PCR analysis and sequencing of immunoglobulin V
gene rearrangement in EPCs in MM patients. A series of 7 VH family-specific primers (VH1–6) common to the most described members of the corresponding VH family, including two separate primers specific for the VH4 family (VH4A and -4B), were used as forward primers. A consensus JH primer was used as reverse primer to amplify rearranged DNA, producing an expected 350-bp PCR product as described by Deane and Norton . (A) Agarose gel analysis of VH4A 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 VH family primers and the constant JH 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 VH1-JH 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 VH5 or VH6 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' VH4A and JH regions identified by BLAST are shown above the sequences along with numbers indicating the length of the sequences analyzed. The VH4A-JH rearrangement in EPCs and BM cells from this patient has a 100% sequence overlap.