestor, F., Girardet, A., Andréo, B., Lefort, G., and Charlieu, J.P. 1997. Incidence of chromosome 1 disomy in human sperm estimated by the primed in situ (PRINS) labeling technique. Cytogenet. Cell Genet. 76:192‐195.
   Serakinci, N. and Koch, J. 1999. Detection and sizing of human telomeric repeat DNA in situ. Nature Biotechnol. 17:200‐201.
   Serakinci, N., Krejci, K., and Koch, J. 1999. Telomeric repeat organization: A comparative in situ study between man and rodent. Cytogenet. Cell Genet. 86:204‐211.
   Serakinci, N., Ostergaard, M., Larsen, H., Madsen, B., Pedersen, B., and Koch, J. 2002. Multiple telomeric aberrations in a telomerase‐positive leukemia patient. Cancer Genet. Cytogenet. 138:11‐16.
   Therkelsen, A.J., Nielsen, A., Koch, J., Hindkjær, J., and Kølvraa, S. 1995. Staining of human telomeres with primed in situ labeling (PRINS). Cytogenet. Cell Genet. 68:115‐118.
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