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CAT #: 24120011

FLT3 ITD Master Mix - 6FAM & HEX

Introduction

Master mixes are components of complete assays. 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:
50µl 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 45µl for each sample + 135µl for the positive, negative and no template controls. We recommend adding an additional 20µl to correct for pipetting errors.
  3. Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase (0.25µl of either AmpliTaq Gold or AmpliTaq @ 5U/µl per 50µl total PCR reaction volume) to each of the master mixes and gently mix by inverting several times or gently vortexing.
  4. Aliquot 45µl of master mix to individual wells of a PCR plate.
  5. Add 5µl 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.

Product Use

Historically, FLT3 Mutation Assays have been used to:

  1. Identify FLT3 mutations in patients with AML. Since FLT3 mutations portend a worse prognosis for AML patients testing positive for FLT3 mutation may be candidates for more aggressive treatment.

Product Details

  • Specimen Requirements

    This product tests genomic DNA. DNA can be extracted from the following specimens:

    1. 5cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; OR,
    2. Minimum 5mm cube of tissue shipped frozen, at room temperature, or on ice in RPMI 1640; OR,
    3. Formalin-fixed paraffin embedded tissue or slides.
  • Description

    Acute myeloid leukemia (AML) in general has a poor prognosis. Recent studies have described mutations of the fms-like tyrosine kinase 3 (FLT3) receptor to be the single most important prognostic factor in AML, with FLT3 mutants having a worse outcome and response to standard chemotherapeutic regimens. All types of AML can have activating mutations in the FLT3 gene and approximately 25-35% of AML patients harbor a FLT3 mutation. Accordingly, identification of a FLT3 mutation in AML may indicate a need to reassess and modify standard treatment options. Mutation, either by internal tandem duplications (ITD) of the juxtamembrane domain or by point mutation of the aspartic acid residue 835 (D835) in the activation loop of the kinase domain, causes constitutive activation of the FLT3 receptor, which is normally expressed on early hematopoietic progenitor cells and plays a key role in stem cell survival and differentiation. The FLT3 gene (also known as STK1, CD135, and Flk2) contains 24 exons and spans at least 96 kb. This assay targets genomic DNA of both the immunoglobulin-like juxtamembrane (JM) domain, known to harbor the ITD mutation, and the second half of the interrupted kinase (TK2) domain, known to harbor the D835 mutation. Detection of the ITD mutation can be determined based on the increased size of the PCR products compared to the wild-type product. However, detection of the D835 mutation requires digest with EcoRV enzyme (not included). After digestion, the wild-type FLT3 produces 3 bands of 21, 49, and 80 basepairs. The D835 mutant loses one restriction site and produces a 129 basepair band and a 21 basepair band.

    Three master mixes are included in this assay. The ITD and D835 master mixes target the juxtamembrane and kinase domain regions, respectively. After amplification of samples with the FLT3 D835 Master Mix, an EcoRV digestion is required (enzyme not included). The Specimen Control Size Ladder master mix targets multiple genes and generates a series of amplicon products of 100, 200, 300, 400, and 600 basepairs to ensure that the quantity and quality of input DNA was adequate to yield a valid result. The presence of a FLT3 ITD mutation is indicated if the FLT3 ITD Master Mix produces an amplicon that is larger than 330 basepairs. The presence of a FLT3 D835 mutation is indicated if, after EcoRV digestion, the FLT3 D835 Master Mix produces a 129 basepair band. This assay is designed to work with all standard DNA extraction methods. PCR products of the FLT3 IDT and D835 master mixes are differentially labeled with 6FAM&HEX and NED, respectively, and can be analyzed using any platform that has the ability to detect these fluorochromes such as the Applied Biosystems ABI 310 and ABI 3100.

Disclaimer

©2019 Invivoscribe, Inc. All rights reserved. The trademarks mentioned herein are the property of Invivoscribe, Inc and/or its affiliates, or (as to the trademarks of others used herein) their respective owners.

For Research Use Only. Not for use in diagnostic procedures.

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