John M. S. Bartlett.pdf - Bio-Nica.info

186 Cremer and Moos
Fig. 1. Rearranged VDJ segment of a mature B-cell and strategies for consensus PCR to
amplify the VDJ segment. The CDR regions of the heavy chain of the immunoglobulin gene
are flanked by highly conserved segments, the framework (FR) regions 1 through 4. Consensus
primers complementary to these FR-regions have been developed that can be used to amplify
the enclosed highly variable CDR regions regardless of their sequence. In this chapter, two
strategies are described using either FR1C or FR3A plus LJH.
diversity of all CDR-regions, and its sequence is a specific marker for each clone
of B-cells (2,3).
In MM, the CDR regions of the malignant cells are somatically hypermutated, and
no further mutations occur, which would lead to an oligoclonal diversification (4). This
is a prerequisite for designing ASO primers complementary to the CDR regions of the
malignant clone that allow the detection of myeloma cells by PCR.
For designing ASO primers, the sequence of the CDR regions of the malignant
clone has to be determined. In a first step, the CDR regions of virtually all B-cells are
amplified with consensus primers flanking the CDRs (see Fig. 1). Besides the strategies
using FR1C (5) or FR3A (6) as sense primers and LJH as antisense primer, consensus
PCR with family-specific primers complementary to the leader segment 5′-end to
the V-region (7), with a mixture of six FR1 family-specific primers (8), with an FR2
consensus primer alone (9) or as a mixture with the FR1 family-specific primers
VH5 and VH6 (5) and a mixture of JH1245, JH3, and JH6 antisense primers (5)
has been described. After consensus PCR, the product of the malignant clone can
be distinguished from normal ones clones by its predominant occurrence among
the polyclonal CDR-regions. After sequencing (directly or after cloning), primers
complementary to the CDR regions can be designed. These ASO primers have to be
tested for specificity and sensitivity before they can be used for the PCR detection of
cells of the malignant clone. PCR is quantified using the method of limiting dilutions.
2. Materials
2.1. Isolation of Nucleic Acids
1. Bone marrow (BM) sample with a high proportion of myeloma cells.
2. Ficoll separating solution (Biochrom, Berlin, Germany).
3. Phosphate-buffered saline (pH 7.4; Gibco BRL, Eggenstein, Germany).
4. TE buffer (10 mM Tris-HCl, pH 7.5, and 1 mM EDTA).
5. Reagents for RNA extraction, for example, Trizol Reagent (Gibco BRL).
6. Reagents for DNA extraction, for example, DNAzol (Gibco BRL).
7. Ethidium bromide-stained agarose gels (0.8 and 2%).