Internal quality control of PCR-based genotyping methods: practical experiences

Vascul Pharmacol. 2002 Aug;39(3):127-9. doi: 10.1016/s1537-1891(02)00299-9.

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.

MeSH terms

  • Genotype
  • Humans
  • Polymerase Chain Reaction / methods
  • Polymerase Chain Reaction / standards*
  • Polymorphism, Restriction Fragment Length
  • Quality Control