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