BCHEM 365

Lecture 7
October 28, 2019

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 Pulsed field gel electrophoresis (PFGE)

• A special technique is used to determine the size of large

DNA. There are two reasons for development of PFGE
• 1. The relationship between the log of DNA size and its
electrophoretic mobility deviates strongly from linearity if
the DNA is very large
• 2. The double-stranded DNA is a very rigid rod, very long and
thin, making it fragile
• 3. The standard gel electrophoresis technique runs on a
unidirectional constant electric field and works best up to
15 kb DNA, beyond which DNA fragments migrate
independent of size and produce a single large MW band
at the top of the gel
• 4. The low concentration of agarose required to separate
large MW DNA fragments is not strong and breaks easily
• Again beyond 15 kb, the large DNA fragments break very easily – swirling in
a beaker or pipetting creates shearing forces sufficient to fracture DNA
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 Pulsed field gel electrophoresis
• PFGE (developed by David Schwartz and Charles Cantor, 1984) is
a variation of gel electrophoresis that can separate DNA molecules
>15 kb to several million base pairs (Megabases, Mb) long and
maintain a relatively linear relationship between the log of their sizes
and their mobilities
• PFGE differs from normal gel electrophoresis. Normal gel
electrophoresis uses constant voltage which runs down the gel in
one direction. In PFGE two things keep changing at regular
intervals: 1) The direction of current, 2) The holding time for each
current direction (may be same or different)
• Direction of current: the current is periodically switched among three
directions- one current runs through the central axis of the gel; the
second current runs at 60o from one side of gel; the third current
runs at 60o from other side of gel. Current may also be changed at
90o to each other
• The pulse times are equal for each direction leading to a net forward
migration of the DNA down the gel
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 Pulsed field gel electrophoresis
The result is that:
• 1. Different DNA sizes respond
differently to the changed
current direction at differing
rates
• 2. DNA molecules elongate
upon application of an electric
field and return to an
unelongated (relaxed) state
upon removal of the electric
field
• The size-dependent relaxation
rate introduces different rates
of migration through the gel to
effect separation
• Technique can be used to
separate chromosomes
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• PFGE of yeast chromosomes
• Identical samples of yeast
chromosomes were
electrophoresed in 10 parallel
lanes and stained with
ethidium bromide. The bands
represent chromosomes
having sizes ranging from 0.2
Mb (bottom) to 2.2 Mb (top).
There are 16 chromosomes in
yeast, no. of genes 6,275 and
genome size 12,156,677 bp

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Short summary

Three gel
electrophoresis
techniques for
separating DNA
molecules by size.
Electrophoresis is
from top to bottom.
Largest and
therefore slowest-
moving DNA are
near the top of the
gel. Smallest and
supercoiled DNA
molecules are near
the bottom

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 Applications of electrophoresis

• The phenomenon of nucleic acid hybridization is very

important in molecular biology

• Nucleic acid hybridization reactions provide a sensitive way to

detect specific nucleotide sequences

• Northern and southern blotting facilitate hybridization with

electrophoretically separated nucleic acid molecules
• Southern blot is used to identify specific DNA fragments
• Northern blot is used to measure gene activiy as RNA
transcripts
• Northern blot is similar to Southern blot but it contains
electrophoretically separated RNAs instead of DNAs
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 Nucleic acid hybridization

• Heat an aqueous solution of DNA at 100oC or expose to a

high pH (pH≥13) and complementary base pairs that hold the
two strands of the double helix together are disrupted, double
helix rapidly dissociates into single strands
• This is DNA denaturation
• When complementary strands reform at a lower temperature
renaturation or annealing occurs
• Similar hybridization can occur between any two
complementary single stranded nucleic acid chains:
DNA/DNA, RNA/RNA, RNA/DNA
• These specific hybridization reactions are widely used to
detect and characterize specific nucleotide sequences in both
RNA and DNA molecules
• The complementary strands which are used for detection of
DNA or RNA on a blot are called probes
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 Nucleic acid hybridization

DNA probes
• These are single-stranded DNA molecules used to detect
complementary sequences. They are usually labeled with a
radioactive or chemical marker for detection. Can range from
15 to 1,000 nucleotides long
• DNA probes are very sensitive. Can detect complementary
sequences present at concentrations as low as one molecule
per cell therefore it can determine the number of copies of a
DNA sequence present in a cell
• Can be used to find related but nonidentical genes
• Can be used to find a gene of interest in an organism whose
genome has not yet been sequenced
• Can be used in hybridization reactions with RNA and allows
one to determine if a particular gene is being transcribed

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 Nucleic acid hybridization

• In this case, a DNA probe that contains part of a gene’s

sequence is hybridized with RNA purified from the cell to see
whether the RNA includes nucleotide sequences matching the
probe DNA, and in what quantities
• After hybridization is complete, the DNA is treated with
specific nucleases to determine the exact regions of the DNA
that have paired with the RNA molecules.
• Through this, one can determine the start and stop sites for
RNA transcription, as well as the precise boundaries of intron
and exon sequences in a gene

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Use of nucleic acid
hybridization to
determine the region
of a cloned DNA
fragment that is
present in an mRNA
molecule

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12
 Southern blot
• Named after its inventor, Edward Southern
• In this method, isolated DNA is first cut into readily separable fragments
with restriction endonucleases
• Double-stranded fragments are then separated on the basis of size by
agarose gel electrophoresis
• Denature the DNA with a base to obtain single-stranded DNA fragments.
• Transfer the DNA single strands from the gel to a nitrocellulose material
– By diffusion, in which the buffer passes through the gel, carrying the
DNA with it
– By electrophoresis
• Hybridize the blot to a labeled probe and detect labeled bands by
autoradiography or phophorimaging
• This probe will form base pairs with its complementary DNA sequence and
bind to form a double-stranded DNA molecule.
• The degree of hybridization of a complementary probe is a measure of the
amount of the specific target sequence in the sample.
• The probe is labeled before hybridization either radioactively or
enzymatically (e.g. alkaline phosphatase or horseradish peroxidase).
• Those fragments complementary to a DNA probe are identified by blotting
and hybridization
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 Southern blot

• Wherever the probe encounters a

complementary DNA sequence, it hybridizes,
forming a labeled band corresponding to the
fragment of DNA containing the gene of interest

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 Blotting by electrophoresis
Blotting by diffusion

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 DNA fingerprinting

• It is a Southern blot technique

• Used in forensic labs to identify perpetrators, that is, suspects
who have left blood or any DNA-containing tissue on a crime
scene
• Makes use of sequences of bases repeated several times:
minisatellites
• Individuals differ in the pattern of repeats of the basic
sequence
• Two individuals have an extremely rare chance of having
exactly the same pattern. Such patterns are like fingerprints
hence DNA fingerprinting
• To do it, first cut the DNA with restriction enzyme such as
HaeIII (GGCC nucleotide sequence)which cuts on either side
of the repeat regions. This produces fragments bearing the
repeated regions
• Perform electrophoresis of the fragments, denature and blot
• Probe with a labeled minisatellite DNA and detect bands with
X-ray film
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17
 Northern blot
• A technique used in molecular biology research to study gene
expression by detection of RNA (or isolated mRNA) in a
sample.
• Northern blotting involves the use of electrophoresis to
separate RNA samples by size, and detection with a
hybridization probe complementary to part of or the entire
target sequence
• Say you have cloned a cDNA and you want to know how the
corresponding gene is expressed in a number of tissues
• Begin by collecting RNA from the tissues, then perform
agarose gel electrophoresis of the RNA and blot them to a
nitrocellulose or nylon support
• Hybridize the northern blot to a labeled cDNA probe.
• Wherever an mRNA complementary to the probe exists on
the blot, hybridization will occur, resulting in a labeled band
that can be detected by X-ray film. If you run the
electrophoresis along with an RNA marker, you can tell the
size of the RNA bands
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 Electrophoresis in the detection of sickle-cell anaemia
• Hemoglobin (Hb) is the iron-containing oxygen-transport
metalloprotein in red blood cells
• In most humans, the hemoglobin molecule is an assembly of
four globular protein subunits (tetramer), each composed of a
polypeptide chain tightly associated with a non-protein heme
group.

• There are variants of hemoglobin

• Normal hemoglobin, called hemoglobin A (HbA) consists of
two alpha chains, and two beta chains as subunits non-
covalently bound, each made of 141 and 146 amino acid
residues, respectively. This is denoted as α2β2.

• Hemoglobin S (α2βS2) –is found in sickle cell anemia. a

variation in the β-chain gene, causing a change in the
properties of hemoglobin which results in sickling of the red
blood cells.

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 Electrophoresis in the detection of sickle-cell anaemia
• HbS arises from a point mutation of the HBB gene on the short arm
of chromosome 11. This gene encodes the beta globin chain.
Mutation in the β-globin which causes codon = GAA and GAG of the
gene (the amino acid glutamic acid at the sixth position ) to be
replaced with codon GUA and GUG (the hydrophobic amino acid
valine)

• HbS therefore assumes a less negative charge. In HbC, glutamic

acid is replaced with lysine making it slightly more positive (or better
still, much less negative than HbA and even HbS
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 Electrophoresis in the detection of sickle-cell
anaemia

• The hydrophobic valine causes Hb to be less

soluble at low oxygen concentration (i.e.,
deoxygenated Hb), enhances hydrophobic
interaction among the β- chains, and leads to
aggregation and distortion in the shape of the
red blood cells

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 Electrophoresis in the detection of sickle-cell anemia

• Hemoglobin C (α2βC2) –variation in the β-chain gene. This

variant causes a mild chronic hemolytic anemia.
• Heterozygotes have one normal and one sickle-cell gene and
their red blood cells contain one HbS and one HbA and are
said to have the sickle-cell trait. Others have one HbS and
one HbC; Homozygotes have two HbS genes and have sickle
cell anemia

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 Electrophoresis in the detection of sickle-cell anemia
• Sickle-cell anemia, is a
disease caused by a defective
gene which produces a life-
long blood disorder
characterized by red blood A
cells that assume an abnormal,
rigid, sickle shape (biconcave
in normal cells).
• Sickle cell anemia affects
about 1 in 500 infants and is
common among blacks
B

A) Normal red blood cells are disc shaped,

biconcave, flexible, move freely through blood
vessels. B) Sickle cells (like crescent), stiff,
elongated, do not move freely, clog and stick to
blood vessels, causing pain in limbs
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 Electrophoresis in the detection of sickle-cell anemia

• During electrophoresis at alkaline pH, HbS with its less negative

charge will migrate more slowly toward the anode than HbA does.
HbC will migrate even more slower than HbS because it is much
less negative
• Vernon Ingram in 1957 used a 2-dimensional fingerprinting method
to find the difference between HbA and HbS. He digested the two
globin subunits to obtain peptides and separated the peptides first
by paper electrophoresis. Then the paper was turned 90o and the
peptides separated further by paper chromatography in the second
dimension
• The peptides appeared as spots on the paper; different peptides
with different amino acid composition gave different pattern of spots.
The patterns were named fingerprints.
• Comparing the fingerprints of HbA peptides and HbS peptides, he
observed that all spots matched except for one, which had a much
slower mobility in the HbS fingerprint than in the normal HbA
fingerprint indicating that it had an altered charge, arising from a
different amino acid composition
• He found that they differed in only one amino acid: the glutamic acid
in the sixth position in HbA becomes valine in HbS
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Fingerprint of hemoglobin A
and hemoglobin S. All spots
are identical except for one
peptide which moves up and
to the left in HbS

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 Electrophoresis in the detection of sickle-cell anemia

• Normal hemoglobin remains soluble under physiological

conditions
• Sickle cell hemoglobin precipitates when blood oxygen level
falls (e.g., during exercise), forming long, fibrous aggregates.
Such cells are impeded in their path through the tiny
capillaries, so they clog and rupture them, starving parts of
the body of blood and causing internal bleeding and pain
• The hemoglobin electrophoresis test precisely identifies the
hemoglobins in the blood by separating them. The separation
of the different hemoglobins is possible because of the unique
electrical charges they each have on their protein surfaces,
causing them each to move characteristically in an electrical
field.

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 Hemoglobin electrophoresis test

• Electrophoresis is carried out on cellulose acetate paper using

an alkaline buffer at pH 8.6 to 9.2 and run for 30 min at 250 to
300 Volts. At the end of the process, the paper is stained and
dried at 60 to 100oC. The pattern of the bands is a function of
the differences in the mobilities of the variants.

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 Paper electrophoresis
of haemoglobin A, S,
+ anode and C

- Cathode as origin

HbA HbS HbC

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 Serum protein electrophoresis (SPEP)

• This is a technique used to investigate the levels of specific

proteins in the blood called albumin and globulins. It is used
clinically to diagnose and monitor a variety of diseases,
especially disorders of the immune system, liver dysfunction,
kidney diseases, impaired nutrition, some genetic disorders,
and cancer
• Blood serum contains two major protein groups: albumin and
globulin. Both albumin and globulin carry substances through
the bloodstream
• In this method, serum samples are placed on electrophoresis
gels and exposed to an electric current to separate the two
major serum protein components into five smaller group which
differ in shape, size, and electrical charge. The intensity of the
fractions are measured with a densitometer

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 Serum protein electrophoresis (SPEP)
• Albumin
– More than half of the protein in blood serum is albumin.
Albumin proteins keep the blood from leaking out of blood
vessels. Albumin also helps carry some medicines and
other substances through the blood and is important for
tissue growth and healing

• Globulins
– Alpha-1 globulin. Made up of α1-antitrypsin and α1-acid
glycoprotein. High-density lipoprotein (HDL), the
"good" type of cholesterol, is included in this fraction. α1-
antitrypsin protects tissues from enzymes of inflammatory
cells, especially elastase. In its absence, elastase is free to
break down elastin, which contributes to the elasticity of
the lungs, resulting in respiratory complications such as
emphysema, or COPD (chronic obstructive pulmonary
disease) in adults and cirrhosis in adults or children.

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 Serum protein electrophoresis (SPEP)

– Alpha-2 globulin. A protein called haptoglobin, that binds

with haemoglobin, is included in this fraction.
– Also present are α2-macroglobin, a large plasma protein
found in the blood and produced by the liver. It functions
as an inhibitor of coagulation by inhibiting thrombin and an
inhibitor of fibrinolysis by inhibiting plasmin.
– Also in this fraction is α2-antiplasmin which is responsible
for inactivating plasmin, an important enzyme that
participates in fibrinolysis and degradation of various other
proteins.
– Ceruloplasmin, officially known as ferroxidase or
iron(II):oxygen oxidoreductase, is the major copper-
carrying protein in the blood, and in addition plays a role in
iron metabolsm is an alpha-2 globulin

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 Serum protein electrophoresis (SPEP)

Beta globulin. This fraction consists of transferrin, LDL and

the complement system. The complement system is a
biochemical cascade (a group of plasma proteins) that
helps clear pathogens from an organism

Transferrin is for iron transport

LDL is a type of lipoprotein that transports cholesterol and
triglycerides from the liver to peripheral tissues. Also
known as bad cholesterol.

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 Serum protein electrophoresis (SPEP)

• Gamma globulins
– This fraction is made up of immunoglobulin (IgA, IgD, IgE,
IgG and IgM) and paraproteins
– The immunoglobulins are also called antibodies. They help
prevent and fight infection. Gamma globulins bind to
foreign substances, such as bacteria or viruses, causing
them to be destroyed by the immune system
– Paraproteins are a group of proteins most often associated
with benign MGUS (monoclonal gammopathy of
undetermined significance), and multiple myeloma

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Each of these five
protein groups moves at
a different rate in an
electrical field and
together form a specific
pattern. The topography
of the pattern helps
identify some diseases.

Classification of serum proteins: (1) Albumin (2) Alpha-1 globulin (3) Alpha-2
globulin (4) Beta globulin (5) Gamma globulin
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 Serum protein levels

• Protein tests may be measured in grams per deciliter (g/dL) or

milligrams per deciliter (mg/dL) depending on the laboratory
performing the test.
Total serum protein: Normal levels are 5.5 to 9.0 g/dL
• Albumin: Normal levels are 3.5 to 5.5 g/dL
• Globulins: Normal levels are 2.0 to 3.5 g/dL
• A/G ratio (albumin to globulin ratio): Normal levels are greater
than one
Normal levels of a serum protein electrophoresis
• Alpha-1 globulin: Normal levels are 0.1 to .4 g/dL
• Alpha-2 globulin: Normal levels are 0.5 to 1 g/dL
• Beta globulin: Normal levels are 0.7 to 1.2 g/dL
• Gamma globulin: Normal levels are 0.5 to 1.6 g/dL

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 Abnormal results

• Albumin levels
Abnormally high may indicate severe dehydration.
A decreased level of albumin is common in many diseases and is
especially important in liver disease Abnormally low may signal
malnutrition, liver or kidney disease, lupus, rheumatoid arthritis,
Hodgkin disease, uncontrolled diabetes, hyperthyroidism or heart
failure.
Globulin levels
Alpha-1 globulin elevated in inflammatory diseases. Absence
indicates a genetic disorder called juvenile pulmonary emphysema
A decrease in alpha-1-globulin is seen in nephrotic syndrome and
absence could indicate possible alpha-1-antitrypsin deficiency which
eventually leads to emphysema

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 Abnormal results

Alpha-2 globulin elevated in nephrotic syndrome of the kidney

(from the macroglobin fraction). Decreased in hyperthyroidism
or severe liver dysfunction and in blood clotting problems
A normal alpha-2 and a raised alpha-1 is typical in hepatic
metastases and cirrhosis.
Beta globulins elevated in hypercholesterolemia and iron
deficiency anemia. Abnormally low levels can indicate
malnutrition
Gamma globulins elevated in chronic inflammatory diseases,
in autoimmune diseases such as lupus or rheumatoid arthritis,
cancer, kidney or liver disease, infection, multiple myeloma (a
bone cancer), or Hodgkin disease (cancer of the lymphatic
system).
Abnormally low levels can indicate , gastrointestinal diseases
such as Crohn’s disease, liver or kidney disease, blood
clotting problems, emphysema, leukemia, hemolytic anemia,
or problems with the immune system.

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Serum protein electrophoresis showing a paraprotein (peak in the
gamma zone) in a patient with multiple myeloma

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 Cardiac markers in myocardial infarction
• Myocardial infarction is the destruction of heart tissue resulting from
obstruction of blood supply to the heart muscle
• It is the medical term for heart attack. This is most commonly
caused by occlusion (blockage) of a coronary artery following
rupture of a vulnerable atherosclerotic plaque, which is an unstable
collection of lipids (like cholesterol) and white blood cells (especially
macrophages) in the wall of an artery.
• The resulting ischemia (restriction in blood supply) and oxygen
shortage, if left untreated for a sufficient period of time, can cause
damage or death (infarction) of heart muscle tissue (myocardium)

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 Cardiac markers in myocardial infarction (MI)
• Heart attack is a leading cause of death for both men and
women all over the world.
• Important risk factors :
-previous cardiovascular disease (such as angina, a previous
heart attack or stroke)
-old age (especially men over 40 and women over 50)
- tobacco smoking
- high blood levels of certain lipids (triglycerides, LDL or "bad
cholesterol")
- low levels of high density lipoprotein (HDL, "good
cholesterol")
-diabetes, high blood pressure, obesity, chronic kidney
disease
-excessive alcohol consumption, the abuse of certain drugs
such as cocaine
- chronic high stress levels
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 Cardiac markers in myocardial infarction (MI)

• Patient will receive a number of diagnostic tests, such as

electrocardiogram (ECG), a chest X-ray and blood tests to
detect elevations in cardiac markers (abnormal protein levels
signifying heart muscle damage).
• Commonly used markers are the creatine kinase-Myocardial
B fraction (CK-MB) fraction and the troponin I (TnI) or troponin
T (TnT) levels.
• These proteins normally reside in the heart cells but are
released into the blood stream after a heart attack, although
elevated levels may also signify problems with other muscles
in the body so these tests are not specific for myocardial
infarction

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 Cardiac markers in myocardial infarction (MI)

• Until the 1980s, the enzymes, serum glutamic oxaloacetic

transaminase (SGOT) and lactate dehydrogenase (LDH)
were used to assess cardiac injury

-Cardiac troponins T and I which are released within 4–6

hours of an attack of MI and remain elevated for up to 2
weeks, have nearly complete tissue specificity and are now
the preferred markers for assessing myocardial damage.
-Elevated troponins in the setting of chest pain may accurately
predict a high likelihood of a myocardial infarction in the near
future. New markers such as glycogen phophorylase isozyme
BB are under investigation

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 Cardiac markers in myocardial infarction (MI)
• Cytosolic creatine kinase (CK) enzymes consist of two
subunits: B (brain type) or M (muscle) type. Three different
isozymes therefore exist: CK-MM, CK-BB and CK-MB. The
genes for these subunits are located on different
chromosomes, B on 14q32 and M on 19q13. Isozyme
determination has been used extensively as an indication for
myocardial damage in heart attacks.
• Isozymes are enzymes that catalyze the same reaction but
differ in their physical properties as a result of differences in
their amino acid sequence which is genetically determined.
• Isozyme patterns differ in tissues. Skeletal muscle expresses
CK-MM (98%) and low levels of CK-MB (1%). The heart
muscle, in contrast, expresses CK-MM at 70% and CK-MB at
25–30%. CK-BB is expressed in all tissues at low levels and
has little clinical relevance.
• Increase in myocardial-specific enzyme CK-MB is considered
an evidence of acute myocardial infarction

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 Cardiac markers in myocardial infarction (MI)

• Troponins are measured in the blood to differentiate between

unstable angina and MI. Cardiac troponin T and I are
measured by immunoassay methods

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 Cardiac markers in myocardial infarction (MI)

• CK-MB is known to exist in two forms: CK-MB2, the gene

product, and CK-MB1, which is modified upon release into the
bloodstream. Carboxypeptidase cleavage of the C-terminal
lysine residue of the M subunit transforms CK-MB2 into CK-
MB1.
• In healthy individuals, CK-MB2 is in equilibrium with the
modified CK-MB1 subform at a ratio of approximately 1:1. In
the early hours of myocardial infarction, the abrupt release of
CK-MB2 from myocardium produces an upward shift in the
serum CK-MB2/CK-MB1 ratio, usually before total CK-MB
(CKMB2 + CKMB1) exceeds normal levels.
• Determinations of the serum CK-MB2/CK-MB1 ratio are also
proving useful in diagnosis of myocardial infarction

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 Cardiac markers in myocardial infarction (MI)

• Electrophoretic separation of the isozymes using agarose,

cellulose acetate or polyacrylamide as support media
achieves distinct bands that can be quantified by fluorescence
densitometry.
• Separation is possible because the M and B subunits have
different charges on them. The mobility of these isozymes at
pH 8.6 toward the anode is BB> MB> MM with the MM
remaining close to the point of application.
The method is sensitive from 2 - 5 U/L

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 Electrophoretic separation of
BB + anode CK-MM, CK-MB, and CK-BB

MB

MM
- cathode

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