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CAT #: 94120020

LeukoStrat® FLT3 Mutation Assay MegaKit - Gel Detection

Intended Use

LeukoStrat® FLT3 Mutation Assay is an in vitro diagnostic product intended for PCR-based detection of FLT3 activating mutations in patients with acute myelogenous leukemia (AML).

Specifically, the FLT3 Mutation Assay can be used to:

  • Identify internal tandem duplications (ITD) in the FLT3 gene.
  • Identify tyrosine kinase domain (TKD) mutations in the FLT3 gene.

Product Details

  • Summary and Explanation of the Test

    Acute myeloid leukemia (AML) in general has a poor prognosis. Assessment of the mutation status of the FLT3 (fms-like tyrosine kinase 3) receptor gene in karyotype normal AML is the most important prognostic indicator of disease outcome, which is often substantial, as many studies in AML have shown that the presence of FLT3 activating mutations portends a poor prognosis. For this reason FLT3 mutation testing is required to stratify disease and determine appropriate treatment options. This LeukoStrat® assay targets regions of the FLT3 gene to identify internal tandem duplication (ITD) mutations and tyrosine kinase domain (TKD) mutations, such as the D835 and I836 mutations.  With this assay, DNA is amplified via PCR, TKD amplicon is enzymatically digested, and the amplicons are detected via gel electrophoresis.

    This product includes everything needed for controlled PCR amplification. Included are PCR master mixes and controls for ITD and TKD targets. Also included, the Specimen Control Size Ladder master mix targets multiple genes and generates a series of amplicons of approximately 100, 200, 300, 400, and 600 base pairs to ensure input DNA quality and quantity is adequate to yield a valid result. This assay reliably detects FLT3 mutations comprising greater than or equal to 5% of the total cell population.

  • Principles of the Procedure

    Internal Tandem Duplication (ITD) Mutations of FLT3

    FLT3 ITD or length mutations are caused by duplication and insertion of a portion of the FLT3 gene that includes the region in and around the juxtamembrane (JM) region of the FLT3 gene. These mutations vary in both the location and the length of the inserted duplicated DNA sequence. ITD mutations result in constitutive autophosphorylation and activation of FLT3. Using this assay, FLT3 mutant alleles are differentiated from wild type based on amplicon size as detected via gel electrophoresis.

    Tyrosine Kinase Domain (TKD) Mutatins of FLT3

    FLT3 TKD mutations are caused by nucleic acid substitutions that result in a change in the amino acid sequence in this highly conserved catalytic center. TKD mutations, such as D835 and I836, result in constitutive autophosphorylation and activation of FLT3.  Wild-type alleles of the FLT3 gene include an EcoRV restriction digest site.  When a nucleic acid substitution occurs, the restriction digest recognition site disappears, and the EcoRV endonuclease is unable to identify and digest the DNA at this site. Our tests use primers that lie on either side of the TKD region. The FLT3 target region is amplified using PCR and then an EcoRV restriction digest is performed. One of the PCR primers contains an EcoRV restriction site, so both wild type and mutant alleles are digested. Using this assay, FLT3 mutant alleles are differentiated from wild type based on amplicon size as detected via gel electrophoresis. Amplicon size further controls for EcoRV digestion.

    Gel Detection

    Gel electrophoresis, such as agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresis (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.

  • Specimen Requirements

    This assay tests genomic DNA from the following sources:

    • 5 cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA (stored at 2°C to 8°C and shipped at ambient temperature)
    • Minimum 5 mm cube of tissue (stored and shipped frozen; or stored and shipped in RPMI 1640 at ambient temperature or on ice)
    • 2 µg of genomic DNA (stored at 2°C to 8°C and shipped at ambient temperature)
    • Formalin-fixed paraffin embedded tissue or slides (stored and shipped at ambient temperature)

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