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Detection of internal tandem duplications in the FLT3 gene by different electrophoretic methods
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Tamás Bubán
Published/Copyright:
November 4, 2011
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.
Keywords: acute myeloid leukemia; agarose gel electrophoresis; capillary electrophoresis; fms-like tyrosine kinase 3 (FLT3); internal tandem duplication (ITD); polyacrylamide gel electrophoresis
Received: 2011-5-15
Accepted: 2011-10-5
Published Online: 2011-11-04
Published in Print: 2012-02-01
©2012 by Walter de Gruyter Berlin Boston
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