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  • Title: Molecular single-cell PCR analysis of rearranged immunoglobulin genes as a tool to determine the clonal composition of normal and malignant human B cells.
    Author: Küppers R.
    Journal: Methods Mol Biol; 2004; 271():225-38. PubMed ID: 15146124.
    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 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.
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