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Clin Chem Lab Med. 2011 Nov 4;50(2):301-10. doi: 10.1515/CCLM.2011.762.

Detection of internal tandem duplications in the FLT3 gene by different electrophoretic methods.

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  • 1Institute of Internal Medicine, 1st Department of Internal Medicine, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary.

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.

PMID:
22053959
[PubMed - indexed for MEDLINE]
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