1. By
Dr Khaled Saleh Algariri

2. Every living thing has DNA. That means
that you have something in common with a
zebra, a tree, a mushroom and a beetle!!!!
DNA stands for:
D: Deoxyribose
N: Nucleic
A: Acid
DNA is too
small to see,
but under a
microscope it
looks like a
twisted up
ladder!

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

5. Examples
 DNA extraction is used
to isolate…
 Mitochondrial DNA
 Genomic DNA
 DNA can be extracted
from…
 Cells or tissues
 Environmental samples
http://www.davidkfaux.org/shetlandislandsmtDNA.html
http://faculty.uca.edu/~benw/biol1400/pictures/

6. Non-examples
 DNA Extraction is not
used to…
 Isolate proteins or RNA
 Give information about
gene expression
http://iwrwww1.fzk.de/biostruct/

7. Nucleic Acid Preparation Application?
 DNA
 Purity and amount of DNA required (and process used) depends
on intended application. Example applications:
 Tissue typing for organ transplant
 Detection of pathogens
 Human identity testing
 Genetic research
 Reverse transcription polymerase chain reaction and Ligase
chain reaction(PCR, LCR)
 RFLP (restriction fragment length polymorphism)
 Hybridization methods (Southern analysis)

8. Nucleic Acid PreparationApplication?
 RNA
 Amplification methods , Reverse transcription
polymerase chain reaction(RT-PCR)
 Hybridization methods (Northern analysis)

9. DNA Purification Challenges
1. Separating DNA from other cellular components
such as proteins, lipids, RNA, etc.
2. Avoiding fragmentation of the long DNA molecules
by mechanical shearing or the action of endogenous
nucleases.
Effectively inactivating endogenous nucleases
(DNase enzymes) and preventing them from
digesting the genomic DNA is a key early step in the
purification process. DNases can usually be
inactivated by use of heat or chelating agents.

10. Nucleic Acid Purification
There are many DNA purification methods. All
must:
1. Effectively disrupt cells or tissues
(usually using detergent)
2. Denature proteins and nucleoprotein complexes
(a protease/denaturant)
3. Inactivate endogenous nucleases
(chelating agents)
4. Purify nucleic acid target away from other nucleic
acids and protein
(could involve RNases, proteases, selective matrix and alcohol
precipitations)

11. Disruption of Cells/Tissues
Most purification methods disrupt cells using lysis buffer
containing:
Detergent to disrupt the lipid bilayer of the cell membrane
Denaturants to release chromosomal DNA and denature
proteins
Additional enzymes are required for lysis of some cell types:
Gram-positive bacteria require lysozyme to disrupt the
bacterial cell wall.
Yeasts require addition of lyticase to disrupt the cell wall.
Plant cells may require cellulase pre-treatment.

12. Disruption of Cells: Membrane Disruption
 Detergents are used to disrupt the lipid:lipid and
lipid:protein interactions in the cell membrane,
causing solubilization of the membrane.
 Ionic detergents (such as sodium dodecyl sulfate; SDS)
also denature proteins by binding to charged residues,
leading to local changes in conformation.

13. Protein Denaturation
Denaturation = Modification of conformation to
unfold protein, disrupting secondary structure but not
breaking the peptide bonds between amino acid
residues.
Denaturation results in:
 Decreased protein solubility
 Loss of biological activity
 Improved digestion by proteases
 Release of chromosomal DNA from nucleoprotein
complexes (“unwinding” of DNA and release from
associated histones)

14. Inactivation of Nucleases
 Chelating agents, such as EDTA, sequester Mg2+
required for nuclease activity.
 Proteinase K digests and destroys all proteins,
including nucleases.
 Some commercial purification systems provide a single
solution for cell lysis, protein digestion/denaturation
and nuclease inactivation.

15. Removal of RNA
 Some procedures incorporate RNase digestion
during cell lysate preparation.
 In other procedures, RNase digestion is
incorporated during wash steps.

16. Basic Protocol
 Most DNA extraction protocols consist of
two parts
1. A technique to lyse the cells gently and solubilize the
DNA
2. Enzymatic or chemical methods to remove
contaminating proteins, RNA, or macromolecules
 In plants, the nucleus is protected within a nuclear membrane
which is surrounded by a cell membrane and a cell wall. Four
steps are used to remove and purify the DNA from the rest of
the cell.
1. Lysis
2. Precipitation
3. Wash
4. Resuspension

17. A comparison of DNA extraction methods used in
research labs as opposed to classroom labs
Research
Lysis: grind in Liquid N2 and use
detergent
Precipitation Part I:
phenol/chloroform extraction to get
rid of proteins
Precipitation Part II: addition of salts
to interrupt hydrogen bonding
between water and phosphates on
the DNA
Precipitation Part III: addition of
ethanol to pull DNA out of solution
Wash and resuspend: DNA is washed
in ethanol, dried, and resuspended
in H20 or TE buffer.
Classroom
Lysis: grind in mortar/pestel and use
detergent
Precipitation Part I: NONE
(chemical are too dangerous!)
Precipitation Part II: addition of salts
to interrupt hydrogen bonding
between water and phosphates on
the DNA
Precipitation Part III: addition of
ethanol to pull DNA out of solution
Wash and resuspend: DNA is washed
in ethanol, dried, and resuspended
in H20 or TE buffer.

18. LYSIS:
In DNA extraction from plants, this step commonly refers to the
breaking
of the cell wall and cellular membranes (most importantly, the
plasma and nuclear membranes)
 The cell wall (made of cellulose) is disrupted by
mechanical force (for example, grinding the leaves)
 Then the addition of a detergent in the which breaks
down the cell membranes
 Detergents are able to disrupt membranes due to the amphipathic
(having both hydrophilic and hydrophobic regions) nature of both
cellular membranes and detergent molecules. The detergent
molecules are able to pull apart the membranes

19. DNA purification: phenol/chloroform extraction
1:1 phenol : chloroform
or
25:24:1 phenol : chloroform : isoamyl alcohol
Phenol: denatures proteins, precipitates form at
interface between aqueous and organic layer
Chloroform: increases density of organic layer
Isoamyl alcohol: prevents foaming

20. PRECIPITATION (In a research lab):
This a series of steps where DNA is separated from the rest of the
cellular components
 In a research lab, the first part of precipitation uses phenol/chloroform
to remove the proteins from the DNA
 Phenol denatures proteins and dissolves denatured proteins.
 Chloroform is also a protein denaturant
THIS STEP CANNOT BE PERFORMED IN CLASSROOM LABS!!
 The second part of research lab DNA precipitation is the addition of
salts
 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.
 The DNA is pelleted by spinning with a centrifuge and the supernatant
removed

21. PRECIPITATION (In a classroom lab):
This a series of steps where DNA is separated from the rest of the
cellular components
 In a classroom lab, DNA precipitation involves the addition
of salts
 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.
 The DNA is pelleted by spinning with a centrifuge and the
supernatant removed
Note: because this protocol does not use phenol/chloroform,
the DNA extracted in a classroom lab is not as “clean” as the
DNA extracted in a research lab!

22. Washing:
The precipitated DNA is laden with acetate salts. It is
“washed” with a 70% ethanol solution to remove salts
and other water soluble impurities but not resuspend
the DNA.
Resuspension:
The clean DNA is now resuspended in a buffer to
ensure stability and long term storage.
The most commonly used buffer for resuspension is
called 1xTE
Washing and Resuspension:

23. cell growth
cell harvest and
lysis
DNA purification
DNA concentration

24. Bacterial genomic DNA prep: cell extract
Lysis:
• Detergents
• Organic solvent
• Proteases (lysozyme)
• Heat
“cell extract”

25. Genomic DNA prep: removing proteins and RNA
Add the enzyme RNase to degrade RNA in the aqueous
layer
Need to mix gently! (to avoid shearing breakage of the
genomic DNA)
chloroform

26. 2 ways to concentrate the genomic DNA
70% final conc.
“spooling” Ethanol precipitation

27. Genomic DNA prep in plants --
how get rid of carbohydrates? CTAB:cetyltrimethyla
mmonium bromide,
hexadecyltrimethylam
monium bromide.
Cationic detergent
(low ionic
conditions)
N+
CH3
Br-
CH3
CH3
C16H33

28. o Successful RNA isolation depends on:
1) Suppression of endogenous RNAases.
2) Avoid contamination with exogenous RNAases
during extraction.
A. Samples should be processed immediately or stored at -
70 degree until required.
B. Inactivation of RNAases by strong denaturing agents
like urea, guanidinium hydrochloride, guanidinium
isothiocyanate.
Suppression of endogenous
RNAases

29. A. Specify glassware, solutions, equipments to be used for
RNA extraction only.
B. Treat water and laboratory utensils with
diethylpyrocarbonate (DEPC) which is a strong
RNAase inhibitor. DEPC is a suspected carcinogen.
C. Autoclave glassware, solutions and equipments if
possible.
D. Use disposable gloves, disposable plastic materials that
must be RNAase free.
Avoid contamination with exogenous RNAases
during extraction

30. 1) Guanidinium isothiocyanate extraction:
Cell lysis.
Protein denaturation by guanidinium
isothiocyanate.
Cell lysate is mixed with cesium chloride.
The density of RNA in cesium chloride is much
greater than of other cellular elements.
During ultracentrifugation, RNA pellets at the
bottom of the tube and becomes separated from
other cellular components.
Methods of RNA extraction

31. 2) RNA extraction by Trizol:
Tizol is a monophasic solution of phenol & chloroform
+ guanidinium isothiocyanate.
The presence of phenol & chloroform will separate cell
lysate into two layers:
o Upper aqueous layer containing RNA.
o Organic layer containing proteins.
RNA is then precipitated from the aqueous layer by
isopropyl alcohol.
3) RNA extraction by spin column:
These columns use RNA adsorbing silica or glass fiber.
RNA is then eluted by elution buffer.

32. RNA extraction by Trizol

33. 4) RNA extraction by magnetic separation
technology:
Couple magnetic beads to silica.
Magnetic silica beads binds RNA in the lysate.
The conjugated magnetic beads are then
collected by applying magnetic field.
RNA is then eluted from the beads.

34. Separate WBCs from RBCs, if necessary
Lyse WBCs or other nucleated cells in presence of protein
denaturants, RNase inhibitors
Denature/digest proteins
Separate proteins, DNA, and contaminants
from RNA
Precipitate RNA if necessary
Resuspend RNA in final buffer
Basic Steps in Isolating RNA from Clinical Specimens

35. RNAses
►RNases are naturally occurring enzymes that degrade
RNA
►Common laboratory contaminant (from bacterial and
human sources)
►Also released from cellular compartments during
isolation of RNA from biological samples
►Can be difficult to inactivate

36. RNAses
►RNAses are enzymes which are small proteins that can
renature and become active.
►MUST be eliminated or inactivated BEFORE isolation.

37. Protecting Against RNAse
►Wear gloves at all times
►Use RNase-free tubes and pipet tips
►Use dedicated, RNase-free, chemicals
►Pre-treat materials with extended heat (180 C for
several hours), wash with DEPC-treated water, NaOH
or H2O2
►Supplement reactions with RNase inhibitors

38. Total RNA
►80-90% of total RNA is ribosomal RNA.
►2.5-5% is messenger RNA
► 15-20% is transfer RNA

39. 2–25 °C 2–8 °C –20 °C –70 °C
Recommended
for long-term
storage in ethanol
<4 Months 1–3 Years <7 Years >7 Years
Nucleic Acid Storage Requirements: Storage of DNA
Specimens

40. BEST WISHES