AGB 254:

METHODS IN MOLECULAR BIOLOGY

S. AMOAH (P h.D)
DEPARTMENT OF CROP AND SOIL SCIENCES, FACULTY
OF AGRIC , KNUST.
COURSE OUTLINE
1. General aspects of DNA isolation and
purification
2. Isolation of genomic DNA from plants
3. Isolation and purification of RNA
4. Agarose gel electrophoresis
5. PCR analysis
6. DNA cloning
7. DNA sequencing
 GENERAL ASPECTS OF DNA ISOLATION AND
PURIFICATION
DNA isolation is the first step in the study of specific
DNA sequences, and in the analysis of genome structure
and gene expression.

Isolation of high-molecular weight DNA is very

important for
 DNA fingerprinting,
 restriction fragment length polymorphism (RFLP),
 construction of genomic or sequencing libraries and
 PCR analysis in research laboratories and industry.
The structure of the plant
cell
The quantity, quality and integrity of DNA directly
affect downstream applications.

DNA constitutes a small percentage (<1%) of the cell

material

In prokaryotic cells, DNA is localized in the nucleoid.

In eukaryotic cells, the bulk of DNA is localized in
the nucleus, which contains about 90% of the total
cellular DNA

The remaining DNA is in other organelles like

mitochondria and chloroplasts.
Composition of typical prokaryotic and eukaryotic cells

Component Percentage of total weight of the cell

Prokaryotic cell Eukaryotic cell

Water 70 70
Inorganic ions 1.0 1.0
Amino acids 0.4 0.4
Nucleotides 0.4 0.4
Lipids 2.2 2.8
Protein 15 22.3
RNA 6.0 1.7
DNA 1.0 0.85
The purpose of DNA isolation is to separate DNA
from all the components of the cell listed in the
Table above.
General requirements for effective DNA isolation

The method used should yield DNA without major contaminants (i.e.
protein and RNA).

The method should be efficient

◦ most of the cellular DNA should be isolated.
◦ It should be nonselective (all species of DNA in the cells should be
purified with equal efficiency).

The method should not physically or chemically alter DNA molecules.

General requirements for effective DNA isolation

The DNA obtained should be of high molecular weight

and with few single-stranded breaks.

The method should be relatively fast and simple

The major steps during DNA isolation

There are several methods for isolation of DNA

that, in general, fulfill most of the requirements
listed above. All methods involve four essential
steps :
• Cell breakage.
• Removal of protein and RNA.
• Concentration of DNA.
• Determination of the purity and quantity of DNA.
 Breaking of cells
The primary ways to break cells are chemical, mechanical
and enzymatic.

Mechanical means of cell breaking includes sonication,

grinding, blending or high pressure.

Mechanical breaking of cells, however, can lead to

shearing of DNA and it’s less preferred method for DNA
preparations.
 Breaking of cells
The preferred method for breaking cells to obtain intact
DNA is through application of chemical (detergents)
and/or enzymatic procedures.

Detergents can solubilize lipids in cell membranes

resulting in gentle cell lysis.

In addition, detergents have an inhibitory effect on all

cellular DNAases and can denature proteins, aiding the
removal of proteins from the solution.
 Breaking of cells
To apply detergent to tissue-derived cells, the tissue is
usually frozen in liquid nitrogen and gently crushed into
small pieces that are accessible to detergent treatment

Often plant and bacterial cells cannot be broken with

detergent alone.

To lyse these cells, they are first treated with enzymes

that make the cell membrane accessible to detergents.
 Breaking of cells
Plant cell walls can be removed by treatment with
enzymes that partially or totally remove the cellulose cell
wall.

Since enzymatic treatment of plant cells is expensive and

time consuming, it can be substituted by gentle grinding
in liquid nitrogen.

Treatment does not mechanically disrupt plant cells but

forms small cracks in the cell wall, permitting detergent
access to the cell lysis of cell membranes
 Removal of Protein and RNA
The second step is the removal of proteins from the cell
lysate. This procedure is called deproteinization.

Removal of proteins from the DNA solution depends on

differences in physical properties between nucleic acids
and proteins.

These differences are: differences in solubility,

differences in partial specific volume and differences in
sensitivity to digestive enzymes.
Removal of Protein and RNA
Methods that exploit solubility differences are:
◦ solubility differences in organic solvents,
◦ solubility differences of salts or detergent
complexes and
◦ differences in absorption to charged surfaces.
 Deproteinization methods using organic
solvents
Nucleic acids are predominantly hydrophilic molecules
and are easily soluble in water.
Proteins, on the other hand, contain many hydrophobic
residues making them partially soluble in organic
solvents.
There are several methods of deproteinization based on
this difference, and they differ largely by the choice of
the organic solvent.
The organic solvents most commonly used are phenol or
chloroform containing 4% isoamyl alcohol.
 Deproteinization methods using organic
solvents
The method that uses phenol as the deproteinizing agent
was first used by Kirby (1957) and is commonly referred
to as the Kirby method.
Use of chloroform isoamyl alcohol mixtures was
introduced by Marmur (1961) and is usually referred to
as the Marmur method.
These methods have undergone many modifications
since their first publication.
 The Kirby method
The application of phenol in the Kirby method is based
on the principle that phenol is crystalline at room
temperature, but in the presence of 20% water, it
forms an aqueous solution containing phenol micelles
surrounded by water molecules.
When an aqueous protein solution is mixed with an
equal volume of phenol, some phenol molecules are
dissolved in the aqueous phase (about 20% water and
80%phenol).
 The Kirby method
As a result, phenol molecules diffuse into the core of
the protein, causing the protein to swell and eventually
to unfold ( become denatured).
Therefore, most of the proteins are partitioned into the
phenol phase or precipitated on the interphase with
water.
Nucleic acids do not have strong hydrophobic groups
and are insoluble in phenol.
The Kirby method
Because the phenol phase, at
saturation, contains 20%water,
every phenol extraction will
remove 20% of the DNA into the
phenol phase.
In addition phenol is highly
toxic and requires special
disposal procedures.
 The Marmur method
Chloroform:Isoamyl (CIA) mixture is also a commonly used
organic solvent for protein removal
The chloroform is not miscible with water and therefore
does not lead to DNA loss in the organic phase.
Isoamyl alcohol is an emulsifier and is added to the
chloroform to increase the water-chloroform surface area.
This helps to remove proteins more efficiently.
The disadvantage of this method is that it requires vigorous
shaking of the mixture and may lead to shearing of the
DNA.
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 Use of detergents
Certain compounds or detergents can be used to remove proteins
Compounds most frequently used are CTAB and potassium or sodium
salt of ethyl xanthogenate.
CTAB (Cetryltimethyl ammonium bromide) or CDA (Cetryldimethyl-ethyl
ammonium bromide) can form complexes with nucleinc acides, proteins
and polysaccharides.
At high concentrations of NaCl (1.0 M or above), CTAB forms complexes
with proteins and polysaccharides but not with nucleic acids.
Such complexes are insoluble in water and are removed by
centrifugation or CIA extraction leaving DNA in aqueous phase

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 Enzymatic removal of proteins
Proteins can be removed from DNA or RNA using protease that can
digest all proteins
Two such enzymes are in use:
◦ Proteinase K and
◦ Pronase
Both enzymes are very stable
Commercial preparation are inexpensive and devoid of DNase
contamination
A major drawback of using these enzymes is that they can only remove
80-90% of proteins present

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Removal of RNA
The removal of RNA from DNA is usually carried out using
an enzymatic procedure.
Two ribonucleases are used
◦ RNase A
◦ RNase T1
RNase A is active under a wide range of conditions such as
the presence of detergents, high temperatures, in the
presence of phenol residues, etc.

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Removal of RNA
RNase A is isolated from bovine pancreas
RNase T1 is isolated from Aspergillus oryzae and is
similar to RNase A
It is recommended that both RNase A and RNase T1
be used together

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3. Concentrating the DNA
Alcohol precipation

Precipitation by dialysis

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 4. Determination of concentration and purity
of DNA
The final step of DNA isolation is to evaluate the DNA extracted.

This involves:
◦ Determination of DNA concentration
◦ Evaluation of the purity of the DNA
◦ Determination of DNA yield
Ultraviolet (UV) spectrophotometry is most commonly used for
the determination of DNA concentration.
DNA has a maximum and minimum absorbance at 260nm and
234 nm respectively

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The relationship between DNA absorbance at 260nm (A260) and DNA
concentration is

Where E260 is the DNA extinction coefficient which is usually taken as

0.02 μg/cm

Thus, absorbance of 1.0 at 260nm gives DNA concentration of 50 μg/ml

(1/0.02 = 50 μg/ml)

DNA concentration can reliably be measured if it falls within the range

of 0.5 to 100 μg/ml

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Concentrations higher than 100 μg/ml must be diluted before
measuring
Concentrations lower than the 0.2 may be affected by light
scattering on dust particles in the solution.
Proteins absorbs maximally at 280 nm and absorption at the
wavelength is therefore useful for the detection of proteins in
DNA samples.
This is usually done by the determination of the A260/A280 ratio.
For pure double stranded DNA the A260/A280 ratio is
approximately 2.0.

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Storage of DNA samples
DNA samples should be stored under the conditions that
limit their degradation.

Even under the best conditions DNA will degrade at

about one phosphodiester bond break / 200kb per year

For long term storage the pH of the buffer should be

above 8.5 to minimize deamination

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The following conditions will increase rate of DNA degradation.
1. DNase contamination
◦ The most frequent source of this contamination is human skin
◦ Avoid direct contact with fingers by wearing gloves
◦ Since DNA is easily absorbed into glass surfaces, only use sterilized plastic
tubes for storage
2. Temperature
◦ For short term storage, the best temperature is between 4 Cͦ and 6 Cͦ
◦ For long-term storage (5 years or more) the best temperature is -70 C ͦ or below
◦ Long or short term storage at -20 is not recommended
◦ Storage at -20 C
ͦ can cause extensive single and double stand breakage.
Because of this temperature, molecular bound water is not frozen.

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3. Presence of heavy metals
◦ Heavy metals promote the breakage of phosphodiester bonds
◦ To prevent degradation due to heavy metals store DNA in EDTA. EDTA
is a heavy metal chelator.
◦ For long term storage store DNA in 10 mM or more EDTA.
◦ For short term storage store DNA in 1 mM to 2 mM EDTA.
 4. Presence of ethidium bromide
◦ The presence of ethidium bromide causes photooxidation of DNA with
visible light in the presence of oxygen.
◦ Since it is difficult to remove all ethidium bromide from DNA, DNA
samples should always be stored in the dark.

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Procedure
1. Breaking of cells
2. Incubation with Proteinase K
3. Treatment with RNase
4. Incubation at 65 C
ͦ
5. Treatment with Phenol : CIA
6. Centrifugation
7. Ethanol precipitation
8. Rehydration of DNA

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Isolation and Purification of RNA
Obtaining pure RNA is an essential step in the analysis of gene
expression

Pure RNA is required for a number of techniques including:

◦ Northern blot
◦ RT-PCR
◦ cDNA library construction
◦ RNA mapping
◦ In vitro translation experiments

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RNA Isolation Strategies
Two strategies of RNA isolation are usually employed:
1. Isolation of total RNA: This includes all species of RNA
(rRNA, mRNA, tRNA, snRNA, snoRNA, etc.)

2. Isolation of mRNA (usually about 1-3% of the cell RNA is

mRNA, that is heterogeneous in size and base composition.

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 Purification of total RNA
The physical and chemical properties of RNA and DNA are very
similar thus the basic procedures used in purification are similar to
those of DNA.
All RNA isolation methods involve the following steps
1. Disruption of cells or tissues
2. Denaturation and removal of proteins
3. Concentration of RNA molecules
4. Determination of purity and integrity of isolated RNA

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 PCR Analysis
The Polymerase Chain Reaction (PCR) is a method of in vitro
DNA synthesis.
Large amounts of a specific segment of DNA, of defined
length can be synthesized from a small amount of template.
PCR is a rapid procedure for amplifying DNA of specific
interest.
PCR involves enzymatic amplification of a DNA fragment
flanked by two oligonucleotides (primers).

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PCR Analysis
DNA polymerase synthesizes new DNA starting from the 3’
end of each primer.
Newly synthesized DNA serves as template for other
primers resulting in doubling the amount of DNA fragments
in each cycle.
This leads to an exponential accumulation of the product.
Theoretically, the amount of products doubles in each cycle.

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 PCR Steps

1. Initial Denaturation
2. Denaturation
3. Annealing Several
cycles
4. Extension
5. Final extension
The amplification is expressed in the following equation:
N = N0 x 2n
◦ Where; N = number of amplified molecules
◦ No = Initial number of molecules and
◦ n = number of amplification cycles.

This equation holds true if efficiency of amplification (E) is one.

E is defined as the fraction of template molecules that take part in

amplification during each cycle.

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Therefore, the equation that better describes the
amplification process is
◦ N = N0 x (1 + E)n
◦ Where, E = amplification efficiency.
Amplification efficiency varies during the course of the
reaction.
At the beginning, the efficiency of amplification is high,
close to 1 (0.8 to 0.97)
- The products proceed exponentially
- This constitutes the exponential phase of the reaction

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Amplification efficiency decreases drastically during the late PCR
cycles.
- This slows down product accumulation
- This effect is usually known as the plateau effect

The number of cycles it takes to reach a plateau depends on the initial

number of molecules present in the reaction (No).

When a plateau is reached, it increases the likelihood of obtaining non-

specific amplification products.

Reaching a plateau should always be avoided.

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Components of a Standard PCR
1. Thermostable DNA polymerase (enzyme)

2. DNA template

3. Primers (forward primer and reverse primer)

4. dNTPs

5. MgCl2

6. Reaction buffer and salt

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 DNA Polymerase
The most commonly used thermostable DNA polymerase is Taq
polymerase.

Taq polymerase is obtained from the thermophilic bacterium, Thermus

aquaticus (Taq).

A variety of thermostable DNA polymerase from other thermophilic or

hyperthermophilic bacteria have been isolated and also used in PCR.

The concentration of the enzyme in a reaction mixture can affect the

fidelity of the product.

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Excessive amount of polymerase can result in
amplification of non-specific PCR products.
Enzyme concentration may vary with respect to
individual DNA template or primers used.
Always use the enzyme concentration recommended
by the manufacturer.

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 DNA
PCR can be performed with a very small amount of impure DNA.

Even degraded DNA can be amplified successfully.

However, DNA must be absolutely pure.

A number of contaminants can decrease the efficiency of amplification.

For example, the presence of urea, SDS, Sodium acetate can interfere
with PCR.

Most impurities can be removed by washing DNA with 70% ethanol or by

precipitation of DNA in the presence of ammonium acetate.

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Primers
Primers should be at least 20 to 25 bases in length.
However, for some applications that require multiple random
initiation (e.g. RAPD analysis), primers are usually 8 to 10
bases in length.
If possible, primers should have GC content similar to that
of the target.
Overall GC content of 40% to 60% works for most reactions.

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 Primers
Primers should not have sequences with significant secondary
structures.
Therefore, primers should not contain simple repeats or
palindromic sequences.
Primer pairs should not contain complementary sequences to
each other. This will reduce the incidence of primer-dimer
formation that severely affects efficiency of amplification.

Many computer programs exist to design primer sets.

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dNTPs
There are four dNTPs
- dATP
- dCTP
- dGTP
- dTTP

The four dNTPs should be used at equivalent concentrations.

An excess of nucleotides inhibits enzyme activity and can

contribute to appearance of false products.

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 MgCl2
Magnesium ion is a required co-factor for all DNA polymerases.

Magnesium ion concentration may also affect primer annealing, product

specificity and formation of primer-dimers.

Many templates require optimization of magnesium ion concentration.

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 Reaction buffer and salt
A standard reaction buffer contains 10 to 50 mM Tris HCl at
pH between 8 and 9.

The reaction buffer also contains potassium or sodium salt.

The K+ or Na+ is usually added to facilitate primer annealing.

When using DNA template with high GC content, the reaction

mixture also includes reagents to lower the Tm of the
template. E.g. DMSO, acetamide or glycerol.

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Thermal Cycling Profile
Standard PCR condition of three steps:
1. Denaturation
2. Annealing
3. Extension

These steps are repeated or cycles 25 to 30 times.

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Most protocols also include a single denaturation step
(initial denaturation) before cycling begins and a single long
extension step (final extension) at the end.
The initial denaturation which last for a few minutes is
needed to ensure complete denaturation of the template.
The final extension step is carried out for 5 to 10 minutes
to ensure completion of partial extension products by the
DNA polymerase.

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Denaturation Step
In this step, DNA template is completely denatured
at 94˚C to 95˚C into single strands.

A standard time of 30 to 60 seconds is usually used.

This is to ensure uniform heating of the entire
reaction volume.

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 Annealing step
In this step, primers anneal (bind) to the template.
For most primers, annealing temperature of 50˚C to 55˚C
without further optimization.
The time used for the step in most protocols is 30 to 60
second.
This is the time required to cool the reaction from
denaturation temperature to the annealing temperature.

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 Extension step
In this step, DNA polymerase synthesizes a new DNA strand by
extending the 3’ ends of the primers.
Time for the extension depends on the length of the sequence
to be amplified.
Most protocols recommend 60 seconds per 1 Kbp DNA.
An extension temperature of 72˚C is used in standard
amplification protocols.

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