Internal quality control of PCR-based genotyping methods: Practical experiences
article
Internal quality control programmes for genetic analyses are needed. We have focused on quality control aspects of selected polymorphism analyses used in thrombosis research. DNA was isolated from EDTA-blood (n=500) and analysed for 18 polymorphisms by polymerase chain reaction (PCR), i.e. restriction fragment length polymorphisms, allele specific amplification, or amplification of insertion/deletion fragments. We evaluated the following aspects in the analytical procedures: sample handling and DNA-isolation (pre-analytical factors), DNA-amplification, digestion with restriction enzymes, electrophoresis (analytical factors), result reading and entry into a database (post-analytical factors). Furthermore, we evaluated a procedure for result confirmation. Isolated DNA was of good quality (42 μg/ml blood, A260/A280 ratio>1.75, negative DNAsis tests). Occasionally, results were reanalysed because of positive reagent blanks (<1%) or because of problems with the controls (<5%). On confirmation, we observed four genotyping discrepancies. Control of data handling revealed 0.1% reading mistakes and 0.5% entry mistakes. Based on our experiences, we propose an internal quality control programme for widely used PCR-based haemostasis polymorphism analyses. © 2002 Elsevier Science Inc. All rights reserved.
Chemicals/CAS: edetic acid, 150-43-6, 60-00-4
Chemicals/CAS: edetic acid, 150-43-6, 60-00-4
Topics
TNO Identifier
236619
ISSN
15371891
Source
Vascular Pharmacology, 39(3), pp. 127-129.
Pages
127-129
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