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. 2009 Jul 7;94(8):1135–1150. doi: 10.3324/haematol.2008.004267

Figure 1.

Figure 1.

IgH rearrangement and heteroduplex clonality studies by polymerase chain reaction (PCR). (A) A schematic representation of the IgH gene rearrangement is shown. Double strand DNA breaks are made to enable the V, D and J heavy immunoglobulin genes rearrangement in cells that are destined to become B lymphocytes and define these cells from others. Clonal population is detected when a group of cells with a unique VDJ spliced sequence expands such that it is detectable above the normal background of B-cell populations that have undergone rearrangement. (B) Heteroduplex analysis. (i) Schematic representation of the IgH rearranged gene (VH-DH-JH) is shown along with position of VH–family specific and JH consensus primers. The yellow lines represent the position of insertion and/or deletion of nucleotides at junctional regions of IgH. (ii) Ethdium stained acrylamide gel with results of a heteroduplex analysis is illustrated. In brief, the junctional region heterogeneity of PCR products of rearranged IgH or TCR genes is exploited to distinguish between monoclonal and polyclonal lymphoid B or T expansion. In heteroduplex studies PCR amplicons are heat denatured and rapidly cooled to induce homo- or heteroduplex. Samples with clonal lymphoid cells the PCR products of rearranged IgH or TCR genes yield homoduplex. In contrast samples with polyclonal lymphoid expansion the single strand PCR fragments leads to formation of heteroduplexes upon re-annealing. In samples with polyclonal and monoclonal expansion, both homoduplex and heteroduplex arise. Thus, because of the conformation differences the homo and heteroduplexes forms can be separated from each other by gel electrophoresis through non-denaturing acrylamide gels as shown. Homoduplexes migrate through the gel faster than the heteroduplexes with imperfect complimentary pairing. The latter form a background smear of slow migrating fragments. The homoduplex yields a relatively sharp discrete amplicon band. (iii) Automated clonal studies (florescent Genescan analysis). Polyclonal VH-DH-JH products form peaks reflecting a Gaussian distribution of average junctional region sizes in normal B lymphocytes. Monoclonal VH-DH-JH gene rearrangements form a discrete fluorescence peak, representing products of identical size. Adapted with permission from Van Dongen et al. Leukemia 2003;17:2257–17.