DNA EXTRACTION

sources of DNA

forensic samples:

• blood, semen, hair etc

mass disaster contexts:

• decomposed tissue

• burnt/exposed remains

• extensive exposure

general principles of DNA extraction

1) cell distruption

• release of DNA from cell

2) deproteination

• separation of protein and DNA

3) purification

• remove protein and other substances

phenol chloroform

1) phenol pre-preaparation

2) proteinase k incubation

3) phenol extraction

4) chloroform extraction

5) incubation with ethanol and sodium acetate

6) purification with ethanol

CENTRIFUGATION

7) drying

8) resuspension in water

phenol chloroform extraction 1

preparation

• depending of sample material 

• liquid blood/saliva- no preprep 

• bone/teeth- decalcification (w/EDTA) 

• dehydrated sample- rehydration 

increasing surface area, removal of impurities 

phenol chloroform 2

protein k-incubation - digestion and lysis

• incubation  of cell suspension with proteinase k 

• optimal- 60-90 mins, 56oc

• proteinase k- destroys peptide bonds  

digestion of proteins, lysis of cell membrane 

phenol chloroform 3

phenol extraction- deproteination 

• incubation with phenol/chloroform 

• phenol:chloroform:water = 70:16:14

• buffered at ph 7.8

• phenol- organic solvent - hazordous

deproteination- dissociation of protein and dna

note: chloroform increases deproteination effectivity 

phenol chloroform 4

chloroform extraction - removal of proteins

• incubation with chloroform

• optimal- chloroform 100%

removal of protein removal of residual phenol 

phenol chloroform 5

precipitation of DNA

• incubation of alcohol and salt 

• optimal: ethanol/ isopropanol, sodium acetate 

alcohol dehydrates DNA

silica (optimal)- adsorptive binding of DNA, supports DNA precipitation

phenol chloroform 6

purification

• washing DNA-silica complex with alcohol

• optimal - 80%

removal of co-precipitated salts 

phenol chloroform 7

drying

• drying of DNA-silica complex

evaporation of residual ethanol

note: ethanol inhibits performance of DNA polymerase during PCR

phenol chloroform 8

elution of DNA

• separation of DNA from silica 

• optimal: sterile water, 50oc, 5 mins 

resuspension of DNA in solution 

storage of extracts 

short term- 4oc, sterile water 

long term- -20oc, presence of silica and sterile water 

low temperature 

• reduced speed of all chemical reactions 

• stabilisation of DNA

minimise freeze-thaw cycles- repeated leads to sheering of DNA

chelex 100

cation chelating resin, removes ionic substances

1) pre-prep

2) add sample materials

• sample material + 5% chelex solution in purified water 

3) incubate 56oc

4) incubate 100oc

5) centrifuge 

6) recover supernatant = DNA extraction

silica based DNA extraction

used in commercial DNA extraction kits

1) proteinase k digestion

2) binding to silica matrix

WASH

3) removal of impurities

4) release of DNA from silica matrix

silica based- 2 ways

1) silica column

• DNA immobilised

• adsorptive binding to stationary silica matrix

• impurities are removed by washing while DNA stays bound

• release DNA by changing salt conc. and ph buffer

2) silica in solution

• DNA immobilised

• adsorptive binding to silica particles in solution

• increases DNA yield precipitation especially when sample contains small amount of fragmented DNA

• release by thermal elution

optimisation of methodology

• SOPs adapt to different types of sample material 

• adaptation to different types of sample material- optimised protocol 

optimisation of methodology e.g.

whole blood stains

• contain PCR inhibitor heme

• wash step prior to chelex intubation  

hair

• keratinised tissue 

• keratin- very stable fibrous protein 

semen

• comparatively strong cell components

• pre-digestion needed

optimisation of methodology e.g.

differential extraction

mixture of samples

• female epithelial cells = male sperm

DNA fraction

• preferential lysis of epithelial cells

  • SDS + proteinase k 

  • nuclei of sperm remains undigested 

• centrifugation to separate fractions

  • supernatant - female epithelial cell debris recovered and extracted 

• pellet- undigested sperm

  • redigest in SDS + proteinase k + DTT (breaks s-s bonds)

• extract DNA- male DNA fraction 

additional pre-prep steps

• e.g. hard tissue 

decalcification 

• releases cells from mineral matrix

• increases amount of DNA accessible for extraction 

pulverisation/milling

• increases surface area

additional pre-prep steps

e.g. low DNA quantity material

proteinase k digestion

• digestion of cell membranes/ proteins

• increases accessible DNA

silica added in precipitation step 

• absorptive binding of DNA 

• increase amount of DNA recovered during precipitation  

additional pre-prep steps

e.g. impurities in sample material

additional purification steps

• repetition of phenol/chloroform steps

• addition of precipitation steps

• silica column purification of DNA extracts

• re-extraction of DNA using kits

decrease amount of DNA extraction of co-extracted impurities

additional pre-prep steps

in general…

• increase in pre-prep steps = increased risks of contamination

  • when opening tubes or addition of reagents 

• increase purification steps = increased loss of DNA