Detection of Isotype Switch Rearrangement in Bulk Culture by PCR

Edward E. Max1, Frederick C. Mills2, Charles Chu3

1 Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, 2 Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, Maryland, 3 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.22
DOI:  10.1002/0471142735.im1022s04
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

When a B lymphocyte changes from synthesizing IgM to synthesizing IgG, IgA, or IgE, this isotype switch is generally accompanied by a unique DNA rearrangement. The protocols in this unit describe two polymerase chain reaction (PCR)‐based strategies for detecting switch rearrangements in bulk culture. The first involves direct PCR across the switch junctions, providing the opportunity for characterizing the recombination products by nucleotide sequence analysis; however, because of characteristics inherent to the PCR methodology this strategy cannot easily be used as a quantitative assay for recombination. A support protocol details the preparation of the 5' Su PCR probe for this protocol. The second basic protocol describes a method known as digestion‐circularization PCR (DCPCR) that is more amenable to quantitation but yields no information on structure of the recombination products. Both techniques should be capable of detecting reciprocal deletion circles as well as functional recombination products remaining on the expressed chromosome.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Direct PCR Across Switch Junctions
  • Support Protocol 1: Preparation of 5′ Sµ Probe by PCR and Random Primer Labeling
  • Basic Protocol 2: Digestion‐Circularization PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Direct PCR Across Switch Junctions

  Materials
  • Mononuclear cells from peripheral blood (PBMC; unit 7.1)
  • Complete IMDM‐10 medium ( appendix 2A)
  • Recombinant human IL‐4 (rhIL‐4; Table 97.80.4711)
  • 1% FCS (heat‐inactivated 1 hr, 56°C) in PBS (FCS/PBS), ice cold
  • Anti‐CD19 magnetic beads (Dynal)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10 mM Tris⋅Cl (pH 8.0)/ 10 mM EDTA
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 3 M sodium acetate, pH 5.0
  • 20 mg/ml proteinase K (store in aliquots at −20°C)
  • Buffered phenol (unit 10.1)
  • 25:1 (v/v) chloroform/isoamyl alcohol
  • 95% and 70% ethanol
  • TE buffer ( appendix 2A)
  • 2.5 µM oligonucleotide primers (Fig. A; 2.5 pmol/µl in sterile water; store at −20°C)
  • recipe10× direct PCR amplification buffer
  • Sterile water
  • Mineral oil
  • 2.5 U Taq DNA polymerase, diluted as in unit 10.20 (AmpliTaq,Cetus)
  • recipePrehybridization solution
  • protocol 2PCR‐amplified 32P‐labeled probe ( protocol 2)
  • 2× SSC/1% SDS/ 10 mM EDTA
  • 0.1× SSC/1% SDS/ 10 mM EDTA
  • 15‐ml conical and round‐bottom polypropylene centrifuge tubes ( Nunc #374632; Falcon 2059)
  • Beckman J‐6 centrifuge and JS‐4.2 swinging bucket rotor, equipped with 15‐ml tube adapters (or equivalent)
  • Motorized rocking platform
  • Rare earth cobalt magnets 0.6 × 0.6 × 2.5 in. (Edmund Scientific #30964)
  • 0.5‐ml microcentrifuge tubes, autoclaved
  • Aerosol barrier pipet tips (Continental Laboratory)
  • Automated thermal cycler
  • Immobilon‐N (Millipore) or equivalent PVDF membrane
  • 65°C shaking water bath
  • Additional reagents and equipment for Ficoll‐Hypaque density gradient centrifugation (unit 7.1), counting viable cells by trypan blue exclusion ( appendix 3B), phenol extraction and ethanol precipitation (unit 10.1), quantitation of DNA ( appendix 3L) agarose gel electrophoresis (unit 10.4), Southern hybridization using nylon filters (unit 10.6), enzymatic labeling of nucleic acids (units 10.10and 10.6), and autoradiography ( 3.NaN)

Support Protocol 1: Preparation of 5′ Sµ Probe by PCR and Random Primer Labeling

  Materials
  • pXE plasmid (Mills et al., ; available from author) or human genomic DNA
  • 2.5 µM oligonucleotide primers NPS‐AR and F14 (Fig. A; 2.5 pmol/µl in sterile H 2O; store at −20°C)
  • recipe10 × direct PCR amplification buffer
  • 2.5 U Taq DNA polymerase, diluted as in unit 10.20 (AmpliTaq, Cetus)
  • Mineral oil
  • Boiling H 2O bath
  • Additional reagents and equipment for PCR (unit 10.20), purification of DNA on low‐gelling/melting temperature agarose (unit 10.5) and labeling DNA by random oligonucleotide‐primed synthesis (unit 10.10)

Basic Protocol 2: Digestion‐Circularization PCR

  Materials
  • Mouse spleens (unit 1.10)
  • Complete RPMI‐5 medium ( appendix 2A) containing 1 U/ml penicillin and 1 µg/ml streptomycin
  • 4 mg/ml Escherichia coli LPS (Difco #011B4; store at −20°C)
  • Recombinant murine IL‐4 (Table 97.80.4711)
  • EcoRI restriction endonuclease and 10× EcoRI buffer (supplied with enzyme)
  • 400 U/µl T4 DNA ligase (New England Biolabs and 10× ligase buffer (unit 10.9)
  • recipe10× DCPCR amplification buffer
  • 2.5 µM oligonucleotide primers (Fig. B; 2.5 pmol/µl in sterile water; store at −20°C)
  • 2.5 U Taq DNA polymerase, diluted as in unit 10.20 (AmpliTaq, Cetus)
  • Sterile water
  • Modified spiking plasmid (optional; available from authors)
  • 10 mCi/ml [α‐32P]dCTP (3000Ci/mmol; Amersham or NEN; optional)
  • Mineral oil
  • Humidified 37°C, 6% CO 2 incubator
  • 70° and 16°C water baths
  • 1.5‐ml and 0.5‐ml microcentrifuge tubes, autoclaved
  • Aerosol barrier pipet tips (Continental Laboratory)
  • Automated thermal cycler
  • Additional reagents and equipment for preparation of cell suspensions from spleen (unit 3.1), T cell depletion by cytotoxic elimination (unit 3.4), isolation of B cells by Percoll gradient centrifugation (unit 3.8), preparation of genomic DNA from mammalian tissue (unit 10.2), quantitation of DNA ( appendix 3L) and digestion of DNA with restriction endonucleases (unit 10.8)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bloch, K.D. and Bartos, B. 1992. Digestion of DNA with restriction endonucleases. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 3.1.1‐3.1.20. Greene Publishing and John Wiley & Sons, New York.
   Chory, J. 1987. Nondenaturing polyacrylamide gel electrophoresis. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp.2.7.1‐2.7.5‐A.3.15. Greene Publishing and John Wiley & Sons, New York.
   Chou, Q., Russell, M., Birch, D.E., Raymond, J., and Bloch, W. 1992. Prevention of pre‐PCR mispriming and primer dimerization improves low‐copy‐number amplifications. Nucl. Acids Res. 1717‐1723.
   Chu, C.C., Paul, W. E., and Max, E.E. 1992. Quantitation of immunoglobulin µ‐γ1 heavy chain switch region recombination by a digestion‐circularization polymerase chain reaction method. Proc. Natl. Acad. Sci. U.S.A. 89:6978‐6982.
   Collins, F.S. and Weissman, S.M. 1984. Directional cloning of DNA fragments at a large distance from an initial probe: A circularization method. Proc. Natl. Acad. Sci. U.S.A. 81:6812‐6816.
   Mills, F.C., Brooker, J.S., and Camerini‐Otero, R.D. 1990. Sequences of human immunoglobulin switch regions; implications for recombination and transcription. Nucl. Acids Res. 18:7305‐7316.
   Mills, F.C., Thyphronitis, G., Finkelman, F.D., and Max, E.E. 1992. Immunoglobulin µ‐ɛ switch in IL‐4‐treated human B lymphoblastoid cells. J. Immunol. 149:1075‐1083.
   Ochman, H., Medhora, M.M., Garza, D., and Hartl, D.L. 1990. Amplification of flanking sequences by inverse PCR. In PCR Protocols (M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White, eds.) pp. 219‐227. Academic Press, San Diego.
   Shapira, S.K., Jabara, H.H., Thienes, C.F., Ahern, D.J., Vercelli, D., Gould, H.J., and Geha, R.S. 1991. Deletional switch recombination occurs in interleukin‐4‐induced isotype switching to IgE expression by human B cells. Proc. Natl. Acad. Sci. U.S.A. 88:7528‐7532.
   Shapira, S.K., Vercelli, D., Jabara, H.H., Fu, S.M., and Geha, R.S. 1992. Molecular analysis of the induction of immunoglobulin E synthesis in human B cells by interleukin‐4 and engagement of CD40 antigen. J. Exp. Med. 175:289.
   Ueda, S., Nakai, S., Nishida, Y., Hisajima, H., and Honjo, T. 1982. Long terminal repeat‐like elements flank a human immunoglobulin epsilon pseudogene that lacks introns. EMBO J. 1:1539‐1544.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library