Evaluation of the methodsEvaluation of the methodsfor DNA extractionfor DNA extraction

from archival blood samplesfrom archival blood samples

Igor Olewiecki M.Sc.Igor Olewiecki M.Sc.

Joanna Karolina Purzycka M.Sc.Joanna Karolina Purzycka M.Sc.

Ireneusz Soltyszewski Ph.D.Ireneusz Soltyszewski Ph.D.

Central Forensic LaboratoryCentral Forensic Laboratory

of the Police Headquarters,of the Police Headquarters,

Warsaw, PolandWarsaw, Poland

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

DNA source

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

DNA extraction

Goals:

1. isolation of nucleic acid from the cell

2. purification of isolated DNA

Yield/quality ratio

should be

as high as possible

Organic Ion-exchange Resin Silica Matrix High Salt Sodium Hydroxide Magnetic Affinity Resin

DNA extraction methods:

Organic

Lysis of the cells:alkaline buffer or chaotropic agents, proteinase K, incubation in 56ºC

Purification:phenol:chloroform

Extraction:ethanol or isopropanol

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

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

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

Methods

Method of Method of extractionextraction

Incubation Incubation TimeTime

Centrifugation Centrifugation TimeTime

Required Required TemperaturesTemperatures

Number of Number of StepsSteps

Biotools

Bloodclean DNA

Purification Kit

Ion-exchange Ion-exchange resinsresins 772 min2 min 6 min6 min

5656ºCºC

100100ºCºC1010

Promega

Amp GenomicIon-exchange Ion-exchange

resinsresins 40 min40 min 4 min4 min5656ºCºC

100100ºCºC1313

Machery-Nagel

NucleoSpin Tissuesilica matrixsilica matrix 770 min0 min 5 min5 min

5656ºCºC

7070ºCºC88

QIAGEN

QIAamp DNA

Mini Kitsilica matrixsilica matrix 21 min21 min 6 min6 min

5656ºCºC

7070ºCºC

8585ºCºC99

Organic Extraction pphenol:chloroformhenol:chloroform O/NO/N 30 min30 min 5656ºCºC 77

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 Slot-blot technique specific to measure only the human DNA with Quantiblot kit only the human DNA with Quantiblot kit (Appl(Applied Biosystemsied Biosystems). ).

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)

  

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

Results

Measurement

Method

Yield of DNA (µg)

for archival samples (from 0,02 g of dried blood)

Mean peak height ratio (RFU)

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 659659

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