A rapid semi automated method for DNA extraction from dried-blood spots: application to the HLA-DR shared epitope analysis in rheumatoid arthritis

J Immunol Methods. 2007 Dec 1;328(1-2):220-5. doi: 10.1016/j.jim.2007.08.001. Epub 2007 Sep 4.

Abstract

Genomic DNA extraction for genotyping analysis is performed from blood samples and is time consuming. We describe a more rapid DNA extraction method, "DBS-miniMAG", that combines filter paper dried blood spots (DBS) with the NucliSens miniMAG semi-automated instrument (bioMérieux). To assess the performance of this method, a post-PCR HLA-DR shared epitope (SE) oligotyping assay was used as a read-out in a cohort of 72 arthritis patients. This new method was compared to the standard manual DBS extraction protocol using FTA reagents (Whatmann Bio-Science), and to a reference phenol-chloroform-based method using EDTA whole blood samples. Higher yield of PCR amplicons was observed with DNA extracts obtained using "DBS-miniMAG" method. The intra- and inter-assay variability of the "DBS-miniMAG" method was similar to that obtained with "DBS-FTA" washing process. Concerning the HLA-DR SE genotyping, "DBS-miniMAG" and "DBS-FTA" methods gave 100% concordance compared to the reference phenol-chloroform method. More importantly, the hands-on time and the turnaround time for "DBS-miniMAG" were both two-times shorter than for "DBS-FTA" protocol. Therefore, the "DBS-miniMAG" combination could facilitate polymorphism analysis in routine clinical practice and the creation of large DNA banks using very small amounts of blood.

MeSH terms

  • Arthritis, Rheumatoid / blood*
  • Arthritis, Rheumatoid / genetics
  • Blood Specimen Collection* / methods
  • DNA / blood
  • DNA / isolation & purification*
  • Epitopes / blood*
  • Epitopes / genetics
  • Female
  • HLA-DR Antigens / genetics*
  • Hematologic Tests / methods*
  • Humans
  • Male
  • Middle Aged
  • Polymerase Chain Reaction
  • Polymorphism, Genetic
  • Reproducibility of Results
  • Time

Substances

  • Epitopes
  • HLA-DR Antigens
  • DNA