b"Gel & Capillary | RUO AssaysGel and CapillaryIGH Somatic Hypermutation Assays v2.0 Research Use Only (RUO) AssayIGH Somatic Hypermutation Assays v2.0Assay Use Background The Research Use Only IGH Somatic Hypermutation Assay v2.0 is usedRearrangements of the antigen receptor genes occur during ontogeny to identify clonal rearrangements of the immunoglobulin heavy (IGH)in B and T lymphocytes.These gene rearrangements are unique chain gene and determine the somatic mutation status of the variablein length and sequence for each cell. Therefore, polymerase chain (V) gene sequence andis useful for the study of: reaction (PCR) assays can be used to identify lymphocyte populations derived from a single cell by detecting the unique V-J gene Identifying clonal rearrangements of the IGH chain gene rearrangements present within these antigen receptor loci. 1This PCR-Assessing the extent of somatic hypermutation in the variablebased assay employs multiple consensus DNA primers that target region of the immunoglobulin heavy chain gene conserved genetic regions within the immunoglobulin heavy chain Evaluating new research and methods in malignancy studies (IGH) gene.This test is used to detect and sequence the majority of clonal IGH rearrangements from either genomic DNA (gDNA) or complementary DNA (cDNA). Clonal products can be detected using Summary and Explanation of the Testa variety of methods, including gel and capillary electrophoresis.These assays amplify either genomic DNA or complementary DNAThe presence of IGH somatic hypermutation (SHM) is defined as (cDNA) that lies between the upstream leader (V H L) or framework 1greater or equal to 2% difference from the germline variable (V) gene sequence, whereas less than 2% difference is considered evidence of (FR1) regions and the downstream joining (J H ) region of the IGH gene.no somatic hypermutation. 3The assays employ two different master mixes: Hypermutation Mix 1 and Hypermutation Mix 2. The Hypermutation Mix 1 targets sequences between the leader (V H L) and joining (J H ) regions. Therefore theSpecimen Requirementsamplicon product(s) span the entire variable (V H ) region, which contains all framework (FR) and complementarity-determiningThis assay tests extracted and purified genomic DNA (gDNA).regions (CDR). The Hypermutation Mix 2 targets sequences between the framework 1 (FR1) and joining (J H ) regions. The resulting amplicons include a portion of the FR1 region to the downstream J Hregion. TheReferencesprimers that target the V H L and FR1 regions have been redesigned to1.P Ghia et al., Leukemia 21: 1-3 (2007).include a universal sequencing tag at the 5'end. This design allows2. P Ghia et al., Blood 105: 1678-1685 (2005).for bi-directional sequencing of clonal PCR products with just one3. F Davi et al., Leukemia 22: 212-214 (2008).sequencing-tag specific forward primer and one J Hreverse primer, thus ensuring a more reliable and complete coverage of clonal products. Positive and negative DNA, positive RNA, as well as an amplification control are included in the assay. Clonality is indicated if any one of the master mixes generates clonal products.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 Leader (L) and Framework 1 (FR1) regions, and the downstream consensus J Hgene segments.126"