b'Gel & Capillary | RUO AssaysGel and CapillaryIGH Gene Clonality AssaysResearch Use Only (RUO) AssayIGH Gene Clonality AssaysAssay Use The FRs are characterized by their similarity among the various V Hsegments, whereas the CDRs are highly different even within the same Immunoglobulin heavy chain (IGH) clonality assays are useful forV Hfamily. The CDRs represent the preferred target sequences for identification of B-cell clonality, studying clonal B-cell populations, andsomatic hypermutations; however, somatic mutations can also occur evaluation of new research and methods in malignancy studies. in the FRs. Therefore, family-specific primers in the three different FRs were designed to increase the detection rate of clonal IGH B-cell populations and decrease the occurrence of false-negative results Summary and Explanation of the Test1due to somatic hypermutation in primer binding sites. In addition to Five master mixes target conserved regions within the variable (V H ),V H -J Hrearrangements, incomplete D H -J Hrearrangements have been diversity (D H ), and the joining (J H ) regions that flank the uniquefound in mature and immature B-cell malignancies. Therefore, D H -J Hhypervariable, antigen-binding, complementarity determiningPCR analysis may be of added value for clonality assessment. 2region 3 (CDR3). Tube A contains six framework region 1 (FR1) primers and a consensus J Hregion primer. Tube B contains seven framework region 2 (FR2) primers and a consensus J Hprimer. Tube C contains seven framework region 3 (FR1) primers and a consensusSpecimen RequirementsJ Hprimer. Tube D contains six D Hregion primers and a consensus J H This assay tests extracted and purified genomic DNA (gDNA).region primer. Tube E contains a D H 7 region primer and a consensus J Hprimer. Positive and negative controls, as well as the Specimen Control Size Ladder Master Mix are included. PCR products can be analyzed by capillary electrophoresis or heteroduplex analysis.ReferencesClonality is indicated if any one of the master mixes generates a1.M Hummel et al., Leukemia 17:2266-2272 (2003).clonal product. 2. AW Langerak et al., Leukemia 17:2272-2275 (2003).3. JJM van Dongen et al., Leukemia 17:2257-2317 (2003).Background The immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.33, formerly 14q32.3) includes 46-52 functional and 30 nonfunctional variable (V H ), 27 functional diversity (D H ), and 6 functional joining (J H ) gene segments spread over 1250 kilobases. 1,2The most frequently used V Hgene segments in normal and malignant B cells belong to the V H 3, V H 4, and V H 1 family, together covering 7595% of V Husage. The V Hgene segments contain three framework regions (FR) and two complementarity determining regions (CDR).This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. Black arrows represent the relative positions of primers that target the conserved framework (FR1-3) and diversity (D H 1-7) regions, and the downstream consensus J Hgene segments. The amplicon products generated from each of these regions can be differentially detected when fluorescent primer sets are used with capillary electrophoresis instruments that employ differential fluorescence detection.102'