Single‐Nucleotide Sequence Discrimination In Situ Using Padlock Probes

Mats Nilsson1, Ulf Landegren1, Dan‐Oscar Antson1

1 Uppsala University, Uppsala, Sweden
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.11
DOI:  10.1002/0471142905.hg0411s34
Online Posting Date:  November, 2002
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Abstract

DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation. This mechanism has been exploited to distinguish DNA sequence variants in situ using so‐called padlock probes. Padlock probes are linear oligonucleotides with target‐complementary sequences at both ends, and an on‐target‐complementary segment in between. The end sequences are brought next to each other upon hybridization to the target DNA sequence, and if the ends are perfectly matched to the target sequence, they can be joined by a DNA ligase. Padlock probes detect target sequences with very high specificity, because both probe segments must hybridize to the target for circularization to occur. This unit presents a protocol for discrimination between closely similar DNA sequences in situ using padlock probes. A discussion of methods for greatly amplifying the signal from circularized probes is also included.DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation.

     
 
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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 Padlock Probe Ligation Reaction at Low Probe Concentration
  • Support Protocol 1: Enzymatic Synthesis of Padlock Probes
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Padlock Probe In Situ Ligation Reaction Using a Thermostable Ligase

  Materials
  • RNase A–treated metaphase chromosome slides (unit 4.1), prepared fresh
  • recipeTth ligation mix (see recipe)
  • recipeStop buffer (see recipe)
  • Deionized formamide (store protected from light for up to 6 months at 4°C)
  • 2× SSC ( appendix 2D)
  • recipeWash 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

  • recipeT4 ligation mix (see recipe)

Alternate Protocol 2: Enzymatic Padlock Probe Ligation Reaction at Low Probe Concentration

  • recipeOvernight 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

  Materials
  • 10× AmpliTaq buffer with 1.5 mM MgCl 2 (PE Biosystems)
  • recipedNTP 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)
  • recipe5010 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 2D)
  • 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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Figures

Videos

Literature Cited

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.
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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