title: Detection of internal tandem duplications in the FLT3 gene by different electrophoretic methods.
abstract: BACKGROUND
  In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis.
  
  
  METHODS
  A complex evaluation of the analytical properties of the three most frequently used detection methods--PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE)--was performed on 95 DNA samples obtained from 73 AML patients.
  
  
  RESULTS
  All the three methods verified the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1%-2% mutant allele, while the detection limit of CE was 0.28%. However, acceptable reproducibility (inter-assay CV <25%) of the mutant allele rate determination was only achievable above 1.5% mutant/total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7% lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes.
  
  
  CONCLUSIONS
  This study suggests the use of PCR+CE in the diagnostics and the follow-up of AML patients. The data further supports the importance of proper analytical evaluation of home-made molecular biological diagnostic tests.
authors: Institute of Internal Medicine, 1st Department of Internal Medicine, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.
authors: Tamás
authors: T
authors: Bubán
authors: Katalin
authors: K
authors: Koczok
authors: Róza
authors: R
authors: Földesi
authors: Gabriella
authors: G
authors: Szabó
authors: Andrea
authors: A
authors: Sümegi
authors: Miklós
authors: M
authors: Tanyi
authors: László
authors: L
authors: Szerafin
authors: Miklós
authors: M
authors: Udvardy
authors: János
authors: J
authors: Kappelmayer
authors: Péter
authors: P
authors: Antal-Szalmás
mesh_terms: D004586:Electrophoresis; D019075:Electrophoresis, Capillary; D015180:Electrophoresis, Gel, Two-Dimensional; D020440:Gene Duplication / Q000235:genetics*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
chemical_list: C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3