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#99
AUTOMATED DNA EXTRACTION FROM FROZEN BLOOD USING THE ABBOTT m1000™ FOR HLA-DRB SEQUENCING-BASED TYPING.
Jeff T. Silva , Christine Y. Wong , JoAnn L. Dileanis , Chad M. Dunn and Chaka C. Impraim . Alameda CA, Celera Diagnostics, 94502, DIS and Abbott Park IL, Abbott Laboratories, 60064, 084K .
The Abbott m1000™ is a fully automated sample preparation system based on magnetic microparticle processes. It is capable of running reagents and protocols to isolate DNA/RNA from a variety of sample types including but not limited to RNA from 1 mL plasma/serum samples, and DNA from 0.2 mL blood samples. The study was to evaluate the performance of the m1000™ for the extraction of genomic DNA from frozen blood samples followed by the HLA-DRB BigDye™ Terminator Sequencing-Based Typing (SBT) Kit. 40 frozen EDTA blood samples were extracted with 4 replicates per sample. DNA yield and purity were quantified by absorbance. The extracted DNA was also tested with the TaqMan® RNase P Detection Reagents on the ABI PRISM® 7000 Sequence Detection System to determine the degree of PCR inhibition from the extraction process. 20 of the extracted DNAs were tested with the HLA-DRB SBT Kit. The sequencing reactions were performed on the ABI PRISM® 3100 Genetic Analyzer and the sequence data were analyzed by MatchMaker™ Allele Identification Software for DRB1, DRB3, DRB4 or DRB5 allele determinations. The results showed that the DNA yield with 0.2 mL sample input was consistently high, with the average concentration above 20 ng/uL. The average A260/A280 ratio was 1.7 and no Ct delay was observed in the Taqman RNase P assay. All 20 DNAs were successfully amplified and sequenced. In summary, the Abbott m1000™ was shown to be an effective system for the automated purification of genomic DNA from frozen blood specimens. The purified DNA is free of enzyme inhibitors, and is ready for use in genomic assays. The m1000™ is suitable for use as part of a fully automated sequencing system.