this slide contain all the contents of DNA extraction

1. DNA EXTRACTION
METHODS & ESTIMATION

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

3. How Much DNA Can We
Recover?
• A Diploid Cell contains approximately 6
pg of DNA
• The average WBC of an adult is 5 - 10 X
106 cells per ml of blood. Therefore, the
theoretical recovery of DNA per ul of
blood is 30 - 60 ng.

4. How Much DNA Do We Need?
• The PCR reactions call for on average 1
ng of DNA (single or double stranded).
• Many of the commercially available kits
are sensitive below 1 ng of DNA
(100-250 pg).

5. Basic steps for DNA extraction
1. Breaking the cells open, commonly referred to
as cell disruption or cell lysis, to expose the
DNA within. This is commonly achieved by
grinding, sonicating or treating the sample with
lysis buffer .
2. Removing membrane lipids by adding
a detergent.

6. Purposes of the Extraction Buffer Detergents
Chaotropic salts
1. Dissolve cellular membranes CTAB
Detergents
Metal chelators
Reducing agents
2. Inactivation of DNase and Rnase
Salts
3. Assist in the removal of contaminants
CTAB
PVP

7. Extraction/Precipitation Method
Use of Detergents to Lyse Cells:
Mixed micelle
Plasma membrane
(phospholipid bilayer) Detergent molecules
+
SDS

8. 3. Removing proteins by adding
a protease (optional but almost always
done).
4. Precipitating the DNA with an alcohol —
usually ice-cold ethanol or isopropanol.
Since DNA is insoluble in these alcohols, it
will aggregate together, giving
a pellet upon centrifugation. This step also
removes alcohol-soluble salt.

9. • Depending on the material and
requirement the extraction process can
be modified with alteration of chemicals
used

10. Most Commonly used DNA
Extraction Procedures
• Organic (Phenol-Chloroform) Extraction
• Non-Organic (Proteinase K and Salting out)
• NCM / Nylon membrane(Collection, Storage,
and Isolation)
The method utilized may be sample dependant,
technique dependant, or analyst preference

11. Extraction/Precipitation Method
Step 1: Disruption of cell walls by grinding
Step 1+2: mechanical disruption
and homogenization in extraction
buffer
Grind sample into a fine powder to
shear cell walls and membranes
Step 2: Lysis of cells in extraction buffer
A homogenizer allows cells to be
mechanically disrupted within the
extraction buffer
Mix thoroughly with extraction
buffer to dissolve cell membranes
and inhibit nuclease activity Crude lysate

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

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

14. RNA extraction
• This method relies on phase separation by
centrifugation of a mix of the aqueous
sample and a solution containing water-
saturated phenol,chloroform and a
denaturing solution (guanidinium
thiocyanate) resulting in an upper
aqueous phase and a lower organic phase
(mainly chloroform).

15. • Nearly all of the RNA is present in the
aqueous phase, while DNA and protein
partition in the interphase and organic
phase, respectively. In a last step, RNA is
recovered from the aqueous phase by
precipitation with 2-propanol or ethanol.
DNA will be located in the aqueous phase
in the absence of guanidinium thiocyanate
and thus the technique can be used for
DNA purification alone.

16. • Guanidinium thiocyanate denatures proteins,
including RNases, and separates rRNA from
ribosomes, while phenol, isopropanol and water
are solvents with poor solubility. In the presence
of chloroform , these solvents separate entirely
into two phases that are recognized by their color:
a clear, upper aqueous phase (containing the
nucleic acids) and a bright pink lower phase
(containing the proteins dissolved in phenol and
the lipids dissolved in chloroform). Other
denaturing chemicals such as 2-mercaptoethanol
may also be used. The major downside is
that phenol and chloroform are both hazardous
and inconvenient materials, and the extraction is
often laborious, so in recent years many
companies now offer alternative ways to isolate
DNA.

17. EXTRACTION
• Perhaps the most basic of all procedures
in genetic engineering is the purification of
DNA. The key step, the removal of
proteins, can often be carried out simply
by extracting aqueous solutions of nucleic
acids with phenol and/or chloroform.

18. EXTRACTION
• Cell Lysis Buffer – This buffer will lyse cell
membrane, nuclei are intact, pellet nuclei.
• The nuclei is Resuspended in a buffer
containing Sodium Dodecly Sulfate (SDS)
and Proteinase K. This will Lyse nuclear
membrane and digest protein.
• DNA released into solution is extracted with
phenol-chloroform to remove proteinaceous
material.

19. EXTRACTION
• DNA is precipitated from the aqueous
layer by the additional of ice cold 95%
ethanol and salt
• Precipitated DNA is washed with 70%
ethanol, dried under vacuum and
resuspended in TE buffer.

20. ORGANIC EXTRACTION
REAGENTS
• Cell Lysis Buffer - Non-ionic detergent ,
Salt, Buffer, EDTA designed to lyse
outer cell membrane, but will not break
down 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.

21. ORGANIC EXTRACTION
REAGENTS
• Proteinase K - it is usual to remove
most of the protein by digesting with
proteolytic enzymes such as 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.

22. 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.

23. ORGANIC EXTRACTION
REAGENTS
• Phenol - often means phenol equilibrated
with buffer (such as TE) and containing 0.1%
hydroxyquinoline and 0.2% b-ercaptoethanol
(added as antioxidants. The hydoxquinoline
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

24. Concentrating DNA by
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.

25. • phenol helps to remove non polar proteins and lipids
from the solution, phenol is used to denature the
proteins.
ethanol change ionic potential of DNA and remove water
molecules, which help in precipitation of DNA.
Salt would attract the phosphate ends of DNA, therefore
it pulls it way from other substances in the sample
(Separation of DNA from surroundings)

26. 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.

27. 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.

28. 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. Vortex briefly,
and recentrifuge. After the 70% ethanol
wash, the pellet does not adhere tightly to
the wall of thetube, so great care must be
taken when removing the supernatant.

29. 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.

30. Concentrating DNA
Centrifugal Filter Unit

31. Resuspension and Storage of DNA
• TE Buffer - Tris-EDTA Buffer: 10 mM Tris-
HCl pH 8.0, 1 mM EDTA, or TE-4 which is
10 mM Tris, 0.1 mM EDTA. DNA is
resuspended and stored in TE buffer. DNA
must be stored in a slightly basis buffer to
prevent depurination, and the EDTA
chelates any Mg2+ helping to inactivate
DNases.

32. • DNA can be stored at 4oC for extended
periods, however for long term storage, -
20oC is preferable.
• Avoid repetitive freeze thawing of DNA,
since this can cause degradation.

33. 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).

34. Non-Phenol Chloroform based extraction of DNA for PCR detection
of Citrus yellow mosaic virus and Citrus greening bacterium
Clear
supernatent
Centrifuge
Supernatant
Centrifuge
Pellet
Cell debris
Add isoprpanol /alcohol
and salt to precipitate
nucleic acids from the
aqueous fraction

35. 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.
• However, if salts are not adequately
removed, problems could occur with the
procedure due to alteration of DNA mobility
(band shifting)

36. Non-Organic DNA Extraction
Procedure
• Cell Lysis Buffer Containing Proteinase -
lyse cell membrane, lyse nuclear
membrane and digest protein at high
temperature eg. 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.

37. 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.

38. Membrane based method
• A spin column using a silica-based
extraction method is used. This does not
require the use of hazardous chemicals.
Nucleic acids are attracted to the silica
bead under high chaotropic salt
concentrations. The sample and lysis
buffer are added to a sterile tube.

39. The lysate is combined with alcohol
and placed into the spin column,
which is inserted into a tube. The
removal of proteins and divalent
cations is accomplished using
multiple buffer washes and
centrifugation steps. Removal of
cations, such as Mg2+, prevents
nucleases from further degrading the
DNA. Pure DNA is eluted from the
membrane into sterile water or TE
buffer.

40. Membrane based Nucleic acid template preparation
Extract in NaOH-EDTA
without use of Liquid N2
Centrifuge 15 min
PCR/RT-PCR 90-100%
Heat at 80 oC 10 min
CG/CMBV/Viroid

41. Assessing the Quality and Yield
of Nucleic Acids

42. Checking for DNA
genomic
DNA
Running nucleic acid 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
RNA
(degraded)
evidence of smearing.

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 of 1 OD at ~260nm.
[dsDNA] ≈ A260 x (50 µg/mL)
[ssDNA] ≈ A260 x (33 µg/mL)
[ssRNA] ≈ A260 x (40 µg/mL)

44. • 1 Optical Density (OD) unit of double-
stranded DNA is 50 micrograms/ml.
• 1 OD unit of single-stranded DNA is 33
micrograms/ml.
• 1 OD unit of single-stranded RNA is 40
micrograms/ml.

45. How pure is nucleic acid sample?
Nucleic acids strongly absorb at 260 nm and less strongly at 280 nm
while proteins do the opposite.
The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. 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).