UNIT 10.22 Detection of Isotype Switch Rearrangement in Bulk Culture by PCR
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Immunology
How to Cite
Max, E. E., Mills, F. C. and Chu, C. 2001. Detection of Isotype Switch Rearrangement in Bulk Culture by PCR. Current Protocols in Immunology. 4:VII:10.22:10.22.1–10.22.16.
- Published Online: 1 MAY 2001
- Published Print: DEC 1992
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 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 details the preparation of the 5' Su PCR probe for this protocol. The second 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.