The 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 in patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).

Specifically, the IGH Somatic Hypermutation Assay v2.0 can be used to:

1. Identify clonal rearrangements of the IGH chain gene
2. Assess the extent of somatic hypermutation in the variable region of the immunoglobulin heavy chain gene
3. Evaluate new research and methods in malignancy studies

InVivoScribe Technologies' assay are standardized PCR-based tests. Each test comes with a Standard Operating Procedure (SOP), an interpretation guide, master mixes, and controls. Master mixes are composed of a buffered magnesium solution, deoxynucleotides, and multiple primers that target the gene segments of interest. Multiple primers are used to ensure a more comprehensive testing approach necessary to reliably identify clonal rearrangements. These tests are complete with the exception of Taq DNA Polymerase, which is not provided. A single thermocycler program and similar detection methods are used within each series of test to improve consistency, reduce human error, and facilitate cross training.

Standard Protocol.
50ul Total PCR Reaction Volume:

1. Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
2. In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube for each of the master mixes). Aliquot volumes should be 45ul for each sample + 135ul for the positive, negative and no template controls. We recommend adding an additional 20ul to correct for pipetting errors.
3. Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase (0.25ul of either AmpliTaq Gold or AmpliTaq @ 5U/ul per 50ul total PCR reaction volume) to each of the master mixes and gently mix by inverting several times or gently vortexing.
4. Aliquot 45ul of master mix to individual wells of a PCR plate.
5. Add 5ul of DNA from the unknown and control samples to individual tubes or wells containing the respective master mix reactions, and pipette up and down several times to mix. Amplify target DNA using the universal thermocycler program.

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This assay tests genomic DNA or complementary DNA (cDNA) from the following sources:

1. 5cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA (stored at 2C to 8C and shipped at ambient temperature)
2. Minimum 5mm cube of tissue (stored and shipped frozen)
3. 2ug of genomic DNA (stored at 2C to 8C or -65C to -85C and shipped at ambient temperature)
4. 5ug of total RNA or mRNA (stored at -65C to -85C and shipped on dry ice)
5. 1ug of cDNA (stored at 2C to 8C or -65C to -85C and shipped at ambient temperature)
6. Formalin-fixed paraffin embedded tissue or slides (stored and shipped at ambient temperature)

This product and the methods employed are covered by United States Letters Patent Numbered 5,296,351 and 5,418,134; Australian Patent Number 626,601 and Japanese Patent Number 2,781,438, all of which are licensed exclusively to InVivoScribe Technologies (“IVS”).

Purchase of this product includes a limited sublicense for non-commercial practice of this technology for use within (or with respect to data or product that are transmitted to) the United States, Japan or Australia only when the purchaser is registered with IVS as an exclusively non-commercial user of IVS products. No sublicense is granted simply by purchase of these products. Non-commercial practice of the technology means sample testing done for teaching and basic research. Non-Commercial practice excludes testing if any of the following apply: (a) test results, products or information derived from the tests are used for or in support of patient care, or are transferred to a healthcare professional involved in patient care; (b) test results are clinically utilized to determine cause of death; (c) compensation, in any form or manner, is received for performing the tests.

To request a form for registration as an exclusively non-commercial product user, to discuss terms for a potential sublicense for broader practice of these methods, or for any questions concerning the scope or content of the non-commercial sublicense please contact our legal department by email at legal@invivoscribe.com, or by telephone at (858) 623-8105.

These methods also require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR), which is covered by patents owned by Hoffmann-LaRoche, Inc. and F. Hoffmann-LaRoche Ltd. No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of these products. The assays described herein are not approved by any regulatory agency for clinical use. These assays are not for diagnostic or therapeutic use. This product is sold FOR RESEARCH USE ONLY; not for use in diagnostic procedures.