Molecular single-cell PCR analysis of rearranged immunoglobulin genes as a tool to determine the clonal composition of normal and malignant human B cells.
Owing to the nearly limitless diversity of immunoglobulin (Ig) variable-region gene rearrangements, such rearrangements represent ideal clonal markers for B-lineage cells. This chapter describes an approach to isolate single cells from frozen tissue sections by microdissection using a hydraulic micromanipulator and the subsequent amplification of rearranged IgH and Igkappa genes from the cells in a seminested polymerase chain reaction (PCR) approach. The amplification of a priori unknown V-gene rearrangements is made possible by the usage of a collection of V-gene family-specific primers recognizing nearly all V-gene segments together with primer mixes for the J-gene segments. By sequence comparison of V-gene amplificates from distinct cells, the clonal relationship of the B-lineage cells can unequivocally be determined. As a large part of the V-gene rearrangements is amplified, the approach is also useful to address additional issues, such as V-, D-, and J-gene usage and the presence and pattern of somatic mutations.
Küppers
Ralf
R
Tumor Research, Institute for Cell Biology, University of Essen Medical School, Essen, Germany.
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D019070:Cell Lineage / Q000235:genetics* / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics* / Q000276:immunology; D015328:Gene Rearrangement, B-Lymphocyte, Light Chain / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007144:Immunoglobulin J-Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D042282:Microdissection; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis
D017931:DNA Primers; D007144:Immunoglobulin J-Chains; D007135:Immunoglobulin Variable Region