Single‐Nucleotide Sequence Discrimination In Situ Using Padlock Probes

Mats Nilsson1, Ulf Landegren1, Dan‐Oscar Antson1

1 Uppsala University, Uppsala
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.8
DOI:  10.1002/0471142956.cy0808s16
Online Posting Date:  May, 2001
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Standard fluorescence in situ hybridization (FISH) techniques using cloned probes are limited in their ability to distinguish between closely similar DNA sequences because long hybridization probes are not detectably destabilized by single mismatched base pairs. This problem has been addressed by using short alleleā€specific oligonucleotide probes whose hybridization to target sequences is more sensitive to mismatches. This revised and expanded unit presents protocols for discrimination between closely similar DNA sequences in situ. The discussion of probe synthesis has been greatly expanded and an added for enzymatic probe ligation at low probe concentration. A new describes enzymatic probe synthesis.

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Table of Contents

  • Strategic Planning
  • Chemical Synthesis of Padlock Probes
  • Enzymatic Synthesis of Padlock Probes
  • Basic Protocol 1: Padlock Probe In Situ Ligation Reaction Using a Thermostable Ligase
  • Alternate Protocol 1: Padlock Probe In Situ Ligation Reaction Using T4 DNA Ligase
  • Alternate Protocol 2: Enzymatic Paslock Probe Ligation Reaction At Low Probe Concentration
  • Support Protocol 1: Enzymatic Synthesis of Padlock Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Padlock Probe In Situ Ligation Reaction Using a Thermostable Ligase

  • RNase A–treated metaphase chromosome slides (unit 8.2), prepared fresh
  • Tth ligation mix (see recipe)
  • Stop buffer (see recipe)
  • Deionized formamide (store protected from light for up to 6 months at 4°C)
  • 2× SSC ( appendix 2A)
  • Wash buffer (see recipe)
  • 10% (w/v) blocking solution (Boehringer Mannheim; stable >1 year at −20°C)
  • 1 mg/ml fluorescein isothiocyanate–conjugated avidin (FITC‐avidin; Vector Laboratories)
  • 200 µg/ml rhodamine‐conjugated anti‐digoxigenin Fab fragments (Boehringer Mannheim)
  • 70%, 85%, and 100% (v/v) ethanol
  • Vectashield mounting medium (Vector Laboratories) containing 100 ng/ml 4′,6‐diamidino‐2‐phenylindole (DAPI) counterstain
  • Programmable heating block (e.g., GeneE from Techne)
  • 24 × 50–mm coverslips
  • Coplin jars
  • Fluorescence microscope with a charge‐coupled device (CCD) camera and a digital imaging system (e.g., IP‐lab, Vysis)

Alternate Protocol 1: Padlock Probe In Situ Ligation Reaction Using T4 DNA Ligase

  • T4 ligation mix (see recipe)

Alternate Protocol 2: Enzymatic Paslock Probe Ligation Reaction At Low Probe Concentration

  • Overnight hybridization mix (see recipe)
  • Rubber cement (e.g., FASTIK, AB Thure Bünger)
  • 37°C humid chamber (e.g., a 1‐liter beaker containing 2× SSC–soaked paper towel and covered with aluminum foil)
  • Tweezers

Support Protocol 1: Enzymatic Synthesis of Padlock Probes

  • 10× AmpliTaq buffer with 1.5 mM MgCl 2 (PE Biosystems)
  • dNTP mix (see recipe)
  • 1 mM labeled dUTP (e.g., digoxigenin‐11‐dUTP, Boehringer Mannheim; or dinitrophenyl‐11‐dUTP, Molecular Probes)
  • 5 µM 5′‐phosphorylated forward primer
  • 5 µM 5′‐biotinylated reverse primer
  • 30 pg bacteriophage λ DNA (e.g., Amersham Pharmacia Biotech)
  • 5 U/µl AmpliTaq DNA polymerase (PE Biosystems)
  • 2% (w/v) agarose gel
  • 3 M sodium acetate, pH 4.6
  • 70% and 100% ethanol, ice cold
  • Streptavidin‐coated paramagnetic particles (Dynabeads M‐280 Streptavidin, Dynal)
  • 5010 buffer (see recipe)
  • 2 M NaCl
  • 0.15 M lithium hydroxide (LiOH)
  • 0.15 M ammonium chloride (NH 4Cl)
  • Glycogen (Boehringer Mannheim)
  • TE buffer ( appendix 2A)
  • 0.5‐ml and 1.5‐ml microcentrifuge tubes
  • PCR thermal cycler
  • Microcentrifuge (e.g., Biofuge 15, Heraeus)
  • Magnetic particle concentrator rack (Dynal)
  • Test‐tube rotator (e.g., Labinco 528)
  • Ultra microvolume cell (Amersham Pharmacia Biotech)
  • Spectrophotometer (e.g., GeneQuant, Amersham Pharmacia Biotech)
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Literature Cited

   Antson, D.‐O., Isaksson, A., Landegren, U., and Nilsson, M. 2000. PCR‐generated padlock probes detect single‐nucleotide variation in genomic DNA. Nucl. Acids Res. 28:e58.
   Banér, J., Nilsson, M., Mendel‐Hartvig, M., and Landegren, U. 1998. Signal amplification of padlock probes by rolling circle replication. Nucl. Acids Res. 22:5073‐5078.
   Connolly, B.A. 1987. Solid phase 5′‐phosphorylation of oligonucleotides. Tetrahedron Lett. 28:463‐466.
   Fire, A. and Xu, S.‐Q. 1995. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. U.S.A. 92:4641‐4645.
   Guzaev, A., Salo, H., Azhayev, A., and Lönnberg, H. 1995. A new approach for chemical phosphorylation of oligonucleotides at the 5′‐terminus. Tetrahedron. 51:9375‐9384.
   Hermanson, G.T. 1996. Bioconjugate Techniques. Academic Press, San Deigo.
   Koch, J., Kølvraa, S., Petersen, K., Gregersen, N., and Bolund, L. 1989. Oligonucleotide‐priming methods for the chromosome‐specific labelling of alpha satellite DNA in situ. Chromosoma 98:259‐265.
   Landegren, U., Kaiser, R., Sanders, J., and Hood, L. 1988. A ligase‐mediated gene detection technique. Science. 241:1077‐1080.
   Liu, D., Daubendiek, S.L., Zillman, M.A., Ryan, K., and Kool, E.T. 1996. Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases. J. Am. Chem. Soc. 118:1587‐1594.
   Lizardi, P.M., Huang, X., Zhu, Z., Bray‐Ward, P., Thomas, D.C., and Ward, D.C. 1998. Mutation detection and single‐molecule counting using isothermal rolling‐circle amplification. Nature Genet. 19:225‐232.
   Luo, J., Bergstrom, D.E., and Barany, F. 1996. Improving the fidelity of Thermus thermophilus DNA ligase.Nucl. Acids. Res. 24:3071‐3078.
   Nilsson, M., Malmgren, H., Samiotaki, M., Kwiatkowski, M., Chowdhary, B.P., and Landegren, U. 1994. Padlock probes: Circularizing oligonucleotides for localized DNA detection. Science. 265:2085‐2088.
   Nilsson, M., Krejci, K., Koch, J., Kwiatkowski, M., Gustavsson, 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.
   O'Keefe, C.L., Warburton, P.E., and Matera, A.G. 1996. Oligonucleotide probes for alpha satellite DNA variants can distinguish homologous chromosomes by FISH. Hum. Mol. Genet. 5:1793‐1799.
   Pellestor, F., Girardet, A., Genevieve, L., Andreo, B., and Charlieu, J.P. 1995. Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y. Hum. Genet. 95:12‐17.
   Raap, A.K., Marijnen, J.G.J., Vrolijk, J., and van der Ploeg, M. 1986. Denaturation, renaturation, and loss of DNA during in situ hybridization procedures. Cytometry. 7:235‐242.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Key References
   Antson et al., 2000. See above.
  A PCR‐based protocol for flexible enzymatic synthesis of padlock probes >100 or so nucleotides is described. The and are taken from this paper.
   Nilsson et al., 1994. See above.
  Describes the padlock probe design, and demonstrates the utility of in situ hybridization using a padlock probe that specifically detects an alpha satellite repeat on human chromosome 12. is taken from this paper, with some modifications.
   Nilsson et al., 1997. See above.
  Single‐nucleotide discrimination ability in situ is demonstrated using two padlock probes that distinguish two alpha satellite repeats on the basis of a single nucleotide substitution present on human chromosomes 13 and 21. The is taken from this paper.
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