FLT3 Mutation Assay - ABI Fluorescence Detection

Product Details

• Summary and Explanation of the Test

  BACKGROUND

  FLT3 is a receptor tyrosine kinase that is normally expressed on many cell types including hematologic stem cells.  Mutation of the FLT3 receptor, by either internal tandem duplication (ITD) of the juxtamembrane domain or point mutation in the activation loop of the tyrosine kinase domain (TKD), causes constitutive activation of the FLT3 receptor.  Such gain-of-function mutations in the FMS related tyrosine kinase 3 (FLT3) gene are the subject of research studies and multiple clinical trials targeting Acute Myeloid Leukemia (AML) subjects. The most prevalent type of FLT3 mutation is an internal tandem duplication in and around the juxtamembrane domain.  The second most common mutation type in the FLT3 gene is a TKD point mutation in aspartate (D835) or isoleucine (I836).

  PRODUCT SUMMARY

  FLT3 Mutation Assays target 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.  DNA is amplified by PCR with fluorophore-labeled primers, TKD amplicon is enzymatically digested, and FLT3 mutations are detected via capillary electrophoresis.

  Test kits include three PCR master mixes, along with positive and negative controls.  FLT3 ITD master mix tests for internal tandem duplication mutations.  FLT3 D835 master mix tests for TKD region mutations.  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 that the quality and quantity of input DNA is adequate to yield a valid result.

  PRINCIPLE OF DIFFERENTIAL FLUORESCENCE DETECTION

  Differential fluorescence detection is commonly used to resolve the different-sized amplicon products using a capillary electrophoresis instrument.  Primers can be conjugated with several different fluorescent dyes (fluorophores) so that they can produce different emission spectra upon excitation by a laser in the capillary electrophoresis instrument.  In this manner, different fluorescent dyes can correspond to different targeted regions.  Therefore, reaction products from several different master mixes can be pooled, fractionated using capillary electrophoresis and detected simultaneously. This detection system results in unsurpassed sensitivity, single nucleotide resolution, differential product detection and relative quantification.  In addition, the use of agarose and polyacrylamide gels, as well as the use of carcinogens such as ethidium bromide, can virtually be eliminated.  Further, differential detection allows accurate, reproducible and objective interpretation of primer-specific products and automatic archiving of data.  Inter-assay and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 nucleotides.

• Specimen Requirements

  This assay tests genomic DNA (gDNA).