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