title: Internal quality control of PCR-based genotyping methods: practical experiences.
abstract: 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 micrograms/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.
authors.affiliation: Department for Thrombosis Research, University of Southern Denmark, Department of Clinical Biochemistry, Ribe County Hospital, Esbjerg, Denmark. emb@ribeamt.dk
authors.forename: Else-Marie
authors.initials: EM
authors.lastname: Bladbjerg
authors.forename: Jørgen
authors.initials: J
authors.lastname: Gram
authors.forename: Jørgen
authors.initials: J
authors.lastname: Jespersen
authors.forename: Moniek P M
authors.initials: MP
authors.lastname: de Maat
mesh_terms: D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012150:Polymorphism, Restriction Fragment Length; D011786:Quality Control