Technical advance
Pitfalls in mutational testing and reporting of common KIT and PDGFRA mutations in gastrointestinal stromal tumors
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* Corresponding author: Sabine Merkelbach-Bruse sabine.merkelbach-bruse@ukb.uni-bonn.de
1 Department of Pathology, University of Bonn Medical School, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany
2 Department of Pathology, University of Regensburg, Franz-Josef-Strauß-Allee 11, D-93053 Regensburg, Germany
3 Department of Gastroenteropathology, Georg-August University Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany
4 Department of Pathology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany
5 Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220/221, D-69120 Heidelberg, Germany
6 Department of Pathology, Otto von Guericke University, Leipziger Str. 44, D-39120 Magdeburg, Germany
7 Department of Pathology, University of Erlangen, D-91054 Erlangen, Germany
8 Department of Pathology, Albert-Ludwig University, D-79098 Freiburg, Germany
BMC Medical Genetics 2010, 11:106 doi:10.1186/1471-2350-11-106
Published: 4 July 2010Additional files
Additional file 1:
Methods used for mutation analysis by the panel labs. This file describes how to extract DNA from formalin-fixed paraffin-embedded tissue, how PCR amplification and purification of PCR products prior to cycle sequencing can be performed and how cycle sequencing and precipitation of sequencing products should be done.
Format: RTF Size: 42KB Download file
Additional file 2:
Proposed standard operation procedure for testing and reporting of common KIT and PDGFRA mutations in GIST. The fundamental rules that should be the gold standard in every diagnostic molecular pathology laboratory are explained. Furthermore, advices are given for the evaluation of the tissue area to be analyzed and about the procedure of sectioning and microdissecting of formalin-fixed, paraffin-embedded tissue blocks. Protocols are given for DNA extraction and quantification, for primers for KIT exons 9 and 11 and for PDGFRA exon 18 and for PCR amplification. Another protocol explains how to purify the PCR fragments and how to perform cycle sequencing.
Format: DOC Size: 36KB Download file
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Additional file 3:
Description of sequence data. This file explains how to report the sequence data. Several examples are given.
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Additional file 4:
Tables AF1-4. The four tables contain technical details about DNA extraction, primer sets, conditions for PCR ampiflications, purification of PDR products and conditions for cycle sequencing. Table AF 1. Kits used for DNA extraction. Table AF 2. Primersets and conditions for PCR amplification of KIT exon 9 and 11 and PDGFRA exon 18. Table AF 3. Purification of PCR products prior to cycle sequencing. Table AF 4. Kits, devices and conditions used for cycle sequencing.
Format: DOC Size: 99KB Download file
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