DNA EXTRACTION
• Have you done DNA extractions with your
class?
1865 presented paper Experiments on Plant Hybridization,
Johann Friedrich Miescher
1844 -1895
1869: Characterizes a new
substance in pus (June 15,
1866-August 23, 1866).
“… the substance was
derived from the nucleus of
the cell. Hence, we call it
nuclein.”
4
Pál Plósz (1871) verified the presence of nuclein in the nucleated
erythrocytes of birds and reptiles and its absence from the
erythrocytes of mammals, which are devoid of a nucleus.
5
1881: (Ludwig Karl Martin
Leonhard)Albrecht Kossel
determines that nucleic acid
is composed of four bases.
Untersuchungen über die
Nukleine und ihre
Spaltungsprodukte
(Investigations into the
nucleins and their
cleavage products)
19106
1893: Albrecht Kossel
determines that nucleic
acid is composed of four
bases.
2 purines:
2 pyrimidines:
7
adenine (A) guanine (G) cytosine (C) thymine (T)
1889: Richard Altmann
finds that nuclein is acidic
and renames it nucleic
acid (nucleïnsäure).
Ueber Nucleinsäuren. Archiv für Anatomie und Physiologie.
Physiologische Abteilung. Leipzig, 1889, 524-536.
8
Phoebus Aaron (Theodore) Levene
(1869-1940)
1909: Phoebus Levene discovers
that DNA is made of 3 basic
components: a sugar, an acid,
and an organic base.
9
1944: Oswald Avery, Colin MacLeod, and Maclyn McCarty establish that Griffith's
transforming principle is DNA, and suggest that it may function as the genetic material.
Avery, O. T., MacLeod, C. M. & McCarty, M. 1944. Studies of the chemical nature of the substance inducing
transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from
Pneumococcus Type III. J. Exp. Med. 79:137-158.
10
1949: Roger Vendrely, Colette Vendrely, and André Boivin find half as
much DNA in the nuclei of sex cells as they find in body cells, thus
paralleling the reduction in the number of chromosomes, making DNA look
like the genetic material.
11
1951
X-ray diffraction of DNA
Rosalind Franklin
12
1952: Alfred D. Hershey & Martha Chase
DNA is
the
genetic
material
13
Martha Chase
Awarded a blender (?)
Alfred D. Hershey
Awarded the Nobel Prize
14
“We have formulated a structure for the nucleic
acids… The structure involves three intertwined
helical polynucleotide chains. Each chain… has
approximately twenty-four nucleotide residues
in seven turns of the helix. The helixes form a
right-handed screw. The phosphate groups are
closely packed about the axis… with the pentose
residues… and the purine and pyrimidine
groups projecting radially...”
Linus Pauling & Robert B. Corey (Nature, 1953,
171:346).
15
Pauling, L. and Corey, R. B. 1953. A proposed structure for the
nucleic acids Proc. Natl. Acad. Sci. USA 39:84-97.
16
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What are the essential
components of a DNA extraction
Procedure?
1.
2.
3.
4.
Maximize DNA recovery
Remove inhibitors
Remove or inhibit nucleases
Maximize the quality of DNA
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 (100250 pg).
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.
Lysis Buffer:
50 ml Lysis Buffer
5 mM EDTA pH 8.0
0.5 M EDTA
= 500 ul
200 mM NaCL
5M NaCl
= 4 mL
100 mM Tris pH 8.0
1 M Tris-HCL ph 8= 5 mL
0.2% SDS Sodium dodecyl Sulfate 10% SDS
= 1 mL
Water
MQ water
= 39.5 mL
Add prior to 55C o/n icubation
0.4 mg/ml Proteinase K
20 mg/mL Prot K = 6 ul per 300ul of Lysis
Buffer
Purposes of the Extraction Buffer
1. Dissolve cellular membranes
2. Inactivation of DNase and Rnase
Detergents
Chaotropic salts
CTAB
Detergents
Metal chelators
Reducing agents
Salts
3. Assist in the removal of contaminants
CTAB
PVP
Extraction/Precipitation Method
Use of Detergents to Lyse Cells:
Plasma membrane
(phospholipid bilayer)
Mixed micelle
Detergent molecules
+
SDS
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.
Most Commonly used DNA
Extraction Procedures
• Organic (Phenol-Chloroform) Extraction
• Non-Organic (Proteinase K and Salting out)
• Qiagen (anion exchange resin)
The method utilized may be sample dependant,
technique dependant, or analyst preference
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.
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.
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.
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.
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
Mix thoroughly with extraction
buffer to dissolve cell membranes
and inhibit nuclease activity
A homogenizer allows cells to be
mechanically disrupted within the
extraction buffer
Crude lysate
Extraction/Precipitation Method
Step 3: Organic extraction
Mix thoroughly with
an equal volume of
organic solvent
e.g. phenol, chloroform,
or phenol:chloroform
Aqueous
Centrifuge
Collect aqueous phase
Interphase
Organic
Perform additional extractions for increased purity
Crude lysate containing
nucleic acids and other
cell constituents
The aqueous phase contains watersoluble molecules, including nucleic
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
Extraction/Precipitation Method
Step 4: Nucleic Acid Precipitation
Before
After
Supernatant
Centrifuge
70% EtOH
Wash
Centrifuge
Pellet
Add alcohol and salt to
precipitate nucleic acids
from the aqueous fraction
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
Dissolve pellet
(H2O, TE, etc.)
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.
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.
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.
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.
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.
Resuspension and Storage of DNA
• TE Buffer - Tris-EDTA Buffer: 10 mM TrisHCl 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.
• 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.
Assessing the Quality and Yield
of Nucleic Acids
Checking for DNA
genomic
DNA
RNA
(degraded)
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
evidence of smearing.
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)
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).