FLT3 Mutation Assay - Gel 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, TKD amplicon is enzymatically digested, and FLT3 mutations are detected via gel 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 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 (gDNA).