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Evaluation of the methods for DNA extraction from archival blood samples

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Central Forensic Laboratory of the Police Headquarters, Warsaw, Poland Evaluation of the methods for DNA extraction from archival blood samples Igor Olewiecki M.Sc. – PowerPoint PPT presentation

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Title: Evaluation of the methods for DNA extraction from archival blood samples


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

2
DNA forensic analysis
  1. Collection and examination of biological samples
  2. DNA extraction
  3. Quantification of isolated DNA
  4. DNA amplification
  5. Capillary electrophoresis
  6. Genotyping

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

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

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
  • phenolchloroform
  • 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
  • contaminants are 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
Method of extraction Incubation Time Centrifugation Time Required Temperatures Number of Steps
Biotools Bloodclean DNA Purification Kit Ion-exchange resins 72 min 6 min 56ºC 100ºC 10
Promega Amp Genomic Ion-exchange resins 40 min 4 min 56ºC 100ºC 13
Machery-Nagel NucleoSpin Tissue silica matrix 70 min 5 min 56ºC 70ºC 8
QIAGEN QIAamp DNA Mini Kit silica matrix 21 min 6 min 56ºC 70ºC 85ºC 9
Organic Extraction phenolchloroform O/N 30 min 56ºC 7
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
Measurement Method Yield of DNA (µg) for archival samples (from 0,02 g of dried blood) Mean peak height ratio (RFU) Mean peak height ratio (RFU)
Measurement Method Yield of DNA (µg) for archival samples (from 0,02 g of dried blood) for fresh blood samples for archival blood samples
Bloodclean DNA Purification Kit (Biotools) 0,15 - 524
Amp Genomic (Promega) 0,05 - 286
NucleoSpin Tissue (Machery-Nagel) 0,07 - 418
QIAamp DNA Mini Kit (QIAGEN) 0,10 2268 612
Organic Extraction 0,18 2704 659
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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