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Organic Extraction
Presented by:
Robert O'BrienTraining Specialist – Forensic Biology
Overview DNA Facts
Has the ability to enter/remain in liquid phase
Has 1 negative charge per nucleotide
Has a molecular Weight of = 1.98 x 10^12 g/mol
There are ~3 pg of genomic DNA in a haploid cell There are ~ 6.6 pg genomic DNA in a diploid cell
Overview The normal number of leukocytes in
human peripheral blood is 5-10 million cells per ml of blood (30-60 g DNA/ml)
The normal number of sperm per ml of semen is 1,500,000 (450g DNA/ml). Additionally each ml of semen can contain approximately 5,000,000 leukocytes which contribute another 30 g DNA/ml, for a total of approximately 480 g DNA/ml
Types of Organic Extraction
In general, there are two types of organic extractions; Stains containing spermatozoa and those which do not contain spermatozoa
The first example is of an organic extraction of a non-sperm stain
Solubilization of Stain
Step 1: Place stain in a microcentrifuge tube with stain extraction buffer. Vortex, centrifuge, incubate.
Solubilization of Stain Water must be replaced and stains must
be resolubilized
It is important to protect the DNA from unnecessary degradation during this process
The process generally involves soaking the stain in buffer, usually at 56ºC for 2 hours
Solubilization of Stain, cont.
The buffer contains EDTA, a chelator of magnesium to prevent the action of the nucleases that could degrade the DNA
Denaturation/Hydrolysis of Proteins The detergent in the stain extraction
buffer causes the lysis of cellular membranes and the disassociation and denaturation of the histone proteins that are tightly attached to the DNA strands
Detergents destroy the secondary and tertiary structures of the proteins which leads to their decreased solubility in the aqueous solution and increased susceptibility to the hydrolytic activity of proteolytic enzymes
Denaturation/Hydrolysis of Proteins, cont. The detergent most commonly used at
this step is sodium dodecylsulfate (SDS).
Proteinase K is added to aid in the hydrolysis of histone proteins This enzyme is active across a wide range of
pH, is active in the presence of SDS, and is unaffected by metal chelators such as EDTA
Denaturation/Hydrolysis of Proteins, cont.
Step 2: Transfer any solid material (substrate) into a spin basket. Centrifuge. Discard basket and substrate.
Denaturation/Hydrolysis of Proteins, cont. The substrate can be placed into a spin
basket to recover the liquid remaining on the substrate
Some laboratories retain the substrate, which can be re-extracted ; while others discard the substrate at this step
The liquid is then carried to the next step, Removal of Denaturation Products
Removal of Denaturation Products
Step 3: Combine the extract with equal volumes of Phenol/Chloroform/Isoamyl Alcohol. Vortex, centrifuge. Remove the aqueous phase (upper level) for purification.
Organic Extraction Phases
The aqueous layer is removed and placed into a new microcentrifuge tube.
DNA(aqueous)
Protein (organic)
Removal of Aqueous Layer It is import to ONLY remove the aqueous
layer and avoid the organic phase and the interface between the two phases.
Phenol can lead to PCR inhibition
If you disturb the interface or pipette some of the organic phase, you can recombine the phases, centrifuge, and start again.
Safety Note Phenol is highly toxic and should be
handled in a fume hood while wearing personal protective equipment. Skin contact and inhalation should be avoided
Chloroform depresses the central nervous system. It is also a suspected teratogen and known carcinogen and should never be handled outside of a fume hood.
Removal of Denaturation Products, cont.
Denatured proteins are removed with phenol and chloroform Phenol is an effective protein
denaturant Chloroform, to a lesser extent also aids in
this process
The products of the denaturation and proteolysis are soluble in phenol
Removal of Denaturation Products, cont. Generally, most procedures use a
phenol/chloroform mixture which includes isoamyl alcohol Isoamyl alcohol reduces the tendency of
proteins to foam when they are denatured during the shaking process with organic solvents
Some procedures use phenol first, followed by one or more treatments with chloroform to ensure complete removal of phenol
Purification Step 4: Place the aqueous phase into a centrifugal filter
unit
Insert a filter unit into a filtrate tube Add sample and buffer, centrifuge Remove the filter unit from the filtrate tube and
discard the filtrate. Insert the filter unit into a filtrate tube and add buffer
to the sample reservoir and cap, centrifuge Add a small volume of buffer to the sample reservoir
to cover the filter with liquid. Invert and transfer the sample reservoir from the
filtrate tube to a retentate tube, centrifuge DNA is in the retentate tube.
Microcon® Components
Centricon® Components
Purification of DNA DNA can be recovered from the
aqueous phase with an ethanol precipitation or using a centrifugal filter unit (Centricon®or Microcon®).
Most protocols use centrifugal filter units since they purify and concentrate DNA
DNA Quantitation
After purification, the DNA is ready for quantitation
Differential Extraction Stains containing spermatozoa undergo a
differential extraction Differential extraction methods are used to
separate spermatozoa from other cell types. Spermatozoa are more difficult to lyse than other cells and conditions can be set so that all cells except spermatozoa are lysed.
The supernatant containing the DNA from these cells is removed from the sperm cells, which can then be lysed separately.
Differential Organic Extraction The steps are similar to those for a non-sperm stain,
except there are two cell lysis steps instead of one
The first step is the non-sperm cell lysis
Extraction buffer, detergent, and Proteinase K are added to the sample and incubated.
The supernatant containing the DNA from the lysed cells (fraction 1) is removed after pelleting the spermatozoa.
The sperm pellet is often washed numerous times with a buffer to remove excess DNA from this lysis step.
If any of the sperm cells are weak or otherwise compromised, these may lyse in the first step, leaving a low level of fraction 2 DNA in fraction 1.
Differential Organic ExtractionThe second step is the sperm cell lysis
The pelleted sperm cells are lysed under more stringent conditions, using a buffer, detergent, DTT, and a higher concentration of Proteinase K (fraction 2), and are subsequently incubated.
Both fractions are extracted separately with the phenol/chloroform/isoamyl alcohol combination and purified in a centrifugal filtration device.
Differential Organic Extraction
Dithiothreitol (DTT) reduces disulfides to dithiols, allowing release of the DNA from its protective proteins and further degradation of the proteins by Proteinase K. DTT is an essential component for sperm cell lysis because the cell membrane contains a high concentration of disulfides.
DNA Quantitation
After purification, the DNA is ready for quantitation