Figure 3

PCR analysis and sequencing of immunoglobulin V _H gene rearrangement in EPCs in MM patients. A series of 7 V_H family-specific primers (V_H1–6) common to the most described members of the corresponding V_H family, including two separate primers specific for the V_H4 family (V_H4A and -4B), were used as forward primers. A consensus J_H primer was used as reverse primer to amplify rearranged DNA, producing an expected 350-bp PCR product as described by Deane and Norton [22]. (A) Agarose gel analysis of V_H4A family-specific IGH gene rearrangement detected in EPCs and BM cells from a MM patient. Briefly, 1 μg of genomic DNA was amplified with each of the V_H family primers and the constant J_H primer, which were added at a concentration of 1 μM each. Products were run on 1% agarose gels and visualized with ethidium bromide staining. DNA from MM cell line U266, which contains a V_H1-J_H rearrangement, was amplified simultaneously as a positive control for rearrangement, shown as a 350-bp PCR product. Primers specific for apolipoprotein B served as a control to monitor the efficacy of PCR. M indicates marker (1 Kb Plus DNA Ladder [Invitrogen, Carlsbad, CA]). No products were detected from this patient's DNA with the V_H5 or V_H6 primers (not shown). (B) Sequence analysis of IGH rearrangement found in EPCs and BM cells from a MM patient. Sequences obtained from PCR amplification of IGH DNA in EPCs and BM cells shown from Case 14 were compared using FASTA. The 5' V_H4A and J_H regions identified by BLAST are shown above the sequences along with numbers indicating the length of the sequences analyzed. The V_H4A-J_H rearrangement in EPCs and BM cells from this patient has a 100% sequence overlap.