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Duplex PCR facilitates the identification of immunoglobulin kappa (IGK) gene rearrangements in acute lymphoblastic leukemia

Leukemia volume 14, pages 218–219 (2000)Cite this article

T cell receptor (TCR) and immunoglobulin (Ig) genes have been widely used as molecular targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL).12 Recent studies of immunoglobulin kappa (IGK) gene rearrangements using Southern blot and PCR strategies have demonstrated that rearrangements of the kappa deleting element (Kde) occur in approximately 50% of B-lineage ALL.34 Almost all IGK gene deletions are mediated via Kde which recombines either to one of the Vκ elements or to the recombination signal sequence in the intron between Jκ5 and Cκ (intron RSS). Kde rearrangements are much more stable during the progression of ALL than other Ig or TCR recombinations and can therefore be regarded as ideal markers for the monitoring of MRD.4 In B-lineage ALL the VκI, VκII, and VκIII elements are preferentially rearranged to Kde, and RSS-Kde recombinations represent approximately one-third of the Kde rearrangements.4 We have now established a duplex PCR strategy which allows the rapid identification of the main Kde rearrangements in ALL patients.

We designed two duplex PCRs for the detection of Kde rearrangements which are frequently observed in B-lineage ALL. One duplex PCR (D-PCR 1) amplifies Vκl-Kde and RSS-Kde rearrangements, the other (D-PCR 2) detects VκII-Kde, VκIII-Kde, and VκIV-Kde recombinations. 100 ng of genomic DNA were were processed in 70 μl of the PCR mixture containing 7 μl of 10× buffer, 2.1 μl of 50 mM MgCl2, 1 U of Taq polymerase (Life Technologies, Karlsruhe, Germany), as well as 200 μM dNTP and 30 pmol of each primer. Primers used for D-PCR 1 were VκI-5′, intron-3′ as well as Kde-3′, and VκII-5′, VκIII-3′ and Kde-3′ for D-PCR 2.5 The mixture was initially heated at 94°C for 4 min and the DNA was amplified using a touch down program whose annealing temperature begins at 66°C and decreases 0.5°C at every cycle on a thermal cycler (Primus96, MWG AG Biotech, Ebersberg, Germany). After the annealing temperature was reached at 52°C in the 28th cycle, 12 additional cycles were performed at 52°C. 10 μl of each PCR product was resolved on an agarose gel (FMC, Rockland, ME, USA) containing ethidium bromide. Buffy coat (BC) DNA from healthy volunteers and water were used in every experiment as negative controls. Heteroduplex analysis was performed as described.6 Homoduplex formations of the expected size were considered as indicators of a clonal cell population. 40 μl of the PCR products were then electrophoresed on an agarose gel and purified with a QIAEXII Kit (Qiagen, Hilden, Germany). For direct sequencing the appropriate internal primer was used.5 Sequence analyses were carried out using a Cy5 Thermo Sequenase Dye Termination Kit (Amersham Pharmacia Biotech, Freiburg, Germany) and an autosequencer (ALFexpress II, Amersham Pharmacia Biotech). In cases exhibiting two homoduplex bands in the heteroduplex analysis the PCR product was cloned into a plasmid vector (TOPO TA cloning kit; Invitrogen, Groningen, The Netherlands) prior to sequence analysis. Ten-fold serial dilutions were made of diagnostic DNA into BC DNA. One μg of each dilution (from 10−1 to 10−6), BC DNA as well as H2O were amplified with the respective outer primers5 using the touch down program described above. 5 μl of each product was dotted on to a nylon filter. ASO design, end-labelling, and hybridization were carried out as described.5

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  1. Institute of Human Genetics, University of Heidelberg, Germany

    M Nakao, JWG Janssen & CR Bartram

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Nakao, M., Janssen, J. & Bartram, C. Duplex PCR facilitates the identification of immunoglobulin kappa (IGK) gene rearrangements in acute lymphoblastic leukemia. Leukemia 14, 218–219 (2000). https://doi.org/10.1038/sj.leu.2401624

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