Lecture 1.
Nucleic acid extraction
(DNA and RNA)
By [Link]
Krida Wacana Christian University
(UKRIDA) Jakarta Indonesia
Definition
Nucleic acid extraction
is the isolation and
purification of DNA
(deoxyribonucleic
acid) or RNA
(ribonucleic acid)
Outline
1. DNA Collection, sample, and storage
2. Extraction
3. Assessment of quality and quantity
4. Nucleic acid storage
5. Electrophoresis
1. DNA collection, sample and storage
Whole blood
Bone marrow
Serum/plasma
Buccal cells
Cultured cells
Blood spots
Body fluids
Bronchial lavage
Amniotic
Semen
Urine
Tissue samples
Fresh/frozen
Paraffin-embedded
Hair (shaft/root)
1. DNA collection, sample and
storage (cont.)
Sample
Two types of tissue: fresh and
preserved.
The damaging action of tissue
endonucleases.
Endonuclease: DNase and Rnase
1. DNA collection, sample and
storage (cont.)
Temperature storage for DNA
Purified DNA may be refrigerated at
4oC for up to 3 years
Samples kept over 3 years should be
frozen at -70oC
Specimen Storage RequirementsDNA
Blood, Bone Marrow, Other Fluids
2225 C Not recommended (<24 hours)
28 C
Suitable condition for up to 72 hours
Not recommended
20 C
NOTE: Do not freeze blood or bone marrow before
lysing red blood cells (RBCs). Leukocyte pellet can
be frozen for up to 1 year.
70 C
Not recommended
NOTE: Do not freeze blood or bone marrow before
lysing red blood cells (RBCs). Leukocyte pellet can
be frozen for >1 year.
Specimen Storage Requirements RNA
Blood, Bone Marrow, Other Fluids
2225 C Not recommended within 2 hours
28 C
20 C
Not recommended within 2 hours
Not recommended 24 weeks
NOTE: Do not freeze blood or bone marrow before
lysing red blood cells (RBCs).
70 C
Preferred storage condition
NOTE: Do not freeze blood or bone marrow before
lysing red blood cells (RBCs)
Blood and bone marrow
Collection tubes are EDTA or ACD
5 15 ml
Sample should not be frozen for transport
4 25oC
Notes:
EDTA: Ethylenediaminetetraacetic acid
ACD: Acid-Citric-Dextrose
Ethylenediaminetetraacetic acid
EDTA
EDTA is used extensively in the analysis of blood.
It is an anticoagulant for blood samples.
In biochemistry and molecular biology, ion
depletion is commonly used to deactivate metaldependent enzymes, either as an assay for their
reactivity or to suppress damage to DNA or
proteins.
ACD
Acid Citrate Dextrose Solution (sometimes
called Anticoagulant Citrate Dextrose Solution) is
a solution of citric acid, sodium citrate and
dextrose in water.
It is mainly used as an anticoagulant to preserve
blood specimens required for tissue typing, it is
also used during procedures such as
plasmapheresis instead of heparin.
Two different solutions (Solution A and B) are
defined by the United States Pharmacopeia.
Chaotropic agent
A denaturating agent is a substance which disrupts the
three dimensional structure in macromolecules such as
proteins, DNA, or RNA and denatures them.
A denaturating agent is a chaotropic agent, but chaotropic
agents aren't necessarily denaturating agents.
Chaotropic agents disrupt the intermolecular forces
between water molecules, allowing proteins and other
macromolecules to dissolve more easily.
Chaotropic agents interfere with stabilizing intramolecular
interactions mediated by non-covalent forces such as
hydrogen bonds, van der Waals forces, and hydrophobic
effects.
Chaotropic reagents include: Urea 6 - 8 mol/l; Thiourea 2
mol/l; Guanidinium chloride 6 mol/l; Lithium perchlorate
4.5 mol/l
GITC (guanidium isothyocyanate)
a general protein denaturant, being a
chaotropic agent,
Serum
Collection tubes with no additives
100 l 1 ml
Transported at 20 25oC
Urine
Urine container should be used for
collection
At least 1 ml should be collected
Transported at 4 25oC
2. Extraction
Chemical treatments cause cells
and nuclei to burst
The nucleic acid is inherently
sticky, and can be pulled out of
the mixture
This is called spooling nucleic
acid
Spooled NA
Extraction
Tissue isolation, membrane
disruptionand cell lysis
Paraffin-embedded tissue require
deparaffinsation (heating or solvents like
xylene)
Blood samples
Organic extraction
Inorganic extraction
RNA extraction
Paraffin-embedded Tissue Sections
Genetic testing, infectious disease
testing, identity testing
Formalin-fixed tissue is suitable.
Mercury or other heavy metal
fixatives are not acceptable.
Tissue sections on glass slides can
be used for in situ applications and
microdissection techniques.
Blood samples
WBCs
RBCs
Plasma/serum
Basic Steps in Isolating
DNA from Clinical Specimens
Separate WBCs from RBCs, if necessary
Lyse WBCs or other nucleated cells
Denature/digest proteins
Separate contaminants (e.g., proteins,
heme)
from DNA
Precipitate DNA if necessary
Resuspend DNA in final buffer
Membrane disruption/ lysis
Detergent SDS: sodium dodecyl
sulfate
Proteolytic agents: Proteinase K
Organic extraction
Phenol-chloroform extraction
Separation of protein into organic
phase and nucleic acid into
aqueous phase.
Phenol pH: 7.8 = 8.0 which prevent
nucleic acid from remaining in the
organic phase.
High MW Genomic DNA Isolation
Typical Procedure
Phenol Extraction
1 Cell Lysis
mix sample with equal volume of sat.
phenol soln
retain aqueous phase
optional chloroform/isoamyl alcohol
extraction(s)
0.5% SDS + proteinase
K (55o several hours)
2 Phenol Extraction
gentle rocking several
hours
3 Ethanol Precipitation
4 RNAse followed by proteinase K
5 Repeat phenol extrac-tion and
EtOH ppt
aqueous phase (nucleic
acids)
phenol phase
(proteins)
27
[Link] Abdi
High MW Genomic DNA Isolation
Typical Procedure
1 Cell Lysis: 0.5% SDS +
proteinase K (55o several
hours)
2 Phenol Extraction: gentle
rocking several hours
3 Ethanol Precipitation
4 RNAse followed by
proteinase K
5 Repeat Phenol Extraction
and EtOH ppt
28
EtOH Precipitation
2-2.5 volumes EtOH, -20o
high salt, pH 5-5.5
centrifuge or spool out
SDS
Sodium dodecyl sulfate (SDS or NaDS),
sodium laurilsulfate or sodium lauryl sulfate
(SLS) is an organic compound with the formula
CH3(CH2)11OSO3Na).
It is an anionic surfactant used in many cleaning
and hygiene products. The salt is of an
organosulfate consisting of a 12-carbon tail
attached to a sulfate group, giving the material
the amphiphilic properties required of a detergent.
Being derived from inexpensive coconut and
palm oils, it is a common component of many
domestic cleaning products.
SDS
DNA purification: overview
cell harvest and
lysis
cell growth
DNA concentration
DNA purification
Bacterial genomic DNA prep: cell extract
Lysis:
Detergents
Organic solvent
Proteases (lysozyme)
Heat
cell extract
Genomic DNA prep: removing proteins and RNA
chloroform
Need to mix gently! (to avoid shearing breakage of the
genomic DNA)
Add the enzyme RNase to degrade RNA in the aqueous
layer
2 ways to concentrate the genomic DNA
70% final conc.
spooling
Ethanol precipitation
Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA
plasmids
Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulate
Plasmid purification: alkaline lysis
Alkaline
conditions
denature
DNA
Neutralize:
genomic DNA
cant renature
(plasmids
CAN because
they never
fully
separate)
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
Phenol extraction
1.
Aqueous volume (at least 200 microliters)
2.
Add 2 volumes of phenol:chloroform, mix well
3.
Spin in centrifuge, move aqueous phase to a new tube
4.
Repeat steps 2 and 3 until there is no precipitate at phase interface
5.
(extract aqueous layer with 2 volumes of chloroform)
Ethanol precipitation (DNA concentration)
Ethanol depletes the hydration shell surrounding DNA
Allowing cations to interact with the DNA phosphates
Reducing repulsive forces between DNA strands
Causing aggregation and precipitation of DNA
Aqueous volume (example: 200 microliters)
-- add 22 microliters sodium acetate 3M pH 5.2
-- add 1 microliter of glycogen (gives a visible pellet)
-- add 2 volumes (446 microliters) 100% ethanol
-- mix well, centrifuge at high speed, decant liquid
-- wash pellet (70% ethanol), dry pellet, dissolve in appropriate volume (then
determine DNA concentration)
DNA purification: overview
cell harvest and
lysis
cell growth
DNA concentration
DNA purification
Inorganic extraction
Salt precipitation
adsorption to silica surfaces, and
anion - exchange chromatography.
Adsorption Methods
nucleic acids selectively absorb to silica or
resins in the presence of certain chaotropic
agents or salts
applications:
plasmid preps
fragments after
electrophoresis
PCR templates
42
Plasmid Miniprep Protocol
1. Solubilize bacteria in alkali solution
2. Neutralize with Na-acetate
3. Centrifuge, discard pellet
4. Mix supernatant with resin +
chaotropic agent
5. Wash resin
6. Elute DNA with low salt buffer
DNA purification: silica binding
Binding occurs in presence of high salt
concentration, and is disrupted by elution with
water
DNA Purification Method Comparison
Liquid Phase
Solid Phase
(Lyse RBCs)
(Pre-lyse cells)
Lyse cells
Apply sample
(Protein digestion-ProK)
Wash
Separate proteins from DNA
Elute DNA
Precipitate DNA-alcohol
Rehydrate DNA
Isolation of RNA
Special Considerations
RNAse inhibitors!
extraction in guanidine salts
phenol extractions at pH 5-6
(pH 8 for DNA)
treatment with RNase-free DNase
selective precipitation of high MW
forms (rRNA, mRNA) with LiCl
oligo-dT column
45
RNA Isolation Methods
Cesium Chloride Gradient
Used mainly to get clean RNA for Northern blots
Homogenize cells in guanidinium isothiocyanate and
b-mercaptoethanol solution.
Add to CsCl gradient and centrifuge for 1220 hours;
RNA will be at the bottom of tube.
Re-dissolve in TE/SDS buffer.
Precipitate RNA with salt and ethanol, then rehydrate.
Advantage: high quality
Disadvantages: extremely time-consuming,
hazardous materials disposal issues
Density Gradient Centrifugation
rate zonal/sucrose (size fractionation)
electrophoresis more common
isopycnic/CsCl (density)
DNA ~1.7 g/cm3
protein ~1.3 g/cm3
RNA > DNA
ssDNA > dsDNA
GC content
1.74
density (g/cm3)
1.72
1.70
1.68
20
47
40
60
% GC base pairs
80
Centrifuge rotors
axis of rotation
Swinging-bucket
At rest
Spinning
Fixed-angle
Differential centrifugation of a
tissue homogenate (I)
Decant supernatant
1000g/10 min
etc.
3000g/10 min
Density gradient centrifugation
Density Barrier
Discontinuous
Continuous
How does a gradient separate
different particles?
Least dense
Most dense
Buoyant
density
banding
Equilibrium
density
banding
Isopycnic
banding
Resolution of density gradients
Density Barrier
I
Discontinuous
II
Continuous
RNA extraction
RNA extraction demands extra care. Most
forms of RNA are labile.
Contaminant RNase: pretreatment with
DEPC (diethylpyrocarbonate) a strong
RNase inhibitor.
Glassware can be baked at 150oC for 4
hours
Plastic materials can be soaked in 0.5 M
NaOH for 10 min.
Autoclave treatment for glassware and
plastic materials.
The problem(s) with RNA:
RNA is chemically unstable -- spontaneous cleavage of
phosphodiester backbone via intramolecular
transesterification
RNA is susceptible to nearly ubiquitous RNA-degrading
enzymes (RNases)
RNases are released upon cell lysis
RNases are present on the skin
RNases are very difficult to inactivate
-- disulfide bridges conferring stability
-- no requirement for divalent cations for
activity
Top 10 sources of RNAse contamination
(Ambion Scientific website)
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
Ungloved hands
Tips and tubes
Water and buffers
Lab surfaces
Endogenous cellular RNAses
RNA samples
Plasmid preps
RNA storage (slow action of small amounts of RNAse
Chemical nucleases (Mg++, Ca++ at 80C for 5 +)
Enzyme preparations
DEPC: diethylpyrocarbonate
RNA Isolation Methods
Guanidinium-based Organic Isolation
Phenol/guanidinium solution disrupts cells,
solubilizes cell components, but maintains
integrity of RNA.
Add chloroform, mix, and centrifuge.
Proteins/DNA remain at interface.
RNA is removed with aqueous top layer.
RNA is precipitated with alcohol and
rehydrated.
Advantage: faster than CsCl method
Disadvantages: fume hood required,
hazardous waste disposal issues
RNA Isolation Methods
Nonorganic Salt Precipitation
Cell membranes are lysed and proteins are
denatured by detergent (such as SDS) in the
presence of EDTA or other RNase inhibitors.
Proteins/DNA are precipitated with a high
concentration salt solution.
RNA is precipitated with alcohol and
rehydrated.
Advantages:
Fast and easy, nontoxic
Produces high quality RNA
3. Assessment of quality and quantity
Maximal absorption of nucleic acid is at wavelength
269 nm.
Proteins absorb well at 280 nm.
OD260 of 1.0 corresponds to approx 50 g/ml of
double-stranded DNA or 40 g/ml for single-stranded
DNA or RNA.
OD 260/280 ratio provides an estimate of nucleic acid
purity, with a pure preparation having a ratio between 1.8
and 2.0.
Dyes that bind nucleic acid are acridine orange,
daminoibenzoic acid (DABA), propidium iodide, and
ethydium bromide.
Double-stranded and single-stranded DNA differ in their
optical absorption at 260 nm
dA
dG
dU
dC
The conjugated p-electron systems of
the purine & pyrimidine bases absorb
strongly in the UV.
nucleotides
ssDNA
dsDNA
The absorbance of double-stranded
DNA (dsDNA) at 260 nm is less than
that of either single-stranded DNA
(ssDNA) or the free bases. This is
called hypochromism.
Using Spectroscopy to analyze DNA
Optical Density
DNA absorbs UV light with a major peak at 260 nm
This absorption is useful because it
varies with the structure of DNA
(&RNA)
i.e. extinction coefficient depends on
the structure
Wave Length
62
[Link] Abdi
dsDNA
ssDNA
Low extinction
coefficient
Higher extinction
coefficient
Resuspending Final Nucleic Acid
Samples
Have some idea of expected nucleic acid yield.
Choose diluent volume according to desired
concentration.
Calculating Expected DNA Yield
Example:
1 X 107 cells X 6 pg DNA/cell X 80% yield= 48 mg DNA
Resuspend DNA in TE buffer or ultra pure
DNAse-free water.
Resuspend RNA in ultra pure RNase-free water.
Quantity from UV Spectrophotometry
DNA and RNA absorb maximally at
260 nm.
Proteins absorb at 280 nm.
Background scatter absorbs at 320
nm.
Quantity from UV Spectrophotometry
[DNA] =
(A260 A320) X dilution factor X 50 g/mL
[RNA] =
(A260 A320) X dilution factor X 40 g/mL
Concentration = g of DNA or RNA per mL of
hydrating solution
Evaluation of Nucleic Acids
spectrophotometrically
quantity
quality
fluorescent dyes
gel electrophoresis
A260
DNA
A260/A280
A260
RNA
A260/A280
66
[Link] Abdi
1.0 50 g/ml
1.6 - 1.8
1.0 40 g/ml
~2.0
Quantity from UV Spectrophotometry
Calculating Yield
Multiply the concentration of the
DNA or RNA sample by the
volume of hydrating solution added.
Example for DNA: 150 g/mL X 0.1 mL = 15 g
Concentration from
UV Spec. (g DNA
per ml of hydrating
solution)
Volume of
hydration
solution
DNA yield
Quality from UV Spectrophotometry
A260/A280 = measure of purity
(A260 A320)/(A280 A320)
1.7 2.0 = good DNA or RNA
<1.7 = too much protein or
other contaminant (?)
4. Nucleic acid storage
To prevent enzymatic or physical damage to the
purified product.
Chelating agents, chaotrophic agents,
refrigeration.
DNA can be stored for long periods in a
TRIS=EDTA buffer at 4oC.
RNA, more labile, should be stored at -80oC in
similar buffer.
DNA and RNA can be stored as an ethanol
precipitate, with -20oC being the optimal storage
temperature.
Nucleic Acid Storage Requirements:
Storage of DNA Specimens
<4 Months 13 Years
225 C
28 C
Not recommended
<7 Years
20 C
>7 Years
70 C
Recommended
for long-term
storage in ethanol
5. Electrophoresis
DNA can be separated based on
size and charge
The phosphate groups are
negatively charged
DNA is placed in a gel and
electricity is run through
71
Separates DNA (or RNA or Protein) fragments on
the basis of charge and size
Because DNA is an acid, it looses protons in basic
buffers; thus it has a negative charge that is uniform
per unit length
Agarose (a polysaccharide) or other gel matrices
are difficult for large DNA fragments to move
through
The larger the fragment, the more difficulty it has
moving through gels
By placing DNA in a gel, then applying a voltage
across the gel, the negatively charged DNA will
move toward the anode (positive electrode)
Large fragments lag behind while small fragments
move through the gel relatively rapidly
Agarose
Agar consists of a mixture of
agarose and agaropectin.
The predominant component
agarose is a linear polymer,
made up of the repeating
monomeric unit of
agarobiose.
Agarobiose is a disaccharide
made up of D-galactose and
3,6-anhydro-Lgalactopyranose.
Agaropectin is a
heterogeneous mixture of
smaller acidic molecules that
gel poorly.
Electrophoresis
Negative DNA moves toward the
positive end
Smaller fragments move farther and
faster
74
Electrophoresis
75
Gel Electrophoresis sorts DNA molecules
by size
Separation technique: separates DNA by size and charge
[Link] enzymes
cut DNA I into fragments
2. The gel
Wells made at one end. Small amounts of DNA are placed in the wells
3. The electrical field
gel placed in solution and an electrical filed is set up with one neg. (-) &
one pos. (+) end
4. The fragments move
negatively charged DNA fragments travel toward positive end. The
smaller fragments move faster.
Mixture of DNA
molecules of
different sizes
Longer
molecules
Power
source
Gel
Shorter
molecules
What is the electrical charge of
DNA?
Negative, so DNA pieces
migrate toward the positive
pole
Smaller fragments move
faster and travel farther
than larger fragments.
Fragments of different sizes
appear as bands on the gel
Agarose Gel
Stained with ethidium bromide (EtBR) to Visualize the DNA
slots where
DNA is loaded
1000 bp
700 bp
600 bp
500 bp
Screening PCR
products to test
for the presence
of specific DNA
sequences
78
molecular
weight
[Link] markers
Abdi
correct
PCR
product
molecular
weight
markers
Gel Electrophoresis
-
Wells
Large
Direction
of
DNA
Travel
Small
Figure 5.1b
DNA Size from Agarose Gel Electrophoresis:
Compares unknown DNA to known size standards
Lambda DNA
1 kb ladder
Lambda DNA cut
with Hind III 12,218 bp
23,130 bp
100 bp ladder
6,018 bp
9,416 bp
3,054 bp
1,500 bp
1,000 bp
6,557 bp
2,036 bp
600 bp
4,361 bp
1,636 bp
48,500 bp
(48.5 kb)
300 bp
1,018 bp
100 bp
2,322 bp
2,027 bp
517 bp
DNA Quality from Agarose Gel
Electrophoresis
Human Whole Blood DNA
Lambda DNA
marker
Whole blood genomic DNA
Lambda DNA cut with
Hind III marker
Text Art Page 91
The electrophoretic mobility of a DNA
fragment is inversely proportional to
the log of its size.
60
50
40
30
20
10
Figure 5.2b
70
3 mm
Agarose gel electrophoresis
Agarose (%)
Standard
NuSieve
0.5
700 bp-25 Kbp
0.8
500 bp-15 Kbp 800 bp-10 Kbp
1.0
250 bp-12 Kbp
400 bp-8 Kbp
1.2
150 bp-6 Kbp
300 bp-7 Kbp
1.5
80 bp-4 Kbp
200 bp-4 Kbp
2.0
100 bp-3 Kbp
3.0
50 bp-1 Kbp
NuSieve 3:1
500 bp-1 Kbp
4.0
100 bp-500 bp
6.0
10 bp-100 bp
Pulsed Field Gel Electrophoresis
agarose gel electrophoresis is a fundamental technique in molecular
biology but is generally unable to resolve fragments greater than 20
kilobases in size (whole microbial genomes are usually greater than
1000 kilobases in size)
PFGE (pulsed field gel electrophoresis) is a adaptation of
conventional agarose gel electrophoresis that allows extremely
large DNA fragments to be resolved (up to megabase size
fragments)
essential technique for estimating the sizes of whole
genomes/chromosomes prior to sequencing and is necessary for
preparing large DNA fragments for large insert DNA cloning and
analysis of subsequent clones
also a commonly used and extremely powerful tool for genotyping
and epidemiology studies for pathogenic microorganisms
Principle of PFGE
two factors influence DNA migration rates through conventional gels
- charge differences between DNA fragments
- molecular sieve effect of DNA pores
DNA fragments normally travel through agarose pores as spherical
coils, fragments greater than 20 kb in size form extended coils and
therefore are not subjected to the molecular sieve effect
the charge effect is countered by the proportionally increased
friction applied to the molecules and therefore fragments greater
than 20 kb do not resolve
PFGE works by periodically altering the electric field orientation
the large extended coil DNA fragments are forced to change
orientation and size dependent separation is re-established
because the time taken for the DNA to reorient is size dependent
Principle of PFGE
the most important factor in PFGE resolution is switching time,
longer switching times generally lead to increased size of DNA
fragments which can be resolved
switching times are optimised for the expected size of the DNA
being run on the PFGE gel
switch time ramping increases the region of the gel in which DNA
separation is linear with respect to size
a number of different apparatus have been developed in order to
generate this switching in electric fields however most commonly
used in modern laboratories are FIGE (Field Inversion Gel
Electrophoresis) and CHEF (Contour-Clamped Homogenous
Electrophoresis)
CHEF
Switch Time
Electric Field 1
Electric Field 2
+
+
+
+
But, what if you want to separate larger fragments, such as entire yeast
chromosomes?
Pulsed-field gel electrophoresis (PFGE) can resolve fragments from 200 Kpb
(0.2 Mbp) to 6000 Kbp (6 Mbp).
91
[Link] Abdi
Isolation of Nucleic Acids
Goals:
Types of Methods:
removal of proteins
DNA vs RNA
isolation of a specific type of
DNA (or RNA)
differential solubility
adsorption methods
density gradient
centrifugation
Types of DNA:
92
genomic (chromosomal)
organellar (satellite)
plasmid (extra-chromosomal)
phage/viral (ds or ss)
complementary (mRNA)
[Link] Abdi
General Features:
denaturing cell lysis (SDS, alkali,
boiling, chaotropic)
enzyme treatments
- protease
- RNase (DNase-free)
- DNase (RNase-free)
Downstream Applications
After DNA is extracted, it is used as a
template in further molecular techniques such
as
PCR (polymerase chain reaction)
RFLP (restriction fragment length polymorphism)
Southern Blotting
What do we need DNA for?
Detect, enumerate, clone genes
Detect, enumerate species
Detect/sequence specific DNA regions
Create new DNA constructs (recombinant DNA)
DEPC: diethylpyrocarbonate
What about RNA?
Which genes are being transcribed?
When/where are genes being transcribed?
What is the level of transcription?