Molecular Single-Cell PCR Analysis of Rearranged Immunoglobulin Genes As a Tool to Determine the Clonal Composition of Normal and Malignant Human B Cells

  • Ralf Küppers
Part of the Methods in Molecular Biology book series (MIMB, volume 271)

Abstract

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 Igκ 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.

Key Words

B cells B-cell lymphoma clonality Hodgkin’s lymphoma micromanipulation V-gene recombination single-cell PCR somatic hypermutation Taq deoxyribonucleic acid (DNA) polymerase errors 

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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Ralf Küppers
    • 1
  1. 1.Tumor Research, Institute for Cell BiologyEssenGermany

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