186-P: COMPARISON OF DIFFERENT EXTRACTION PROTOCOLS ON DNA YEILD AND PURITY

Abstract

DNA extraction is the first step of sample transformation before HLA typing analysis. This manipulation is thought very important and is crucial in terms of yield and purity of the DNA, especially when working with mouth swabs, dry blood from FTA paper, or old blood sample as starting material. An urban legend in our laboratory was that one mouth swab could not give sufficient amount of DNA to allow a low resolution HLA-A, B, C and high resolution DRB1 typing. Another urban legend was suggesting that blood older than 7days must inevitably be extracted manually on a column. We wanted to challenge these popular beliefs. We report here the comparative study we made in order to reorganize our DNA collection strategy. Three different protocols were used to extract and purify DNA from samples of different origins. The Maxwell 16 system (Promega), the GenoM-6 system (Qiagen) and the manual QiaAmp Column kit (Qiagen) were compared for DNA extraction from mouth swabs, whole blood (either fresh and up to 21 day old), cord blood, buffy coat and dry blood on FTA paper. The DNA collected was first evaluated for quantity and quality and should be sufficient to allow the HLA-A, B, C, DRB1 and DQB1 typing by SSO, SSP and/or SBT methods. The second step was to verify that these analyses could be performed without any problem. The first step of the study revealed that the concentration of DNA extracted from mouth swabs using the Maxwell 16 system was the highest among the different extraction systems and that only one mouth swab was sufficient to generate a complete HLA typing. Also, old blood (as old as 21 days) can be extracted without any problem by any of the 3 methods. The second step of the study demonstrated that the quality and quantity of the DNA collected was suitable for HLA-A, B, C, DRB1 and DQB1 typing using SSO, SSP and SBT techniques. Urban legends were proven wrong and the DNA collection strategy was able to be revised with the collaboration of all the techs.