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Evaluation of the methods for DNA extraction from archival blood samples

Central Forensic Laboratory of the Police Headquarters, Warsaw, Poland. Evaluation of the methods for DNA extraction from archival blood samples. Igor Olewiecki M.Sc. Joanna Karolina Purzycka M.Sc. Ireneusz Soltyszewski Ph.D. DNA forensic analysis.

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Evaluation of the methods for DNA extraction from archival blood samples

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  1. Central Forensic Laboratory of the Police Headquarters, Warsaw, Poland Evaluation of the methodsfor DNA extractionfrom archival blood samples Igor Olewiecki M.Sc. Joanna Karolina Purzycka M.Sc. Ireneusz Soltyszewski Ph.D.

  2. DNA forensic analysis • Collection and examination of biological samples • DNA extraction • Quantification of isolated DNA • DNA amplification • Capillary electrophoresis • Genotyping

  3. DNA source • Saliva • Blood • Tissue (i.e. muscle, liver, brain) • Bone marrow • Hair • Sperm

  4. Goals: isolation of nucleic acid from the cell purification of isolated DNA Yield/quality ratio should be as high as possible DNA extraction

  5. DNA extraction methods: • Organic • Ion-exchange Resin • Silica Matrix • High Salt • Sodium Hydroxide • Magnetic Affinity Resin

  6. Organic • Lysis of the cells: alkaline buffer or chaotropic agents, proteinase K, incubation in 56ºC • Purification: phenol:chloroform • Extraction: ethanol or isopropanol

  7. Ion-exchange Resin • Lysis of the cells: sample is heated in the presence of resin to 100ºC • Purification: resin binds cellular components other than DNA • Extraction: centrifugation removes resin leaving DNA in the supernatant

  8. Silica matrix • Lysis of the cells: chaotropic agents, proteinase K, incubation in 56ºC • Purification: DNA binds to the silica matrix contaminantsare eliminated by washing steps • Extraction: DNA is rinsed with water from the matrix

  9. Why to use a commercial kits? • Non-toxic methods • Short incubation and centrifugation times • Easy procedure steps • Able to get rid of inhibitors that are solvable in the aqueous phase • Ready for automatisation

  10. Methods

  11. DNA measurement • DNA concentration was measured fluorometrically using PicoGreen dsDNA Quantitation Reagent (Invitrogen - Molecular Probes) and Fluoroscan Ascent FL (Labsystems) • Slot-blot technique specific to measure only the human DNA with Quantiblot kit (Applied Biosystems).

  12. SGM plus analysis • 1,25 µg of DNA in volume of 25 µl was taken for PCR reaction • PCR reaction was performed in a standard conditions • STR analysis was done with GenScan 3.7 and Genotyper 3.7 software (default settings)

  13. Material • Blood samples were collected in 1955 from 30 non-related individuals • Dried samples were packed in a paper envelopes, and stored at the room temperature and constant humidity • For DNA extraction 0,02 g of dried blood was used from each sample

  14. Results

  15. Conclusion • It is possible to isolate DNA from archival blood samples that are almost 50 years old using different methods • It is possible to obtain a full genetic profile form such a DNA • QIAamp was the best commercial kit for DNA extraction from archival blood samples

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