Detection of isotype switch rearrangement in bulk culture by PCR.
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.
Max
Edward E
EE
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland.
Mills
Frederick C
FC
Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, Maryland.
Chu
Charles
C
National Institute of Allergy and Infectious Diseases, Bethesda, Maryland.
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002478:Cells, Cultured; D017578:Immunoglobulin Class Switching / Q000235:genetics* / Q000276:immunology; D007136:Immunoglobulins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*
D007136:Immunoglobulins