A rapid semi automated method for DNA extraction from dried-blood spots: application to the HLA-DR shared epitope analysis in rheumatoid arthritis.
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.
Pachot
Alexandre
A
Hospices Civils de Lyon, BioMérieux Joint Unit, Hôpital Edouard Herriot, 5, Place d'Arsonval, 69003, Lyon, France.
Barbalat
Véronique
V
Marotte
Hubert
H
Diasparra
Jennifer
J
Gouraud
Aurore
A
Mougin
Bruno
B
Miossec
Pierre
P
D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000235:genetics; D001800:Blood Specimen Collection / Q000379:methods; D004247:DNA / Q000097:blood / Q000302:isolation & purification*; D000939:Epitopes / Q000097:blood* / Q000235:genetics; D005260:Female; D006684:HLA-DR Antigens / Q000235:genetics*; D006403:Hematologic Tests / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D013995:Time
D000939:Epitopes; D006684:HLA-DR Antigens; D004247:DNA