MLS 312 – MOLECULAR BIOLOGY AND DIAGNOSTICS

DNA ISOLATION AND PURIFICATION

2nd SEMESTER | S.Y. 2022-2023 TRANSCRIBED BY: CAILENE S. INFANTE
LECTURER: SIR ROSTHON GARDOCE
TOPIC OUTLINE ➔ DNA was first isolated in 1869 by chemist
FRIEDERICH MIESCHER from eukaryotic nuclei
I. DNA Isolation and Purification ➔ Routinely done for various molecular methods
a. DNA Isolation or Extraction including clinical diagnostic applications such as:
b. Lysis of Cells ➢ Pathogen testing
c. Separation of DNA From Other Cell Components ➢ Mutation identification
II. Isolation of the DNA ➢ Whole-genome sequencing.
a. Liquid-Liquid Extraction
b. Solid-Phase Extraction
III. DNA Extraction from Different Sources
IV. DNA Extraction from Whole Blood
a. Ficoll-Directed Density Gradient Through
Centrifugation
b. Quick Extraction Through Proteinase K And Phenol
c. Phenol-Chloroform Extraction
V. DNA Extraction from Dry Blood Spots
VI. Noninvasive Human DNA Isolation
VII. DNA Preparation from Microorganisms
Figure 1 Friedrich Miescher
a. Chemical Method
b. Chelex-100 Method ➔ In a typical cell, the DNA is tightly packed into
VIII. DNA Purification chromosomes by coiling around histone proteins
a. Organic Purification forming NUCLEOSOMES
b. Inorganic Purification ➔ DNA is also NEGATIVELY CHARGED as opposed to
c. Salting Out Procedure the POSITIVE CHARGE of HISTONES and is stabilized
d. Solid Phase Nucleic Acid Extraction by MAGNESIUM which also plays a part in the
IX. DNA Preparation From Formalin Fixed, Paraffin function of enzymes that cut up DNA
Embedded (FFPE) Tissues
a. Automated DNA Isolations
b. Storage Of Extracted DNA
X. Storage Of Extracted DNA
XI. Agarose Gel Electrophoresis
XII. Fluorometry

DNA ISOLATION AND PURIFICATION

LECTURE OUTCOMES

➔ Learn the importance of DNA isolation/extraction

➔ Understand the basis of DNA isolation/extraction
➔ Comprehend the different steps involved in DNA
isolation/extraction
➔ Learn the different techniques used in DNA
isolation/extraction and purification

DNA ISOLATION / EXTRACTION

➔ One of the most critical part in molecular techniques Figure 2 National Human Genome Research Institute Genetics
Glossary
because downstream procedures and analysis relies
on the quality of the DNA used for the assay
➔ DNA ISOLATION requires the removal various
➔ Refers to the process of separating DNA from other
cellular materials to free up the DNA material inside
cellular materials such as proteins and membranes.
the cell.
➔ SOURCES OF DNA
➔ DNA needs to be separated from other cellular
➢ Plasmid DNA circular
materials because these inhibit DNA analysis
➢ Viral Nucleic Acids
➔ The process of DNA isolation removes potential
➢ Genomic DNA from Blood and Biological Fluids
inhibitors to the polymerase chain reaction (PCR)
➢ Genomic DNA from Tissue and Cells
amplification and produces a stable solution of high-
➢ Genomic DNA from Forensic Samples
quality DNA that can be stored for prolonged
➢ Genomic DNA from Plant and Fungi
durations without degrading
➢ Genomic DNA from Food and Feed
➢ Ancient DNA fossils

Infante, Cailene S. [BS MLS 3-E] 1

➔ PRINCIPLES OF DNA ISOLATION
gel matrix 3x
➢ Break the cells open, commonly referred to as
cell disruption or cell lysis, to expose the DNA
within.
➢ Remove membrane lipids by adding a
DETERGENT.
➢ Remove proteins by adding a PROTEASE
◼ Optional but almost always done
➢ Precipitate the DNA with an alcohol
◼ Usually ice-cold ethanol or isopropanol.
Figure 6 DNA Isolation / Extraction. Typical workflow for the
➢ Resolubilize DNA in a slightly alkaline buffer or in isolation of DNA in a blood sample using affinity
ultra-pure water (optional). cryopreservation chromatography.
◼ Adding a chelating agent to bind divalent
cations (Mg2+ and Ca2 +) to stop DNase LYSIS OF CELLS
activities.
◼ Cellular and histone proteins bound to the ➔ DNA must be released from cells, nuclei or
DNA can be removed by adding a protease organisms as the first step of the DNA isolation
or by having precipitated the proteins with process.
sodium /ammonium acetate, or extracted ➔ Lysis can be a single step or involve multiple steps.
them with a phenol-chloroform mixture ➔ When using a blood sample, for example, red blood
prior to the DNA-precipitation. cells need to be lysed first, yielding white blood cells
➔ THE EXTRACTION OF DNA GENERAL FOLLOWS which contain the DNA.
THREE BASIC STEPS: ➔ The WBCs are then lysed to free the DNA from the
➢ Lysis of cells to free the DNA material, cells
➢ Separation of DNA from other cell components TECHNIQUES THAT CAN BE EMPLOYED TO LYSE
➢ Isolation of the DNA. THE CELLS INCLUDE
➔ CHEMICAL LYSIS
➢ The simplest and cheapest method that can be
employed.
➢ THESE INCLUDE:
◼ Chaotropic Agents
◼ Detergents
◼ Salts
◼ Strong Bases.
➔ ENZYMES
➢ Enzymes such as PROTEINASE K and LYSOZYME
can target proteins in the cell or organism to
Figure 3 Three basic steps of DNA extraction induce lysis.
➔ EXTERNAL PHYSICAL FORCES
➢ Can also be used to lyse cells such as through:
◼ Milling
• Particularly bead milling
◼ Sonication
• Using high-intensity sound waves
◼ Boiling
◼ Freeze-thaw cycling.
➔ It is important to select the most optimal method of
lysis depending on the complexity of the cells to be
lysed
➔ Most human cells and pathogens are susceptible to
simple chemical lysis but some samples are more
resistant to lysis such as bacterial spores, fungal
Figure 4 DNA Extraction
cells, and protozoan oocysts

SEPARATION OF DNA FROM OTHER CELL

COMPONENTS

➔ The isolation of DNA is prone to protein

contamination due to the presence of histones and
other accessory proteins
➔ Because the presence of proteins is detrimental to
nucleic acid preparation as well as to downstream
analysis, it is imperative to either denature or
remove proteins during the extraction of DNA
➔ For example, nucleases can break down nuclei acids,
proteases can act as an inhibitor to enzymatic
procedures while large amounts of proteins can
affect the affinity of nucleic acids in some testing
Figure 5 Basic Steps in Isolating DNA procedures

Infante, Cailene S. [BS MLS 3-E] 2

PROTEIN REMOVAL ➔ Other chemicals employed as precipitating agents
include:
➔ DETERGENTS such as SODIUM DODECYL SULFATE ➢ Acetone
(SDS) and TRITON X-100 can be used to remove ➢ Lithium chloride.
proteins from DNA through solubilization. ➔ After precipitation, the sample is centrifuged to
➔ CHEMICALS such as CHAOTROPIC ACIDS can be concentrate and separate the DNA into a pellet and
used to denature proteins. lyse hydrogen bonds is then dried and resuspended either in a buffer or
➢ They can also be employed to lyse bacteria and ultrapure water.
yeasts.
➢ CHAOTROPIC ACIDS INCLUDES: SOLID-PHASE EXTRACTION
◼ Guanidine Hydrochloride
◼ Guanidinium Thiocyanate ➔ Involves DNA separation either by size or affinity
➔ Unwanted proteins can also be removed by ➔ It is commonly employed because:
PROTEASE DIGESTION. ➢ It is less hazardous
➢ A common protease used is the serine protease ➢ Easier to perform
PROTEINASE K originally extracted from the ➢ Adaptable for automation
fungus Engyodontium album. ➢ Suitable for high-volume sample preparation
◼ It breaks down proteins into smaller ➔ THE BASIC STEPS IN A SOLID PHASE EXTRACTION
molecules by cleaving peptide bonds and is ARE:
thus useful in reducing the protein ➢ Lysis
background as well as in the lysis of cells ➢ Binding
➢ Washing
ISOLATION OF THE DNA ➢ Eluting
➔ TECHNIQUES
➔ After lysis, the DNA needs to be isolated from other ➢ Gel filtration
sample or cellular materials ➢ Ion-exchange chromatography
➔ There are basically two separation methods that can ➢ Affinity chromatography
be employed:
➢ Liquid-liquid extraction
➢ Solid-phase extraction

LIQUID-LIQUID EXTRACTION

➔ A method which involves liquid phase separation

and precipitation
➔ The use of liquid phase extraction is based on the
differential solubility of DNA from other molecules in
immiscible liquids to remove
residual
➔ A primary organic solvent employed for this contaminats
Figure 8 Solid-phase extraction
technique is phenol usually mixed with
CHLOROFORM and ISOAMYL ALCOHOL. GEL FILTRATION

➔ Separates DNA by size exclusion

➔ The gel matrix in a spin column allows larger
molecules to pass while retaining smaller molecules
➔ A common gel matrix used is SEPHADEX

Figure 7 Conventional Chemical Liquid/Liquid Extraction

➔ The addition of chloroform or isoamyl alcohol aids in

the partitioning of the different phases and prevents
foaming
➔ DNA is then precipitated from the aqueous phase
using alcohol
➔ Although an effective method for DNA separation, it
is manually laborious and poses risks due to the
hazards posed by the chemicals used and the
wastes generated by the procedure
➔ Precipitation of DNA is a liquid phase method that
yields a relatively pure product and concentrates the
DNA ice cold
➔ Alcohols such as ETHANOL and ISOPROPANOL are
Figure 9
usually used to precipitate DNA
➔ A high concentration of salt (0.1-0.5 M) can also be
used to precipitate DNA such as:
➢ Sodium Chloride
➢ Ammonium Chloride
➢ Ammonium Acetate

Infante, Cailene S. [BS MLS 3-E] 3

ION-EXCHANGE CHROMATOGRAPHY SILICA-GEL-MEMBRANE TECHNOLOGY
PROCEDURE:
➔ Based on the selective binding of negatively charged
➔ Is based on a simple BIND-WASH-ELUTE procedure
DNA to surfaces with charged groups
➔ Nucleic acids are adsorbed to the silica-gel
➔ Charged DNA exchange places with the ions and
membrane in the presence of chaotropic salts,
bind to the surface by charge and unbound
which remove water from hydrated molecules in
substances are washed away
solution
➔ DNA is released by displacing it with a flood of free
➔ Polysaccharides and proteins do not adsorb and are
ions that replace the DNA molecules
removed
➔ A common anion exchange resin used for this
➔ After a wash step, pure nucleic acids are eluted
technique is DIETHYLAMINOETHYL CELLULOSE
under low- or no-salt conditions in small volumes,
(DEAE-C)
ready for immediate use without further
concentration.
ADVANTAGES:
➔ Fast, convenient, and economical
➔ No time-consuming phenol-chloroform extractions
(toxic reagents), or alcohol precipitations.

DNA EXTRACTION FROM DIFFERENT

SOURCES

➔ The simplest cells, such as BACTERIA, are

PROKARYOTES
➔ These cells are composed of a lipid bilayer outer
membrane and a cytoplasm containing a:
➢ Circular chromosome
Figure 10 Ion Change ➢ Proteins
➢ Inorganic salts
AFFINITY CHROMATOGRAPHY ➢ Metal ions
➢ Carbohydrates
➔ Uses reversible adsorption of DNA to surfaces such ➢ Other cellular components
as SILICA and is the most common method for DNA ➔ Lysis of prokaryotic cells releases chromosomal
extraction/isolation material where DNA can be extracted
➔ It is used for many DNA preparation procedures and ➔ Differences in the structure of the cell wall of
commonly used in automated methods Gram-positive and Gram-negative bacteria plays a
➔ DNA binds to silica surfaces when specific binding role in the optimal selection of extraction/isolation
conditions are met, especially upon the addition of methods to be used
chaotropic salts ➔ The isolation of genomic DNA from bacteria is
➔ Linear DNA adsorbs lengthwise to silica surfaces traditionally achieved using organic extraction of the
because of complex hydrogen bond formation soluble DNA while the insoluble cell debris is
between the silica and DNA surfaces in the presence removed
of chaotropic salts or alcohols at high concentration ➔ The DNA is then purified from soluble proteins and
and low pH (below pH 7) resulting to binding RNA by ETHANOL PRECIPITATION
➔ Silica and DNA surfaces are both negatively charged, ➔ Commercial kit-based extraction methods are
thus, the binding is due to adsorption in high ionic currently available for the convenient and rapid
strength conditions and hydrogen bonding when extraction of genomic DNA from bacteria.
water is removed from the surfaces ➔ Eukaryotic cells such as those found in humans,
➔ The DNA is released when the salt or alcohol is animals and plants are also composed of a lipid
removed and the surfaces are hydrated bilayer outer membrane and a cytoplasm containing
various proteins, carbohydrates, lipids and other
inorganic materials
➔ Differences in cell structure often presents difficulties
in extracting the DNA
➔ The cell walls of plants for example complicate the
DNA extraction process by presenting a barrier
which makes it more difficult to lyse the cells
➔ Many plant species have a high content of
polysaccharides and polyphenols which are not
removed by phenol extraction.
➔ It is much easier to lyse and extract genomic DNA
from human and animal cells because of the
absence of cell walls and chloroplasts
➔ Most animal cells do not have a cell wall like
Figure 11 Column DNA Extraction
microbial cells, and consequently, are easier to lyse
and can be lysed using only detergents
➔ Another major difference from prokaryotes lies
largely on the presence of membrane-enclosed
organelles in the cytoplasm

Infante, Cailene S. [BS MLS 3-E] 4

➔ The nucleus of a typical eukaryotic cell in a human ➔ Viral DNA can be extracted from the tissue cultures
being is an organelle with 23 pairs of chromosomes used for propagation
while the mitochondria contain a circular DNA ➔ DNA can be extracted from virus particles by lysis of
chromosome, all of which direct the production of the virions and purification of the genome using
proteins typical techniques similar to other samples
➔ The mitochondrial proteins are used as some of the ➔ In cases where the virus is very cell associated, such
metabolic machinery for digestion of carbohydrates as varicella zoster, or some strains of human herpes
and lipids and production of most of the energy for virus, it may be necessary to extract the DNA from
the cell the cells
➔ Other organelles are involved in the synthesis and ➔ For viruses which cannot be propagated in cell
modification of proteins, carbohydrates, and lipids, culture, or for viruses with low molecular weight DNA,
and other molecules used by the cell or in its their genomes can be extracted directly from cells
signaling activities avoiding contamination from high molecular weight
DNA such as chromosomal DNA
➔ PLASMID DNA is used for a number of downstream
procedures such as:
➢ Transfection
➢ Sequencing
➢ Screening clones
➢ Restriction digestion
➢ Cloning
➢ PCR
➔ Plasmids are typically designed to contain an
antibiotic resistant gene, allowing selection of
bacteria containing the plasmids during growth of
colonies or cultures
➔ A number of methods have been developed for the
➔ The genes that code for heritable traits such as purification of plasmid DNA from bacteria
height, blood type, hair color, eye color, skin color, ➔ Extraction of plasmids is typically performed from
and temperament are found on chromosomes in the bacterial liquid cultures, and there are many
nucleus of the eukaryotic cell methods available for plasmid DNA isolation that
➔ Although the basic principles and steps in the DNA are capable of isolating large amounts of high-
extraction in eukaryotic cells are essentially the quality DNA
same to those of prokaryotic cells, there are slight ➔ The most common method used for plasmids is
modifications owing to the differences in cellular based on the ALKALINE LYSIS METHOD
structure ➔ PRINCIPLE of this procedure is to take advantage of
➔ For example, the lysis steps need to destroy both the the alkaline denaturation of plasmid and bacterial
outer membrane of the cell as well as the chromosomal DNA and the selective renaturation of
membranes enclosing the organelles particularly plasmid DNA following neutralization of the solution
that of the nucleus ➔ The size of the bacterial culture used defines the
➔ Furthermore, complex samples such as organs and plasmid preparation as miniprep, midiprep,
tissues require breakdown of complex structures maxiprep etc
through physical (grinding, freezing-thawing, etc.) or ➔ This largely affects also the plasmid DNA yield
enzymatic means prior to DNA extraction/isolation ➔ The small-scale mini preparation of plasmid DNA is
➔ Typical amounts of DNA extracted depends on the commonly used to screen bacterial clones for the
type of the sample used presence of recombinant DNA inserts
➔ For example, oral swab typically yields 100 to 1500
ng of DNA per swab while tissue samples may yield DNA EXTRACTION FROM WHOLE BLOOD
50 to 500 ng of DNA per gram of sample
➔ Ficoll-Directed Density Gradient Through
TYPICAL DNA AMOUNTS THAT MAY BE Centrifugation
EXTRACTED FROM BIOLOGICAL MATERIALS ➔ Quick Extraction Through Proteinase K and Phenol
TYPE OF SAMPLE AMOUNT OF DNA
LIQUID BLOOD 20,000 ng/mL to 40,000 ng/mL FICOLL-DIRECTED DENSITY GRADIENT
BLOOD STAIN 250 ng/cm2 to 500 ng/cm2 THROUGH CENTRIFUGATION
LIQUID SEMEN 150,000 ng/mL to 300,000 ng/mL
POST-COITAL ➔ Fresh blood is collected in the presence of
10 ng/swab to 3,000 ng/swab
VAGINAL SWAB anticoagulants such as EDTA or CITRATE
PLUCKED HAIR ➔ After centrifugation, whole blood is separated into
1 ng/root to 750 ng/root four layers, with plasma at the top followed by white
(WITH ROOT)
SHED HAIR blood cells containing peripheral blood mononuclear
1ng/root to 10 ng/root cells, a Ficoll-Hypaque medium layer, and a bottom
(WITH ROOT)
LIQUID SALIVA 1,000 ng/mL to 10,000 ng/mL layer containing erythrocytes and granulocytes
ORAL SWAB 100 ng/swab to 1,500 ng/swab ➔ This is because red blood cells (RBCs) and
URINE 1 ng/mL to 20 ng/mL granulocytes have a higher density than Ficoll and
BONE 3 ng/mg to 10 ng/mg will sediment at the bottom of the FicollHypaque
TISSUE 50 ng/mg to 500 ng/mg layer

Infante, Cailene S. [BS MLS 3-E] 5

 ➔ This method relies on phase separation by
centrifugation of a mix of the aqueous sample and a
solution containing watersaturated phenol,
chloroform and a chaotropic denaturing solution
(guanidinium thiocyanate) resulting in an upper
aqueous phase and a lower organic phase (mainly
chloroform).
➔ Nearly all of the RNA is present in the aqueous phase,
Figure 12 Ficoll-Directed Density Gradient Through while DNA and protein partition in the interphase
Centrifugation. Blood specimen via Ficoll gradient centrifugation and the organic phase, respectively.
and diagram representation of granulocyte isolation for DNA ➔ In a last step, RNA is recovered from the aqueous
extraction.
phase by precipitation with 2-propanol or ethanol.
➔ DNA will be located in the aqueous phase in the
QUICK EXTRACTION THROUGH PROTEINASE absence of guanidinium thiocyanate and thus the
K AND PHENOL technique can be used for DNA purification alone.

➔ For the proteinase K protocol, whole blood is mixed

with Tris, EDTA, sodium dodecyl sulfate (SDS), MgCl2,
and proteinase K in the presence of high salt for
overnight digestion at 37°C
➔ After digestion is complete, samples can be further
purified through a standard purification protocol as
described later in the section of DNA purification

PROTEINASE K

➔ ALSO KNOWN AS:

➢ Protease K Figure 13 Phenol-chloroform extraction
➢ Endopeptidase K
➔ Proteinase K is a BROADSPECTRUM SERINE DNA EXTRACTION FROM DRY BLOOD SPOTS
PROTEASE
➔ Proteinase K is commonly used in molecular biology
CHELEX-100
to digest protein and remove contamination from
preparations of nucleic acid. ➔ Place punches in 1 mL 0.5% saponin
➔ Addition of proteinase K to nucleic acid preparations ➔ Incubate at 4°C overnight
rapidly inactivates nucleases that might otherwise ➔ Remove saponin and add 1 mL PBS
degrade the DNA or RNA during purification. ➔ Incubate at 4°C for 30 min
➔ The enzyme is active in the presence of chemicals ➔ Remove PBS and place punches in 100
SOAKING IN
that denature proteins, such as SDS and UREA, as mL 5% Chelex- 100
SAPONIN
well as TRYPSIN or CHYMOTRYPSIN INHIBITORS. ➔ Incubate at 100°C for 8 min
➔ Proteinase K is also stable over a wide pH range of ➔ Centrifuge at 10,600 g for 2 min
➔ Carefully remove and store supernatant
4-12.
at -20°C if the extract is not used
➔ The enzyme's activity towards native proteins is
promptly
stimulated by denaturants such as SDS.
➔ Place punches in 100 uL PBS
PHENOL METHOD ➔ Incubate at 4°C overnight
➔ Centrifuge at 18,000g for 2 min
➔ Remove PBS and place punches in 100
➔ For the phenol method, whole blood is mixed with
uL PBS
Tris-HCl (pH 8.0)-saturated phenol and water
➔ Centrifuge at 18,000g for 2 min
followed by shaking for 4 h at room temperature SOAKING IN
➔ Remove PBS and place punches in 100
➔ After centrifugation, the aqueous phase is collected PBS
uL 5% Chelex-100
for further standard purification as described later in
➔ Incubate at 100°C for 8 min
the section on DNA purification
➔ Centrifuge at 10,600g for 2 min
➔ Carefully remove and store supernatant
PHENOL-CHLOROFORM EXTRACTION at -20°C if the extract is not used
promptly
➔ Is a liquid-liquid extraction technique used in ➔ Place punches in 180 uL 5% Chelex-100
molecular biology for isolating DNA, RNA and already heated to 100°C
PROTEIN ➔ Vortex 30 s
➔ PRINCIPLE: ➔ Incubate at 99°C for 10 min
➢ Equal volumes of a phenol:chloroform mixture ➔ Centrifuge at 12,000g for 1.5 min
and an aqueous sample are mixed, forming a ➔ Remove the supernatant and transfer to
biphasic mixture. NO SOAKING
a clean tube
➔ This method may take longer than a columnbased ➔ Centrifuge the supernatant at 12,000 g
system such as the silica-based purification, but has for 1.5 min
higher purity and the advantage of high recovery of ➔ Carefully remove and store supernatant
RNA at -20°C if the extract is not used
promptly

Infante, Cailene S. [BS MLS 3-E] 6

 INSTAGENE MATRIX
➔ Place punches in 100 uL PBS
SALIVA
➔ Incubate at 4°C overnight
➔ Centrifuge at 18,000 g for 2 min ➔ BUCCAL SWABS and MOUTHWASH PROTOCOLS are
➔ Remove and discard supernatant and the most commonly used protocols for buccal cell
add 100 uL PBS collection
➔ Centrifuge at 18,000 g for 2 min ➔ The buccal swab samples are first suspended in
➔ Remove and discard supernatant LYSIS BUFFER that includes Tris, EDTA, SDS, and
SOAKING IN ➔ Add 200 pL InstaGene Matrix Incubate proteinase K
PBS at 56°C for 30 min. Vortex carefully ➔ The sample is incubated 1–3 h at 56°C until the
after 15 min and after completed tissue is totally dissolved.
incubation ➔ The DNA is then extracted from the solution
➔ Boil samples at 100°C for 8 min ➔ For the mouthwash method, samples from saline
➔ Centrifuge at 15,000 g for 2 min rinses need to be processed or frozen immediately
➔ Carefully remove and store supernatant after collection
at -20°C if the extract is not used
promptly URINE
➔ Place punches in a clean tube
➔ Add 200 uL InstaGene Matrix ➔ Urine specimen is inverted or swirled in a specimen
➔ Incubate at 56°C for 30 min. Vortex cup to create a homogenous suspension of cells
carefully after 15 min and after followed by the centrifugation
completed incubation ➔ The supernatant is removed, and a dry pellet
NO SOAKING
➔ Boil samples at 100°C for 8 min containing cells is chilled at 20°C for 15min followed
➔ Centrifuge at 15,000 g for 2 min by the addition of lysis buffer that includes Tris, EDTA,
➔ Carefully remove and store supernatant SDS, and proteinase K
at -20°C if the extract is not used ➔ The sample is incubated 2 h at 56°C
promptly ➔ The DNA is then extracted from the solution

COMPARISON:
➔ Although DNA from all the samples are suitable for
PCR, the blood and hair samples provided a good-
quality DNA for restriction analysis of the PCR
product compared with the buccal swab and urine
samples

DNA PREPARATION FROM

MICROORGANISMS

CHEMICAL METHOD

➔ A combination of enzymes and chemical reagents

are commonly used
➔ Detergent cell lysis is a milder and easier alternative
to physical disruption of cell membranes, although it
is often used in conjunction with homogenization
and mechanical grinding
➔ Detergents break the lipid barrier surrounding cells
by solubilizing proteins and disrupting lipid-lipid,
protein-protein, and protein-lipid interactions
NONINVASIVE HUMAN DNA ISOLATION ➔ Detergents self-associate and bind to hydrophobic
surfaces.
HAIR ➢ They are composed of a polar hydrophilic head
group and a nonpolar hydrophobic tail and are
➔ A hair with root is incubated at 95°C for 10min in categorized by the nature of the head group as
NaOH buffer, and the supernatant is subjected to ionic (cationic or anionic), nonionic, or
DNA purification after centrifugation zwitterionic.
➔ Alternatively, a smooth chemical digestion method ➢ Their behavior depends on the properties of the
using DITHIOTHREITOL (DTT) can be employed head group and tail.
➔ DTT is a strong reducing agent with relatively high ➔ In general, nonionic and zwitterionic detergents are
salt content and also an anionic detergent milder and less denaturing than ionic detergents
➔ A hair sample can be incubated 2 h at 56°C with and are used to solubilize membrane proteins where
buffer that contains Tris-HCl, EDTA, NaCl, SDS, DTT, it is critical to maintain protein function and/or
and proteinase K, followed by gentle mixing and retain native protein-protein interactions for enzyme
incubation at 60°C for 2 h or until the hair has assays or immunoassays
dissolved completely ➔ CHAPS, a ZWITTERIONIC DETERGENT, and the
➔ The genomic DNA can then be extracted from the TRITON-X series of NONIONIC DETERGENTS are
solution commonly used for these purposes

Infante, Cailene S. [BS MLS 3-E] 7

➔ In contrast, IONIC DETERGENTS such as SDS are DNA PURIFICATION
strong solubilizing agents and tend to denature
proteins, thereby destroying protein activity and ➔ The purification of DNA from crude cell extract
function involves the removal of proteins, carbohydrates,
➔ Animal cells, bacteria, and yeast all have different lipids, and cell debris
requirements for optimal lysis because of the ➔ Various techniques have been developed, including:
presence or absence of a cell wall ➢ Organic
➔ Because of the dense and complex nature of animal ➢ Inorganic
tissues, they require both detergent and mechanical ➢ Spin column methods.
lysis
ORGANIC PURIFICATION
➔ For bacteria, lysozyme is very efficient for breaking
down the cell walls of Gram-positive bacteria ➔ For the organic method, phenol and
because their cell walls have a high portion of chloroform/isoamyl alcohol (25:24:1) are mixed with
peptidoglycan an equal volume of samples by vortexing
➔ Although Gram-negative bacteria are less ➔ Although phenol, a flammable, corrosive, and toxic
susceptible to lysozyme, the combination of carbolic acid, can denature proteins rapidly, it does
lysozyme and EDTA can effectively break the cell not completely inhibit ribonuclease (RNase) activity
walls and membranes ➔ A mixture of PHENOL:CHLOROFORM:ISOAMYL
➔ MECHANICAL DISRUPTION METHOD can be used to ALCOHOL (25:24:1) is normally used to inhibit RNase
disrupt Gram-positive bacterial cells and spores activity during the purification process
efficiently ➔ Furthermore, both proteinase K and RNase can be
➔ Fungal cells are difficult to disrupt because the cell added to the sample at this step to remove lipids
walls may form capsules or resistant spores and degrade RNA, respectively
➔ LYTICASE ➔ After centrifugation, a biphasic emulsion forms
➢ ALSO KNOWN AS: ➔ The organic hydrophobic layer of the emulsion is
◼ Zymolyase settled on the bottom and the aqueous hydrophilic
➢ Can be used to digest the cell walls of fungal layer on top
cells ➔ The UPPER aqueous hydrophilic layer contains DNA,
➔ CELLS CAN ALSO BE BROKEN BY: and the BOTTOM organic layer contains the
➢ Alkaline chemicals precipitated proteins
➢ Detergents,
➢ Xanthogenates
➔ Fungal genomic DNA can also be extracted with the
CTAB (CETYLTRIMETHYLAMMONIUM BROMIDE)
METHOD
➔ Usually, CTAB mixes with Tris, EDTA, high-salt buffer,
and the conidial suspension followed by incubation
at 65°C for 1 h.
➢ This can break fungal cell walls very efficiently
Figure 14 Phenol extraction to isolate nuclei acids free of protein
contamination
CHELEX-100 METHOD
➔ The upper aqueous layer is mixed with an equal
➔ Chelex-100, a chelating resin that has a high affinity volume of chloroform, which can remove the
for polyvalent metal ions, can also be used to isolate residual phenol from the aqueous layer
fungi DNA quickly and efficiently ➔ The upper aqueous DNA-containing layer is now
➔ It is frequently used to release DNA from cells by a pure and can be further concentrated
boiling treatment, at the same time protecting the ➔ A high concentration of salt, such as 0.3M SODIUM
DNA from the boiling effects with resin beads ACETATE in the presence of 2.5 volumes of 100%
➔ Typically, conidial suspension from samples is ethanol or 1 volume of isopropanol at 20°C or below
extracted by adding 5% Chelex-100 resin can precipitate DNA
➔ The mixture is incubated at 90°C for 30min followed ➔ DNA precipitate is collected by centrifugation, and
by centrifugation excess salt is rinsed with 70% ethanol and
➔ Samples are then incubated at 90°C again for 15min. centrifuged to discard the ethanol supernatant
Subsequently, the supernatant is collected after ➔ The DNA pellet is then dissolved with Tris-EDTA
centrifugation. solution or sterile distilled water for long-term
➔ The Chelex extraction method is quick to obtain DNA storage
from spores ➔ With some cell extracts, the protein content is so
➔ Chelex-100 efficiently makes extraction of the DNA great that a single phenol extraction is not sufficient
from spores available for direct use in molecular to completely purify the nucleic acids
analyses ➔ This problem can be solved by carrying out several
➔ The yield is about 28 ng per μL from 200μL conidial phenol extractions one after the other, but this is not
suspension which contains 1–5 x 105 spores/mL for desirable, as repeated mixing and centrifugation
Chelex extraction may break the DNA molecules into small fragments
➔ CHELEX METHOD is usually recommended for fungal ➔ The other alternative is to treat the cell extract with
genomic DNA extraction considering its simplicity a protease such as proteinase K before phenol
and cost effectiveness, especially in the routine extraction
processing of large amounts of samples. ➔ The proteinase can break the polypeptides down
into smaller peptides, which can be easily removed
by phenol

Infante, Cailene S. [BS MLS 3-E] 8

 INORGANIC PURIFICATION ➔ Column conditioning can be done by using a buffer
at a particular pH or salt concentration to convert
➔ This involves the incubation of nuclei with only the surface or functional groups on the solid into a
proteinase K at 65°C particular chemical form
➔ It has been shown that proteinase K is more active ➔ Next, the cell extract is applied to the column
on denatured protein and that after prolonged ➔ The desired nucleic acid will adsorb to the column
incubation at 65°C it can be auto-inactivated with the aid of high pH and salt concentration of the
➔ As a result, following incubation for more than 2 h binding solution
with Tris, EDTA, and proteinase K in a low-salt buffer, ➔ Other compounds, such as protein, may have strong
the extracted DNA can be used directly for specific bond with the column surface as well
diagnostic analysis without any additional ➔ These contaminants can be removed in the washing
purification step by using washing buffer containing a
➔ The yield is greater than 90% of theoretical with an competitive agent or high salt concentration
average size greater than 300 kb ➔ For the elution step, TE buffer or water is introduced
➔ Salting out is another simple inorganic DNA isolation to release the desired nucleic acid from the column,
method so that it can be collected in a purified state
➔ In this procedure, saturated NaCl is used to
precipitate protein DIATOMACEOUS EARTH EXTRACTION
➔ Next, the DNA is purified from the supernatant by
addition of 1–2 volumes of 20°C chilled absolute ➔ DIATOMACEOUS EARTH
(100%) ethanol ➢ ALSO KNOWN AS:
➔ In these approaches, pure DNA is obtained and ◼ Kieselguhr
nontoxic substances are used during sample ◼ Diatomite
processing. ➢ Has silica content as high as 94%
➔ This technique is FAST and INEXPENSIVE for use in ➔ It has been used for filtration and in
laboratory settings chromatography
➔ It is useful for the purification of plasmid and other
SALTING OUT PROCEDURE DNA by immobilizing DNA onto its particles in the
presence of a chaotropic agent
➔ Rapid, safe and inexpensive method ➔ The resulting diatomaceous earth-bound DNA can
➔ Was developed to simplify the deproteinization then be washed with an alcohol-containing buffer
procedure by S. A. Miller et al. ➔ The alcohol-containing buffer is then discarded and
➔ After red cell lysis and a subsequent Proteinase K DNA is eluted out in a low-salt buffer or in distilled
digestion the method involves a salting out of the water.
cellular proteins by dehydration and precipitation
with a saturated NaCl solution. AFFINITY EXTRACTION

SOLID PHASE NUCLEIC ACID EXTRACTION ➔ Magnetic separation is a simple and efficient
method that can also be applied in the purification
➔ A solid-phase system will absorb nucleic acid in the of nucleic acid
extraction process depending on the pH and salt ➔ Often, magnetic carriers with immobilized affinity
content of the buffer ligands or prepared from biopolymer showing
➔ The absorption process is based on the following affinity to the target nucleic acid are used for the
principles: isolation process
➢ Hydrogen-bonding interaction with a ➔ Magnetic particulate materials such as beads are
hydrophilic matrix under chaotropic conditions preferable as supports in the isolation process
➢ Ionic exchange under aqueous conditions because of their larger binding capacity
through an anion exchanger ➔ The nucleic acid binding process may be assisted by
➢ Affinity and size exclusion mechanisms the nucleic acid “wrapping around” the support
➔ Solid-phase purification is normally performed by a
spin column through centrifugation.
➢ This method can purify nucleic acid rapidly
compared to conventional methods
➔ COMMONLY USED SOLID SUPPORTS IN SOLID-
PHASE EXTRACTION:
➢ Silica matrices
➢ Glass particles
➢ Diatomaceous earth
➢ Anion-exchange carriers Figure 15 Magnetic beads positively charged and forms an ionic
bond with the negatively charged DNA backbone at low pH. The
➔ THE MAJOR KEY STEPS INVOLVED IN SOLID-PHASE
magnetic beads loose their charge and DNA binding ability at
EXTRACTION ARE: higher pH. After a standard lysis procedure, the genomic DNA
➢ Cell lysis can be isolated in a 15 minute procedure that involves binding
➢ Nucleic acid adsorption the genomic DNA to the magnetic beads in a low pH buffer,
➢ Washing immobilizing the beads with a magnet, washing and finally
➢ Elution eluting in a higher pH buffer.
➔ The INITIAL STEP in a solid-phase extraction process
is to condition the column for sample adsorption

Infante, Cailene S. [BS MLS 3-E] 9

 ANION-EXCHANGE RESIN COMPARISON OF NUCLEIC ACID PURIFICATION
TECHNOLOGIES
➔ Anion-exchange resin uses the anion-exchange ANION- SILICA-GEL- MAGNETIC-
principle EXCHANGE MEMBRANE PARTICLE
RESIN TECHNOLOGY TECHNOLOGY
➔ It is based on the INTERACTION between positively
Selective
charged diethylaminoethyl cellulose (DEAE) groups
adsorption to
on the RESIN’S SURFACE and negatively charged silica-gel Binding to
phosphates of the DNA BACKBONE Solid-phase,
SEPARATION membranes magnetic silica
➔ The anion-exchange resin consists of defined silica anion-exchange
PRINCIPLE under particles
chromatography
beads with a large pore size, a hydrophilic surface controlled
coating, and a high charge density ionic
➔ The large surface area of the resin allows dense conditions
coupling of the DEAE groups Binding: variable
Binding: high Binding: high
salt and pH
➔ The resin works over a wide range of pH conditions salt Elution: salt Elution:
Elution: variable
(pH 6– 9) and/or salt concentration (0.1–1.6M) which PROCEDURE
salt and pH
low salt low salt
can optimize the separation of DNA from RNA and Ready-to-use Ready-to-use
Alcohol
other impurities eluate eluate
precipitation
➔ Resin is a macroporous silica-based resin with a Delivers high-
high density of diethylaminoethyl (DEAE) groups . Delivers high- purity nucleic
➔ PURIFICATION Delivers purity nucleic acids for use in
➢ Based on the interaction between negatively ultrapure, acids for use most
transfection in most downstream
charged phosphates of the NUCLEIC ACID
grade DNA for downstream applications
BACKBONE and positively charged DEAE groups optimal results in applications Fast,
on the SURFACE OF THE RESIN . ADVANTAGES sensitive Fast, inexpensive
➔ The salt concentration and pH conditions of the applications inexpensive Available in
buffers used in each step control binding, wash Available in No silica- versatile
stringency, and elution of nucleic acids versatile formats slurry carry formats for all
for all scales of over, no scales of
purification alcohol purification
precipitation Easy
automation

DNA PREPARATION FROM FORMALIN FIXED,

PARAFFIN EMBEDDED (FFPE) TISSUES

INFLUENCE OF TISSUE FIXATIVES ON NUCLEIC ACID

QUALITY
RELATIVE AVERAGE
Figure 16 Anion-Exchange Resin
QUALITY FRAGMENT
FIXATIVE OF SIZE
➔ DNA PURIFICATION NUCLEIC RANGE
➢ Based on the interaction between negatively ACID (kb)
charged phosphates of the DNA BACKBONE 10% buffered neutral formalin Good 2.0-5.0
and positively charged DEAE groups on the Acetone (acetone-methylbenzoate-xylene
Good 2.0-5.0
SURFACE OF THE RESIN . technique)
➔ The salt concentration and pH conditions of the HOPE (Hepes-Glutamic acid buffer
buffers used determine whether DNA is bound or mediated Organic solvent Protection Good 2.0-5.0
eluted from the column . Effect)
➔ DNA remains tightly bound to the DEAE groups over Zamboni’s (2% Paraformaldehyde; 15% Not as
0.2-2.0
Picric acid) good
a wide range of salt concentrations .
Clarke’s (75% Ethanol; 25% Glacial acetic Not as
➔ Impurities such as RNA, protein, carbohydrates, and acid) good
0.8-1.0
small metabolites are washed from the resin with Paraformaldehyde (4% Paraformaldehyde Not as
medium -salt buffers, while genomic DNA remains 0.2-5.0
in PBS) good
bound until eluted with a high -salt buffer . Metharcan (60% Methanol; 30% Not as
0.7-1.5
➔ The separation range of resins depends on the Chloroform; 10% Glacial acetic acid) good
manufacturer but can extend from 0.1 M to 1.6 M Formalin-alcohol-acetic acid (85% Ethanol; Not as
1.0-4.0
salt . 4% Formaldehyde; 5% Glacial Acetic Acid) good
B-5 (4% formaldehyde; 6% mercuric Less
<0.1
chloride; 1.25% sodium acetate) desirable
Carnoy’s (60% ethanol; 30% chloroform; Less
0.7-1.5
10% glacial acetic acid) desirable
Zenker’s(5% mercuric chloride; 2.5%
Less
potassium dichromate;1% sodium sulfate; 0.7-1.5
desirable
5% glacial acetic acid)
Bouin’s (75% picric acid; 10% Less
<0.1
formaldehyde; 5% glacial acetic acid) desirable

Infante, Cailene S. [BS MLS 3-E] 10

➔ Routinely used methods for extracting DNA from ➔ Magnetic Glass Particles (MGP) are added and the
FFPE consist of many steps, including: DNA is bound to their surfaces.
➢ Deparaffinization in xylene ➔ Unbound substances are removed by several
➢ Washing in a descendent series of ethanols washing steps, then the purified DNA is eluted.
➢ Protein digestion ➢ Lysis/Binding Buffer is added to the sample,
➢ DNA purification resulting in complete cell lysis and release of
➔ Typically, the FFPE materials are deparaffinized by nucleic acids. Nucleases are denatured
incubating the samples with xylene followed by Proteinase K is added to the samples and
centrifugation proteins are digested.
➔ The pellets are washed with ETHANOL ➢ DNA binds to the silica surface of the added
➔ After dewaxing, the samples are subject to lysis and MGPs due to the chaotropic salt conditions,
thorough homogenization followed by incubation at isopropanol, and the high ionic strength of the
65°C for 1 h and 95°C for 15min. Lysis/Binding Buffer.
➔ Proteinase K is added for 48 h digestion at 56°C. ➢ MGPs with bound DNA are magnetically
➢ This proteinase K digestion is important to separated from the residual lysedsample.
increase the DNA yield ➢ MGPs with bound DNA are washed repeatedly
➔ After centrifugation, the supernatant can be further with Wash Buffer to remove unbound
purified through: substances like proteins (nucleases), cell
➢ Phenol/ Chloroform membranes, PCR inhibitors such as heparin or
➢ Spin Column hemoglobin, and to reduce the chaotropic salt
➢ Inorganic Methods concentration.
➔ Another method uses glycine in an alkaline ➢ Again MGPs with bound DNA acid are
environment magnetically separated from the Wash Buffer
➔ Glycine reacts with formaldehyde to form either containing residual sample debris.
NMETHYLENEGLYCINE or DIMETHYLGLYCINE and ➢ The purified DNA is eluted at 70°C from the
shifts the equilibrium toward formaldehyde, thereby MGPs in the wells of the Elution Cartridge,
depleting the store of methylene glycol, the source of whereas the MGPs are retained in the reaction
formaldehyde, in the tissue tip and d iscarded.
➔ Alkalinity of the buffer further aids in reversing the
crosslinking of DNA and proteins AUTOMATED DNA ISOLATIONS USING VACUUM
➔ The method involves heat treatment and DEVICES
proteinase K followed by purification of the nucleic
acids ➔ Uses silica-based resin and columns which get
➔ These extraction methods can yield DNA amounts of automatically loaded.
1–9μg/mg of tissue and allow stable amplification of ➔ Flow through of solutions is enhanced by a vacuum
PCR products device
➔ 96 preparations can be performed in parallel
AUTOMATED DNA ISOLATIONS

➔ ADVANTAGES:
➢ Rapid, fully automated isolation of high-quality
DNA, RNA
➢ Reliable, reproducible results
➢ High flexibility in sample type and volume as
well as elution volume and post-elution
processes
➢ Automatic pipetting of PCRs into generic
96-well PCR plates or strips, or tubes
➢ Available from different companies
◼ e.g. Beckman-Coulter
◼ PerkinElmer
◼ Roche
◼ TECAN
◼ Thermo etc.

Figure 18 Automated DNA isolations using vacuum devices

STORAGE OF EXTRACTED DNA

Figure 17 Automated DNA isolations using magnetic particles ➔ DNA can be stored for MONTHS in a REFRIGERATOR
or stored FROZEN for YEARS
➔ The samples are lysed by incubation with a special ➔ Extracted DNA is typically stored at -20°C and up to
buffer containing chaotropic salts and Proteinase K. -80°C for long-term storage, to prevent nuclease
activity

Infante, Cailene S. [BS MLS 3-E] 11

➔ Nucleases are protein enzymes found in cells that ➔ Calculate the concentration of DNA in the sample by
degrade DNA to allow the cells to recycle nucleotide the O.D. at 260 nm.
components ➢ Diluting the sample 1 ul in 100 ul
➔ NUCLEASES need magnesium to work properly so ◼ The dilution factor is 100
one of the measures to prevent them from digesting ➢ OD260 = 1.6.
DNA in blood is the use of EDTA ➢ 1 O.D. at 260 nm for double-stranded DNA = 50
➔ The EDTA chelates, or binds up, most of the free ng/ul of dsDNA.
magnesium and thus helps prevent the nucleases ➢ OD260 * 50 ng/ul * dilution factor.
from destroying the DNA in the collected blood ➢ The concentration is:
sample ◼ 1.6 * 50 ng/ul * 100 = 8000 ng/ul or 8 ug/ul.
➔ Highly concentrated DNA is usually more stable over
time. AGAROSE GEL ELECTROPHORESIS
➢ DNA can gradually hydrolyze in acidic
➔ AGAROSE GEL ELECTROPHORESIS
conditions.
➢ A method used in biochemistry and molecular
➢ As such, storage buffers are slightly basic such
biology to separate DNA, or RNA molecules by
as Tris buffer
size.
➔ This is achieved by moving negatively charged
nucleic acid molecules through an agarose matrix
with an electric field (electrophoresis).
➔ Shorter molecules move faster and farther than
longer ones

Figure 19 Storage of DNA Specimens

QUANTITY FROM UV SPECTROPHOTOMETRY form bands

➔ DNA and RNA absorb maximally at 260 nm

➔ Proteins absorb at 280 nm.
➔ Background scatter absorbs at 320 nm.
➔ [DNA] Figure 20 Agarose Gel Electrophoresis
➢ (A260 – A320) x dilution factor x 50 µg/Ml
➔ [RNA] APPLICATIONS
➢ (A260 – A320) x dilution factor x 40 µg/mL
➔ CONCENTRATION = µg of DNA or RNA per mL of ➔ Estimation of the size of DNA molecules following
hydrating solution restriction enzyme digestion
➢ e.g. in restriction mapping of cloned DNA.
A260 ➔ Analysis of PCR products
MEASURE OF PURITY = ➢ e.g. in molecular genetic diagnosis or genetic
A280
A260 – A320 fingerprinting
A280 - A320 ➔ Separation of restricted genomic DNA prior to
Southern transfer, or of RNA prior to Northern
➔ 1.8 – 2.0 transfer, or of a plasmid backbone in cloning.
➢ Good DNA or RNA FACTORS AFFECTING MIGRATION
➔ <1.8 ➔ AGAROSE CONCENTRATION
➢ Too much protein or other contaminant ➢ By using gels with different concentrations of
agarose, one can resolve different sizes of DNA
260 nm 260 / 280 fragments.
0.151 1.987 ➢ Higher concentrations of agarose facilitate
0.113 2.093 separation of small DNAs, while low agarose
0.338 0.498 concentrations allow resolution of larger DNAs

USING SPECTROPHOTOMETER TO QUANTITATE

DNA AND RNA

➔ Take readings at wavelengths of 260 nm and 280

nm.
➔ Pure preparation of DNA has OD260/OD280 values
of 1.8 to 2.0.
➢ A ratio of <1.8
Figure 21 The image shows migration of a set of DNA fragments
◼ There may be proteins. in three concentrations of agarose, all of which were in the same
➢ A ratio of >2.0 gel tray and electrophoresed at the same voltage and for
◼ Samples may be contaminated with identical times. Notice how the larger fragments are much
chloroform or phenol. better resolved in the 0.7% gel, while the small fragments
separated best in the 1.5% agarose, the 1000 bp fragment is
indicated in each lane.

Infante, Cailene S. [BS MLS 3-E] 12

AGAROSE GEL ELECTROPHORESIS ➔ Do not use glass (spectrophotmetry) cuvettes in a
VISUALIZATION fluorometer because the frosted glass on the side of
the cuvette interferes with detection of fluorescent
➔ Formerly, most common dye for agarose gel light.
electrophoresis is ETHIDIUM BROMIDE (EtBr).
➢ It fluoresces under UV light when intercalated
into DNA (or RNA).
➢ By running DNA through an EtBr-treated gel and
visualizing it with UV light, any band containing
more than ~20 ng DNA becomes distinctly
visible.
➢ EtBr is a known mutagen!!
➔ SYBR Green is another dsDNA stain
➢ More expensive, but 25 times more sensitive

Figure 24

Figure 22

DNA DEGRADATION IN AGAROSE GEL

ELECTROPHORESIS

Figure 23 Agarose gel analysis (0.8% agarose gel, 1x TBE buffer,

90 V, 75 minutes) of approximately 500 ng DNA from 24
samples. DNA was eluted in Buffer AE or water, and stored for 8
years at 2– 8ºC or –20ºC. M: marker (λ HindIII- taken from
Qiagen.de)

FLUOROMETRY

➔ Fluorometry utilizes fluorescent dyes which

specifically bind DNA or RNA.
➔ It requires a NEGATIVE CONTROL (to set the zero
point on the fluorometer) and a STANDARD of known
concentration.
➔ The fluorometer shines light on the sample
(excitation) and then measures level of fluorescent
light being emitted to the side (at a 90º angle) of the
excitation light beam.
➔ The fluorescent dyes are relatively specific to nucleic
acids as opposed to protein and other cellular
components.
➔ The fluorescence of the dyes increases when they
bind nucleic acids.
➔ Fluorometry is about 1,000x more sensitive than
spectrophotometric absorbance (i.e. measurement
of A260) and less susceptible to protein and RNA
contamination.
➔ However it also does not give a crude measurement
of purity (like an A260/A280 ratio) nor does it assure
that the DNA or RNA is not degraded (e.g. like size
determination by gel electrophoesis).

Infante, Cailene S. [BS MLS 3-E] 13