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CAT #: 14120010

FLT3 Mutation Assay - Gel Detection

Product Use

FLT3 Mutation Assays are useful for the study of:

  • Identifying FLT3 mutations in specimen with AML.

Product Details

  • Summary of Explanation of the Test

    BACKGROUND:

    Acute myeloid leukemia (AML) in general has a poor prognosis. Recent studies have described mutation of the FLT3 (fms-like tyrosine kinase 3) receptor to be the most important prognostic factor in AML, with FLT3 mutants having a worse outcome and response to standard chemotherapeutic interventions. Accordingly, identification of an FLT3 mutation in AML may indicate a need to reassess and modify standard treatment options.

    The FLT3 gene (aliases: STK1; CD135; FLK-2) contains 24 exons and spans at least 96 kb. FLT3 is a receptor tyrosine kinase that is normally expressed on many cell types including hematologic stem cells.

    All types of AML can have activating mutations in the FLT3 gene. Mutation of the FLT3 receptor, either by internal tandem duplication (ITD) of the juxtamembrane domain or by point mutation of the aspartic acid residue D835 in the activation loop of the kinase domain, causes constitutive activation of the FLT3 receptor.

    Gel electrophoresis, such as agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresisis (PAGE), is commonly used to resolve the different amplicon products based on their size, charge and conformation. Since DNA is negatively charged, when an electrical potential (voltage) is applied across the gel containing PCR products, the electrical field causes the amplicons to migrate through the gel. Smaller DNA fragments are able to easily migrate through the gel matrix, whereas larger DNA fragments migrate more slowly. This causes a separation of the amplicon products based on size. Ethidium bromide or other DNA intercalating dyes can then be used to stain and detect these products in the gel.

    This test kit includes 3 master mixes. The ITD and D835 master mixes target the juxtamembrane and kinase domain regions (respectively). The third master mix, the Specimen Control Size Ladder, targets multiple genes and generates a series of amplicons of 100, 200, 300, 400, and 600 base pairs to ensure that the quality and quantity of input DNA is adequate to yield a valid result. A single thermocycler program and similar detection methodologies are used with all of our standard assays. Many of our customers have remarked that this improves consistency and facilitates cross training on a broad range of different assays. These robust Invivoscribe assays can be used to test DNA extracted from virtually any source.

  • Specimen Requirements

    This assay tests genomic DNA

    • 5 cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA. Ship at ambient temperature; OR
    • Minimum 5mm cube of tissue shipped frozen; or at room temperature or on ice in RPMI 1640; OR
    • 2 µg of genomic DNA; OR
    • Formalin-fixed paraffin embedded tissue or slides.

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