Assessing Quality and Functionality of DNA from Fresh and Archival Dried Blood Spots and Recommendations for Quality Control Guidelines.
Författare
Summary, in English
Background: Dried blood spots (DBS) are a convenient and inexpensive method for biobanking. Although many countries have established population-based DBS biobanks from neonatal screening programs, the quality and usefulness of DNA from DBS have not been extensively assessed.
Methods: We compared 4 common DNA extraction methods (Qiagen, EZNA, Chelex 100, and alkaline lysis) in a pilot study using fresh DBS with known lymphocyte count. We assessed suitability for multiple displacement amplification (MDA) and subsequent single-nucleotide polymorphism (SNP) analyses. We selected the EZNA method for DNA extraction from archival samples up to 27 years old, stored at room temperature or –20 °C, and SNP analyses were performed after MDA.
Results: Extraction using alkaline lysis failed in most tests, and Chelex 100 was unsuccessful in real-time PCR, whereas the EZNA and Qiagen methods were successful by all evaluated quality indices. DNA extraction by EZNA, MDA, and SNP analyses were successful for the archival samples stored at –20 °C.
Conclusion: Routine protocols for evaluation of the quality and functional integrity of DNA based on DNA yield, DNA size, and quantification of amplifiable DNA allow use of sufficient template for MDA and successful SNP analyses from both primary DBS extract and MDA product. A single 3-mm disc can yield sufficient DNA for several thousand SNP analyses. DNA from DBS is thus suitable for genetic epidemiology studies.
Methods: We compared 4 common DNA extraction methods (Qiagen, EZNA, Chelex 100, and alkaline lysis) in a pilot study using fresh DBS with known lymphocyte count. We assessed suitability for multiple displacement amplification (MDA) and subsequent single-nucleotide polymorphism (SNP) analyses. We selected the EZNA method for DNA extraction from archival samples up to 27 years old, stored at room temperature or –20 °C, and SNP analyses were performed after MDA.
Results: Extraction using alkaline lysis failed in most tests, and Chelex 100 was unsuccessful in real-time PCR, whereas the EZNA and Qiagen methods were successful by all evaluated quality indices. DNA extraction by EZNA, MDA, and SNP analyses were successful for the archival samples stored at –20 °C.
Conclusion: Routine protocols for evaluation of the quality and functional integrity of DNA based on DNA yield, DNA size, and quantification of amplifiable DNA allow use of sufficient template for MDA and successful SNP analyses from both primary DBS extract and MDA product. A single 3-mm disc can yield sufficient DNA for several thousand SNP analyses. DNA from DBS is thus suitable for genetic epidemiology studies.
Avdelning/ar
Publiceringsår
2007
Språk
Engelska
Sidor
1401-1407
Publikation/Tidskrift/Serie
Clinical Chemistry
Volym
53
Dokumenttyp
Artikel i tidskrift
Förlag
American Association for Clinical Chemistry
Ämne
- Biochemistry and Molecular Biology
Status
Published
Forskningsgrupp
- Clinical Microbiology, Malmö
- Clinical Chemistry, Malmö
ISBN/ISSN/Övrigt
- ISSN: 0009-9147