1. Organic Extraction Presented by: Robert O'Brien Training Specialist – Forensic Biology

2. 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

3. 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 (450  g 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

4. 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

5. Solubilization of Stain Step 1: Place stain in a microcentrifuge tube with stain extraction buffer. Vortex, centrifuge, incubate.

6. 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

7. Solubilization of Stain, cont. The buffer contains EDTA, a chelator of magnesium to prevent the action of the nucleases that could degrade the DNA

8. 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

9. 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

10. Denaturation/Hydrolysis of Proteins, cont. Step 2: Transfer any solid material (substrate) into a spin basket. Centrifuge. Discard basket and substrate.

11. 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

12. 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.

13. Organic Extraction Phases The aqueous layer is removed and placed into a new microcentrifuge tube. DNA (aqueous) Protein (organic)

14. 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.

15. 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.

16. 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

17. 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

18. 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.

19. Microcon ® Components

20. Centricon ® Components

21. 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

22. DNA Quantitation After purification, the DNA is ready for quantitation

23. 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.

24. 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.

25. Differential Organic Extraction The 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.

26. 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.

27. DNA Quantitation After purification, the DNA is ready for quantitation