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CAT #: 51010040

IGH Somatic Hypermutation Assay MegaKit v2.0 for Gel Detection

Assay Use

The Research Use Only IGH Somatic Hypermutation Assay v2.0 is used to identify clonal rearrangements of the immunoglobulin heavy (IGH) chain gene and determine the somatic mutation status of the variable (V) gene sequence and is useful for the study of:

  • Identifying clonal rearrangements of the IGH chain gene
  • Assessing the extent of somatic hypermutation in the variable region of the immunoglobulin heavy chain gene
  • Evaluating new research and methods in malignancy studies

Product Details

  • Summary and Explanation of the Test

    Rearrangements of the antigen receptor genes occur during ontogeny in B and T lymphocytes.  These gene rearrangements are unique in length and sequence for each cell.  Therefore, polymerase chain reaction (PCR) assays can be used to identify lymphocyte populations derived from a single cell by detecting the unique V-J gene rearrangements present within these antigen receptor loci.1  This PCR-based assay employs multiple consensus DNA primers that target conserved genetic regions within the immunoglobulin heavy chain (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 a variety of methods, including gel and capillary electrophoresis.

    The primers that target the leader (VHL) and framework 1 (FR1) regions have been designed to include a universal sequencing tag at the 5’end.  This design allows for bi-directional sequencing of clonal PCR products with just one sequencing-tag specific forward primer and one JH reverse primer.  The presence of IGH somatic hypermutation (SHM) is defined as greater or equal to 2% difference from the germline variable (V) gene sequence, whereas less than 2% difference is considered evidence of no somatic hypermutation.

    This test amplifies either gDNA or cDNA that lies between the upstream leader (VHL) or framework 1 (FR1) regions and the downstream joining (J) region of the IGH gene.  The test utilizes two (2) different master mixes: Hypermutation Mix 1 v2.0 and Hypermutation Mix 2 v2.0.  The Hypermutation Mix 1 v2.0 targets sequences between the leader and joining regions.  Therefore the amplicon product(s) span the entire variable (V) region, which contains the FR1, CDR1 (complementarity-determining region 1), FR2, CDR2, FR3 and CDR3 regions.  The Hypermutation Mix 2 v2.0 targets sequences between the framework 1 (FR1) and joining (J) regions.  The resulting amplicons include a portion of the FR1 region to the downstream J region.  Accordingly products do not include the complete FR1 sequence.

  • Specimen Requirements

    This assay tests extracted and purified gDNA or cDNA derived from RNA.  Common sources of gDNA and RNA include:

    • 5 cc of peripheral blood, bone marrow biopsy or bone marrow aspirate anti-coagulated with heparin or EDTA (stored at 2ºC to 8ºC and shipped at ambient temperature)
    • Formalin-fixed paraffin embedded tissue or slides (stored and shipped at ambient temperature)

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