ANALYTICAL MICROBIOLOGY

MIC402

K. M. Mazharul Alam
Lecturer,
MNS Department,
BRAC University
1
ELECTROPHORETIC
TECHNIQUES

K.M. Mazharul Alam

Lecturer,
MNS Department,
BRAC University
2
Factors Affecting Migration Of Nucleic Acids

Size of DNA
◼ The gel sieves the DNA by the size of the DNA molecule whereby smaller
molecules travel faster.

Conformation of DNA
◼ Supercoiled DNA (such as plasmid) usually moves faster than relaxed DNA
because it is tightly coiled and hence more compact.
◼ In a normal plasmid DNA preparation, multiple forms of DNA may be present.
Therefore, running plasmid on gel will give multiple bands.
Factors Affecting Migration Of Nucleic Acids

The medium
◼ The inert medium can exert adsorption or molecular sieving effects on
the particle influencing its rate of migration.
◼ Adsorption: retention of the component on the surface of supporting
medium.
◼ Molecular sieving: media such as agar, polyacrylamide, sephadex have
cross linked structures giving rise to pores within the gel beads.
Factors Affecting Migration Of Nucleic Acids

Gel concentration
◼ The concentration of the gel determines the pore size of the gel which affect
the migration of DNA.
◼ The more the concentration of agarose, the finer/smaller will be the pores.
◼ For a standard agarose gel electrophoresis, a 0.7% gives good separation or
resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution
for small 0.2–1kb fragments.
Factors Affecting Migration Of Nucleic Acids

Concentration of ethidium bromide

◼ Circular DNA are more strongly affected by ethidium bromide concentration
than linear DNA
◼ Ethidium bromide which intercalates into circular DNA can change the charge,
length, as well as the super helicity of the DNA molecule
◼ Increasing ethidium bromide intercalated into the DNA can change it from a
negatively-supercoiled molecule into a fully relaxed form.
Advantages

1. Easy to prepare and small concentration of agar is required.

2. Resolution is superior to that of filter paper.
3. The gel is easily poured, does not denature the samples.
4. The samples can also be recovered
Disadvantages

◼ Electro osmosis is high.

◼ Gels can melt during electrophoresis, the buffer can become exhausted,
and different forms of genetic material may run in unpredictable forms
◼ Resolution is less compared to polyacrylamide gels.
◼ Different sources and batches of agar tend to give different results and
purification is often necessary.
Special Type Of Agarose Gel Electrophoresis
Special Type Of Agarose Gel Electrophoresis
🞂 Agarose gel electrophoresis is the standard technique for separating nucleic acids,
however, if the fragments are long then it may be necessary to employ a special
type of agarose gel electrophoresis to study them.
🞂 This is because the relationship between the length of a DNA molecule and its
migration rate in a conventional electrophoresis gel is not linear, the resolution
decreasing as the molecules get longer. This means that it is not possible to
separate molecules more than about 50 kb in length.
🞂 To separate them it is necessary to replace the linear electric field used in
conventional gel electrophoresis with a more complex field.
Pulsed-Field Gel Electrophoresis (PFGE)
🞂 Large DNA fragments are unable to migrate through the gel matrix by
the same sieving mechanism that smaller fragments do.
🞂 In fact, the only way that large DNA fragments can even enter an
agarose gel is by "squirming" through the gel like a snake in a parallel
direction relative to the electric field
🞂 The technique of pulsed-field gel electrophoresis (PFGE) takes advantage
of this electrophoretic property of large DNA fragments. PFGE resolves
DNA molecules of 100-1000 kb by intermittently changing the direction
of the electric field in a way that causes large DNA fragments to re-
align more slowly with the new field direction than do smaller molecules.
Pulsed-Field Gel Electrophoresis (PFGE)

A & B represents two sets of

electrodes, switched on and off
alternatively. When A is on, DNA is
driven toward the bottom right corner
of the gel where the anode of that pair
is situated. When A is switched off,
and B is on, DNA moves toward the
bottom left corner. The arrows thus
show the path followed by the DNA as
electrophoresis proceeds
Pulsed-Field Gel Electrophoresis (PFGE)

Since there is a length dependent

relaxation behavior when a long-chain
molecule undergoes conformational
change in an electric field, the smaller
a molecule, the quicker it realigns itself
with the new field and is able to
continue moving through the gel
Pulsed-Field Gel Electrophoresis (PFGE)
Agarose Gel Plug
Parameters: PFGE
◼ Voltage
Typically, the best resolution of chromosomal-sized DNA
fragments (>3 Mb) is achieved when using low-voltage
gradients.

◼ The angle
Separations of larger sizes of DNA, such as
chromosomal DNA, are greatly improved by the use of a
smaller included angle (106°). As the angle is decreased,
the resolution of the largest fragments generally
improves and the smaller fragments become crowded.
The Angle
Parameters: PFGE

◼ The switching time

◼ critical parameter in achieving the best pulsed field gel electrophoresis (PFGE)
resolution.

◼ It is the length of time the electrical field is pulsed in a single direction. For example, a
60 sec switch time means that the electrical field will be pulsed in one direction for 60

sec and then switched to the other direction for 60 sec.

◼ generally shorter for samples with small DNA fragments and longer for samples with
large DNA fragments.
The Switching Time

The ramp of the switch time can be linear, that is, increasing in exact
increments over the course of a run, or nonlinear, so that the user can
concentrate the switch times on the largest or smallest regions as
necessary to achieve better resolution. The nonlinear switch time
ramping expands the linear range of fragment separation to 50–700 kb,
yielding more precise fragment size measurements
Parameters: PFGE

◼ Temperature
Buffer recirculating during the electrophoresis run can be chilled to as low as 4°C or not
chilled at all. Buffer temperature will affect run time. The warmer the buffer
temperature, the faster the run. Resolution decreases as the temperature increases.
https://youtu.be/RcKTqy-7wxc

https://youtu.be/k_QAXLGuQ5w
Orthogonal Field Alternation Gel Electrophoresis (OFAGE)

◼ The electric field alternates between two pairs of electrodes, each

positioned at an angle of 45o to the length of the gel
◼ The DNA molecules still move down through the gel, but each change in the
field forces the molecules to realign. Shorter molecules realign more quickly
than longer ones and so migrate more rapidly through the gel. The overall
result is that molecules much longer than those separated by conventional
gel electrophoresis can be resolved
Orthogonal Field Alternation Gel Electrophoresis
(OFAGE)
Field Inversion Gel Electrophoresis (FIGE)

◼ 1986, Carle, Frank and Olson

◼ Orientation angle : 180°
◼ For FIGE, no special equipment is needed; a conventional power supply and gel
box can be used.
◼ A rather simple and therefore cheap relay box, controlled by a computer or a
timer, is sufficient to achieve good separation.
◼ Separation resolution : a range from several hundred bases up to at least 6 Mbp
Field Inversion Gel Electrophoresis (FIGE)

◼ Works by periodically reversing the direction of the electric field using a

standard gel box apparatus.
◼ Each time the direction of the field is inverted (a 180o field alteration);
larger molecules take longer to reverse direction than do smaller
molecules.
◼ Net movement in one direction is accomplished by keeping the time of
field inversion in the forward direction greater than in the reverse
orientation.
The Field Inversion Gel Electrophoresis (FIGE)

◼ is a special pulsed field gel electrophoresis technique that is based on the periodic
inversion of electric field in one dimension
◼ A net migration is achieved since the product of the duration and amplitude of the
“forward” pulse is larger than that of the pulse in the “backward” direction.
◼ Electrode polarity was reversed at intervals, with a longer forward than reverse pulse
time to generate a net forward sample migration. Net forward migration is achieved by
increasing the ratio of forward to reverse pulse times to 3:1.
◼ To improve the resolution of the bands by FIGE, the duration of pulse times is
increased progressively during a run. This is called “switch time ramping”. By changing
pulse durations continually during the course of an experiment, FIGE has the advantages
of straight lanes and simple equipment.
Field Inversion Gel Electrophoresis (FIGE)
Contour-clamped Homogeneous Electric Fields
(CHEF)

◼ a technique of pulsed-field gel electrophoresis that enables the

resolution of large fragments of DNA that cannot be resolved by
conventional gel electrophoresis.
◼ involves the application of controlled electric fields that change direction
at a predetermined angle to samples of DNA that have been embedded in
an agarose gel matrix and digested with a restriction endonuclease.
◼ Adjustment of the electrophoresis conditions enables the separation of
DNA fragments with lengths from 10 kilobases up to 9 megabases in a
size-dependent manner in agarose gels.
Contour-clamped Homogeneous Electric Fields
(CHEF)

This system uses a

hexagonal gel box that
alters the angle of the
fields relative to the
agarose gel. Electric field
alternates 120o every 90
sec for 18 to 24 h at 14oC
in CHEF.

Electric field alternates 120o every 90 sec for 18 to 24 h at 14oC in CHEF

Contour-clamped Homogeneous Electric Fields
(CHEF)

◼ The banding patterns can be used for epidemiological typing, the

separated DNA can be immobilized onto a membrane and used for genetic
mapping, or individual fragments can be extracted and used for
downstream genetic manipulations.
◼ The protocol requires specialized equipment and can be completed in a
maximum of 7 days.
Contour-clamped Homogeneous Electric Fields (CHEF)
SUMMARY

◼ Basics of Gel electrophoresis

◼ Basics, principles and uses
◼ Agarose Gel Electrophoresis
◼ Pulse Field Gel Electrophoresis
◼ Orthogonal Field Alteration Gel Electrophoresis
◼ Field-inversion Gel Electrophoresis
◼ Contour-clamped Gel Electrophoresis
Restriction Fragment Length Polymorphisms (RFLPS)

◼ It is a difference in homologous DNA sequences

◼ can be detected by the presence of fragments of
different lengths after digestion of the DNA samples in
question with specific restriction endonuclease
◼ They are polymorphisms in the patterns of fragments
produced when DNA molecules are cut with the same
restriction enzyme.
◼ When mutations change the nucleotide sequences in
restriction enzyme cleavage sites, the enzymes no longer
recognize them
◼ This is an RFLP, and its position on a genome map can be
worked out by following the inheritance of its alleles.
◼ There are about 10^5 RFLPs in a mammalian genome.
Application

◼ Individuality in humans and other species derives from their high degree of genetic
polymorphism. Homologous human chromosomes (e.g. the pairs of maternally and
paternally inherited chromosomes) differ in sequence, on average, every 200 to 500 bp.
These genetic differences create or eliminate restriction sites.
◼ RFLPs are particularly valuable for diagnosing inherited diseases for which the
molecular defect is unknown. If a particular RFLP is closely linked to a defective gene,
detecting that RFLP in an individual indicates that there is a high probability that the
individual has also inherited the defective gene. For example, the identification of an
RFLP that is closely linked to the defective Huntington’s gene has permitted the
children of Huntington’s disease victims to make informed decisions in ordering their
lives.
◼ It is used in paternity cases or criminal cases to determine the source of a DNA
sample
SSLP: Simple Sequence Length Polymorphism

◼ SSLPs are arrays of repeat sequence that display length

variations, different alleles* containing different numbers of
repeat units.
◼ Unlike RFLPs, SSLPs can be multiallelic as each SSLP can have
a number of different length variants.
Types Of SSLP

◼ There are two types of SSLP:

◼ Minisatellites, or variable number of tandem repeats (VNTRs).
◼ repeat unit: up to 25 bp in length.
◼ tend to be found more frequently in the telomeric regions at the ends of chromosomes
◼ Microsatellites or simple tandem repeats (STR).
◼ repeat unit: 13 bp or less.
◼ more conveniently spaced throughout the genome
◼ There are 5×105 microsatellite with repeat units of 6 bp or less in the human genome
 Telomere
A telomere is a region of
repetitive nucleotide sequences at
each end of a chromosome, which
protects the end of the
chromosome from deterioration or
from fusion with neighboring
chromosomes
Centromere

◼ Centromeres are stretches of

repeated sequences that lie within
chromosomes. They act as attachment
points for the chromosomes to the
mitotic spindles during cell division
How to Score a SSLP : by AGE following PCR

• The STR and part of the surrounding sequence is

amplified by PCR, and the size of the product
determined by AGE electrophoresis.

• In agarose gel, lane A contains the PCR product and

lane B for DNA markers that show the sizes of the
bands given after PCR of the two alleles. The band in
lane A is the same size as the larger of the two DNA
markers
SSLP: Application

◼ The number of times that a sequence is repeated varies between different individuals
and between maternal and paternal loci of an individual.
◼ The likelihood of two individuals having the same band pattern is extremely improbable.
Therefore, VNTR analysis has become useful in genetics research, forensics, and DNA
fingerprinting.
◼  For example, an individual may inherit a certain number of repeats at one locus from
their mother, and a different number of repeats at the same locus, from their father.
Through PCR amplification of material between conserved sites on either side of the
VNTR locus, one can obtain a banding pattern of the products on a gel.
Banding Pattern Of VNTR

VNTR often show variations in length

between individuals.
•Each variant acts as an inherited allele,
allowing them to be used for personal or
parental identification
Opportunities

◼ Individuals have different number of these repeat motifs. So,

by studying only 10 microsatellite markers, 1 person can be
uniquely distinguished in 1 billion people. Different commercial
STR kits are available to perform this fingerprinting
techniques
DNA Act, 2014, Gob

◼ Recently, the Bangladesh government passed the Deoxyribonucleic Acid

(DNA) Act, 2014, which is regarded as a remarkable instrument in the
field of criminal investigation system for crime detection.
◼ Forensic evidence provides precision in proving the connection of a person
to the crime, either it proves someone is guilty or it proves their
innocence.
◼ The use of forensic evidence is a very efficient method, utilizing it
properly will only contribute to the development of the criminal justice
system.
DNA Act, 2014, Gob

◼ In a very recent case, where Oishee Rahman was proven as guilty, as the
DNA of Mahfuzur and his wife matched the samples taken from Oishee’s
bloodstained clothes and ornaments.
◼ A case sited as the Bangladesh Jatiyo Mahila Ainjibi Samity v.
Bangladesh, Writ Petition No. 5359 of 2006: the Court found that, the
result of Sibling DNA Test shows that all the seven children are unlikely
to be related to each other whom were claimed as children of the Former
DIG of Bangladesh and his wife Mrs. Anwara Rahman. They actually
procured those children to traffic them out of country.
Thank You