1. DNA EXTRACTION METHODS
DNA Isolation
DNA EXTRACTION METHODS
Dr Nikola Tanić

2. Definition
DNA Extraction is the isolation and purification of DNA (deoxyribonucleic acid)

3. Examples DNA extraction is used to isolate… DNA can be extracted from…
Mitochondrial DNA
Genomic DNA
DNA can be extracted from…
Cells or tissues
Environmental samples
...
any nucleated cell

4. Purpose of DNA Extraction
To obtain DNA in a relatively purified form which can be used for further investigations such as:
PCR (polymerase chain reaction)
RFLP (restriction fragment length polymorphism)
Southern Blotting

5. DNA Analysis Downstream techniques can:
Reveal how organisms are related
Identify cryptic species
Locate mutations in DNA

6. James Watson and Francis Crick (1953)

7. 5' 3' 3' 5'

8. What are the essential components of a DNA extraction Procedure?
Maximize DNA recovery
Remove inhibitors
Remove or inhibit nucleases
Maximize the quality of DNA

9. How Much DNA Can We Recover?
A Diploid Cell contains approximately 6 pg of DNA
Sperm contains approximately 3 pg of DNA
The average WBC of an adult is X 106 cells per ml of blood. Therefore, the theoretical recovery of DNA per ul of blood is ng.

10. How Much DNA Do We Need?
The RFLP (or PCR) procedure on requires a minimum of 50 ng of high molecular weight double stranded DNA. This is the equivalent of approximately 2 ul of blood. The number of intact sperm ( 3 pg/sperm) is approximately 20,000.

11. Basic Protocol Most DNA extraction protocols consist of two parts
A technique to lyse the cells gently and solubilize the DNA
Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules

12. What are the Most Commonly used DNA Extraction Procedures?
Organic (Phenol-Chloroform) Extraction
Non-Organic (Proteinase K and Salting out)
Adsorption method (silica-gel membrane)
The method utilized may be sample dependant, technique dependant, or analyst preference

13. Nucleic Acid Extraction Requirements
1. Disruption of cell wall and membranes to liberate cellular components.
2. Inactivation of DNA- and RNA-degrading enzymes (DNases, RNases).
3. Separation of nucleic acids from other cellular components.
• Extraction/Precipitation method
• Adsorption Chromatography method

14. Creating a Nuclease-Free Environment
There are several things you can do to minimize the risk of exposing your samples to external DNases and RNases.
• Autoclave solutions. This is usually sufficient for getting rid of DNases,
and most RNases as well.
• Treat solutions with 0.1% DEPC. DEPC inactivates nucleases by covalently
modifying the His residues in proteins. Generally considered unnecessary
for DNA extraction. Not compatible with solutions containing Tris or HEPES.
• Have a dedicated set of pipettors or use aerosol barrier tips.
• Wear gloves. You should be doing this anyway for
safety reasons, but skin cells also produce RNase7,
a potent RNA-degrading enzyme.
• Bake glass, metal, or ceramic equipment at high temp.

15. ORGANIC EXTRACTION
Perhaps the most basic of all procedures in molecular biology is the purification of DNA by organic extraction.

16. ORGANIC EXTRACTION PROCEDURE
Typical Procedure
Harvest cells
2 Cell Lysis
0.5% SDS + proteinase K (55o several hours)
3 Phenol Extraction
gentle rocking several hours
4 RNAse followed by proteinase K
5 Repeat Phenol Extraction
6 Ethanol Precipitation

17. ORGANIC EXTRACTION REAGENTS
Cell Lysis Buffer - Non-ionic detergent (SDS), Tris-Cl, EDTA - designed to lyse outer cell membrane and nuclear membrane.
EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.

18. What does the detergent do?
Each cell is surrounded by a cell membrane
DNA is in the nucleus of a cell, which is also surrounded by a membrane

19. The membranes must be broken open in order to get the DNA out of the cell

20. Detergent:
Breaks apart membranes by attaching to the lipids (fats) & proteins in the membranes

21. The cell and nuclear membranes have been broken apart, as well as all of the organelle membranes, such as those around the mitochondria and chloroplasts. So what is left?
Proteins
Carbohydrates (sugars)
DNA

22. ORGANIC EXTRACTION REAGENTS
Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as Pronase or proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.

23. ORGANIC EXTRACTION REAGENTS
Phenol/Chlorform - The standard way to remove proteins from nucleic acids solutions is to extract once with phenol, once with a 1:1 mixture of phenol and chloroform, and once with chloroform. This procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one.
Also, the final extraction with chloroform removes any lingering traces of phenol from the nucleic acid preparation.
Phenol is highly corrosive and can cause severe burns.

24. ORGANIC EXTRACTION REAGENTS
Phenol - often means phenol equilibrated with buffer (such as TE) and containing 0.1% hydroxyquinoline and 0.2% b-mercaptoethanol (added as antioxidants). The hydroxyquinoline also gives the phenol a yellow color,making it easier to identify the phases (layers).
Chloroform - often means a 24:1 (v/v) mixture of chloroform and isoamyl alcohol. The isoamyl alcohol is added to help prevent foaming.
The Phenol/Chloroform/Isoamyl Alcohol ratio is 25:24:1

25. Extraction/Precipitation Method
Step 3: Organic extraction
Mix thoroughly with an equal volume of organic solvent
Aqueous
Centrifuge
Collect aqueous phase
e.g. phenol, chloroform, or phenol:chloroform
Interphase
Organic
Perform additional extractions for increased purity
Crude lysate containing nucleic acids and other cell constituents
The aqueous phase contains water-soluble molecules, including nucleic acids. Proteins and lipids become trapped in the organic phase, and are thus separated away. Insoluble debris become trapped in the interphase between the two layers

26. Phenol denatures proteins and dissolves denatured proteins.
Chloroform is also a protein denaturant

27. Using Nucleases to Remove Unwanted DNA or RNA
Add DNase
+ DNase (protein)
Add RNase
+ RNase (protein)
Depending on when nuclease treatment is performed, it may be necessary to repeat purification steps for protein removal (e.g. phenol/chloroform extraction).

28. Concentrating DNA Alcohol Precipitation
The most widely used method for concentrating DNA is precipitation with ethanol. The precipitate of nucleic acid, forms in the presence of moderate concentrations of monovalent cations (Salt, such as Na+), is recovered by centrifugation and redissolved in an appropriate buffer such as TE.
The technique is rapid and is quantitative even with nanogram amounts of DNA.

29. The salts interrupt the hydrogen bonds between the water and DNA molecules
The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol
In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.

31. Concentrating DNA Alcohol Precipitation
The four critical variables are the purity of the DNA, its molecular weight, its concentration, and the speed at which it is pelleted.
DNA a concentrations as low as 20 ng/ml will form a precipitate that can be quantitatively recovered.
Typically 2 volumes of ice cold ethanol are added to precipitate the DNA.

32. Concentrating DNA Alcohol Precipitation
Very short DNA molecules (<200 bp) are precipitated inefficiently by ethanol.
The optimum pelleting conditions depend on the DNA concentration. Relatively vigorous microcentrifuge steps such as 15 minutes at or below room temperature at 12,000 rpm are designed to minimized the loss of DNA from samples with yields in the range of a few micrograms or less.

33. Concentrating DNA Alcohol Precipitation
Solutes that may be trapped in the precipitate may be removed by washing the DNA pellet with a solution of 70% ethanol. To make certain that no DNA is lost during washing, add 70% ethanol until the tube is 2/3 full. Recentrifuge. After the 70% ethanol wash, the pellet does not adhere tightly to the wall of the tube, so great care must be taken when removing the supernatant.

34. Concentrating DNA Alcohol Precipitation
Isopropanol (1 volume) may be used in place of ethanol (2 volumes) to precipitate DNA. Precipitation with isopropanol has the advantage that the volume of liquid to be centrifuged is smaller.
Isopropanol is less volatile than ethanol and it is more difficult to remove the last traces; moreover, solutes such sodium chloride are more easily coprecipitated with DNA when isopropanol is used.

35. Extraction/Precipitation Method
Step 4: Nucleic Acid Precipitation
Before
After
After
Supernatant
70% EtOH
Centrifuge
Wash
Centrifuge
Pellet
Dissolve pellet (H2O, TE, etc.)
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Pellet down nucleic acids.
Add alcohol and salt to precipitate nucleic acids from the aqueous fraction

36. Non-Organic DNA Extraction
Does not use organic reagents such as phenol or chloroform.
Digested proteins are removed by salting out with high concentrations of LiCl (NaCl).
However, if salts are not adequately removed, problems could occur with the RFLP procedure due to alteration of DNA mobility (band shifting)

37. Non-Organic DNA Extraction Procedure
Cell Lysis Buffer - lyse cell membrane, nuclei are intact, pellet nuclei.
Resuspend nuclei in Protein Lysis Buffer containing a high concentration of Proteinase K. Lyse nuclear membrane and digest protein at 65oC for 2 hours. Temperature helps denature proteins, and Proteinase K auto digests itself
To remove proteinaceous material, LiCl is added to a final concentration of 2.5 M, and incubated on ice. Proteins precipitate out and are pelleted by centrifugation.

38. Non-Organic DNA Extraction Procedure
4. DNA remains in solution. Transfer supernatant to a new tube, care must be taken not to take any of protein pellet.
5. DNA is precipitated by the addition of room temperature isopropanol. LiCl will not precipitate with DNA.
6. Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in TE buffer.

39. Nucleic acids within a crude lysate are bound to a silica surface
Adsorption method
Basic Principle
Nucleic acids within a crude lysate are bound to a silica surface
The silica surface is washed with a solution that keeps nucleic acids bound, but removes all other substances
The silica surface is washed with a solution unfavorable to nucleic acid binding. The solution, containing purified DNA and is recovered.

40. Adsorption Method Step 1: Prepare crude lysate
Step 2: Adsorb to silica surface
Apply to column
Centrifuge
Nucleic acids
Silica-gel membrane
Extraction Buffer composition favors DNA adsorption to silica:
• Low pH
• High ionic strength
Flow through
(discard)
Nucleic acids bind to the membrane, while contaminants pass through the column.
Having the ability to destabilize hydrogen bonding and hydrophobic interactions.

41. Adsorption Method Step 3: Wash away residual contaminants
Centrifuge
Wash buffer
Nucleic acids
Nucleic acids
Flow through
(discard)
Step 4: Elute nucleic acids
Centrifuge
Elution buffer
Nucleic acids
Elution Buffer composition is unfavorable to surface binding:
High pH
Low ionic strength
Nucleic acids

42. Evaluation of Nucleic Acids
spectrophotometrically
quantity
quality
fluorescent dyes
gel electrophoresis

43. Nucleic Acid Analysis via UV Spectrophotometry
DNA Absorption Spectra
By measuring the amount of light absorbed by your sample at specific wavelengths, it is possible to estimate the concentration of DNA and RNA. Nucleic acids have an absorption peak at ~260nm.
[dsDNA] ≈ A260 x (50 µg/mL)
[ssDNA] ≈ A260 x (33 µg/mL)
conc. DNA (mg/ml) = (A260 x R x 50)/1000

44. How pure is your sample?
The A260/A280 ratio is ~1.8 for dsDNA. Ratios lower than 1.7 usually indicate significant protein contamination.
The A260/A230 ratio of DNA and RNA should be roughly equal to its A260/A280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates).
Turbidity can lead to erroneous readings due to light interference. Nucleic acids do not absorb light at the 320 nm wavelength. Thus, one can correct for the effects of turbidity by subtracting the A320 from readings at A230, A260 and A280.

45. Checking for Degradation
Running your sample through an agarose gel is a common method for examining the extent of DNA degradation. Good quality DNA should migrate as a high molecular weight band, with little or no evidence of smearing.

46. Intercalating Agents Distort the Double Helix
Several hydrophobic
molecules containing
flat aromatic and fused
heterocyclic rings can
insert between the
stacked base pairs
of DNA. These
molecules are called
intercalating agents.
Intercalating agents
are potential
Cancer-inducing
reagents.

47. Gel Electrophoresis of DNA
Separation of DNA fragments according to size, based on movement through a gel medium when an electric field is applied
Agarose - a polysaccharide made from seaweed. Agarose is dissolved in buffer and heated, then cools to a gelatinous solid with a network of crosslinked molecules
Some gels are made with acrylamide if sharper bands are required

48. A bit like electronic chromatography
The fragments are seperated according to size in process called gel electrophoresis

49. Make up the gel which the DNA will be put into
Square tray
2-3 cm of agarose gel which is left to set, can also be made of starch or polyacrylamide, special comb put in so that there are small wells left in the gel

51. A comb is put in the gel to create holes which we call wells

53. Dye added to the DNA
Makes the sample visible when it is put into the agarose wells

54. Buffer solution added to the tank
This ensures that the electric current goes through the whole tank and that maintains that ions can move in the solution

55. DNA samples loaded into wells
Glycerol also in the loading dye

56. Electrical current applied to the chamber
Safety cover is put over the top and the current is switched on
The dye will migrate through the gel toward the positive electrode, as will the DNA
Depending on how much voltage is applied and how warm the gel is and size and shape of molecules will depend on how fast the mols move through the gel
Smaller fragments will move easier so they will be closer to the positive electrode
Once the dye has moved through the gel to the buffer, the electrical current is switched off and gel is removed from the tray