Lecture 1.

Nucleic acid extraction

(DNA and RNA)
By K.H.Timotius
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

Dr.Saba 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

Dr.Saba 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

Dr.Saba 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
1.Restriction 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
Dr.Saba 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

Dr.Saba 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)

Dr.Saba 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?