CH 4306

Bioanalytical Techniques
Semester 1
AY2015-2016
Instructor: Chan Vincent
Dept: Chemical and Biomolecular Engineering
Office Hours for CH4306: Every Monday (2pm - 4pm)
Or e-mail: mvchan@ntu.edu.sg (to fix another time)
Requirements: One CA (Take Home) and Final Exam
CA: Around Mid-October, 2015

Course Objectives

To introduce students to modern bioanalytical methods

and techniques used in the study of a host of analytes
such as DNA, protein, sugars, drugs, cells, drug
discovery, diagnostics, etc.

To illustrate each modern bio-instrumental techniques

covered functions (i.e. what are the components of the
instrument and how does each component operate) and
the basic principles underlying the operation of each of
the instruments discussed.
To possess the ability to select an appropriate genome
and proteomic tools for solving a given problem in
diagnostics and research.

Why Bioanalytical Techniques

important?
Most of classical analytical techniques (GC,
LC etc.) are not suitable for the analysis of
biomolecules.
The enormous progress in genomics (DNA
based) and proteomics (Protein based) would
have been impossible without the
development of modern bioanalytical
techniques, e.g., DNA microarray, mass
spectrophotometer.

Learning Outcomes (Continued)

Develop a solid understanding in data interpretation
in each of the techniques covered (i.e. what does the
data by the instrument mean, and how is the signal
quantified).
Gain understanding of the principle underlying the
operation of each of the instruments discussed (which is
useful in diagnosing problems with instrumentation and
in optimizing performance).
An ability to select an appropriate analytical methods
for solving a given problem in the field of biochemical
engineering, food sciences, etc.

Some Established Tools

Glucose biosensor

Pregnancy test

iCycler Thermal
Cycler (PCR)

DNA sequencer

One Typical Rationale behind

Bioanalytical Technique
Genetic Origin of Diseases

Genomics: Point Mutation

truncate the
protein

A point mutation, or single base substitution, is a type of mutation that causes the
replacement of a single base nucleotide with another nucleotide of the genetic
material, DNA or RNA.

Specific diseases caused by point mutations

Cystic fibrosis. A defect in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene causes cystic fibrosis
(CF). A protein made by this gene controls the movement of
the water and salt in and out of the body's cells. Genes in
people with CF incorrectly code proteins. This causes thick,
sticky mucus and very salty sweat.
Neurofibromatosis. Neurofibromatosis is caused by point
mutations in the Neurofibromin 1 or Neurofibromin 2 gene.
Sickle-cell anemia. Sickle-cell anemia is caused by a point
mutation in the -globin chain of haemoglobin, causing the
hydrophilic amino acid glutamic acid to be replaced with the
hydrophobic amino acid valine at the sixth position.

Clubbing in the fingers of a person

with cystic fibrosis

Back of an elderly patient with

neurofibromatosis

Abnormal, sickled red blood cells log jamming, sticking

and accumulating at the branching point in a vein

Disease Caused by Mutation

Cancer. Point mutations in multiple tumor
suppressor proteins cause cancer. For
instance, point mutations in Adenomatous
Polyposis Coli promote tumorigenesis.
A novel assay, Fast parallel proteolysis
(FASTpp), might help swift screening of
specific stability defects of specific
proteins in individual cancer patients.

Mutation: Insertion

Mutation: Insertion
An insertion (also called an insertion mutation) is
the addition of one or more nucleotide base pairs
into a DNA sequence.
This can often happen in microsatellite regions
due to the DNA polymerase slipping.
Insertions can be anywhere in size from one base
pair incorrectly inserted into a DNA sequence to a
section of one chromosome inserted into another.

Mutation: Deletion

Mutation: Deletion
Deletion (also called gene deletion, deficiency, or
deletion mutation) (sign: ) is a mutation (a genetic
aberration) in which a part of a chromosome or a
sequence of DNA is missing.
Deletion is the loss of genetic material. Any number
of nucleotides can be deleted, from a single base to an
entire piece of chromosome.
Deletions can be caused by errors in chromosomal
crossover during meiosis. This causes several serious
genetic diseases. Deletion also causes frameshift.

Specific diseases caused by insertions/deletions

Tay-Sachs Disease. Tay-Sachs Disease is a fatal
disease affecting the central nervous system. It is
most frequently found in infants and small children.
Symptoms do not appear until approximately 6
months of age. The child becomes blind, deaf, unable
to swallow, atrophied, and paralytic.
Death usually occurs before the age of four. There is
no cure for the disease.
Mutations in the -hexosaminidase A (Hex A) gene
are known to affect the onset of Tay-Sachs.

Cherry-red spot as seen in Tay Sachs disease: the

fovea's center appears bright red because it is
surrounded by a milky halo

 Cancer. Insertion/deletion mutations cause

colorectal cancer as well as other cancers with
microsatellite instability.
While there are environmental factors that
contribute to the progression of prostate cancer,
there is also a genetic component.
There are over 500 mutations on chromosome 17
that seem to play a role in the development of
breast and ovarian cancer in the BRCA1 gene,
many of which are Insertion/deletion.

Breast Cancer

Proteomics
Proteomics is the large-scale study of proteins,
particularly their structures and functions.
Proteins are vital parts of living organisms, as they are the
main components of the physiological metabolic pathways
of cells.
The proteome consists of the entire complement of
proteins, including the modifications made to a particular
set of proteins, produced by an organism or system.
This will vary with time and distinct requirements, or
stresses, that a cell or organism undergoes.

Number of Proteins in Human

It is clear now that analyzing genome sequences alone will not
lead to new therapies to fight human diseases.
Whereas the human genome has approximately 35,000 genes and
theoretically the ability to encode up to 35,000 corresponding
proteins.
The occurrence of alternative RNA splicing and posttranslational
modifications (PTM), such as phosphorylations, acetylations, and
glycosylations, or protein cleavages may increase the expression of
proteins to 500,0001,000,000.
The proteins reflect more accurately the intrinsic genetic
mechanisms of the cell and their impact on the microenvironment,
since they are the effectors and characterize more accessible
therapeutic targets than the nucleic acids.

Our Targets:
Biological Macromolecules
Nucleic Acids
Amino Acids
Peptides
Proteins
Polysaccharide (sugars)

Biological Macromolecules
DNA

RNA

Polysaccharide

Protein

Nucleic Acids
Two classes of nucleic acids:
deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA)
Cells use DNA to determine and control the
synthesis of proteins with the help of
messenger RNA (mRNA).
mRNA dictates the synthesis of protein from
amino acids delivered by transfer RNA.
Made up from three components:
nucleobases, sugars and phosphoric acid.

Building blocks of nucleic acids

nitrogenous
base

phosphate
pentose sugar
(ribose for RNA)
(H for DNA)
(AMP)

The pentose sugar

H
(in RNA)

(in DNA)

2. The bases

3. The phosphate

Attached to the 5 carbon of the

sugar

Nucleosides = Base + sugar

adenine

ribose

thymine

deoxyribose
H

The nucleotide
Note: to
distinguish
between sugar
and base,
positions in the
sugar are
designated with
a prime ( )

Nucleoside plus phosphate = ?

nucleotide

A trinucleotide

3
5

3-D structure of DNA

Discovered by Francis Crick and James
Watson in 1953
AT or GC base-pairing

A continuous
spectrum of x-ray is
produced as a result
of the interaction
between the
incoming electrons
and the nucleus of
the target element.

http://www.youtube.com/watch?v=3fe6rHnhkuY

http://www.youtube.com/watch?v=n9FkLBaktEY

Double helix structure

Secondary (2-D)

Tertiary (3-D)

Double helix structure (contd.)

1. DNA molecule consists of two polynucleotide
chains in a double helix configuration.
2. The two strands are anti-parallel.
3. The sugar-phosphate backbone is on the outside
of the helix, bases are on the inside.
4. A always pairs with T; G always pairs with C. The
sequence of one strand (5 3) dictates the
sequence of the other strand.

5 GCATGCAATGCCGAATG 3
3 CGTACGTTACGGCTTAC 5

Double helix structure (contd.)

5. 2nm wide diameter: perfect for purine-pyrimidine
bond.
6. Base pairs are 3.4 apart: a complete 360
turn of the helix is 34 , which equals 10 base
pairs.
7. The helix has a major groove and a minor
groove.
8. When heated or when deviating from
physiological conditions, hydrogen bonds
between the two DNA strands are cleaved and
the strands are separated from each other to
form single string DNA (ssDNA).

This process is usually reversible

3-D structure of RNA

GCAU instead of GCAT
Due to the additional OH group on the
ribose sugar, steric hindrance is too great
to allow for the formation of a double
strand. So, RNA exists as a
single stranded molecule.

Amino Acids
General Structure: -carbon connected to four
groups--amino group, carboxylic group,
hydrogen atom and a substituent group (R
group)

R=side chain

The names for amino acids are often

abbreviated to either three symbol or a one
symbol short form (eg: Glycine, Gly, G).
20 amino acids found in living organisms.

Amino Acids (contd.)

Building blocks of peptides and proteins.
Linear chain of amino acids forms peptide/protein.
Peptides - Small peptides with fewer than about ten
constituent amino acids are called oligopeptides
and peptides with more than ten amino acids are
termed polypeptides.
Proteins Chain of amino acids with molecular
weights of more than 10,000 (50100 amino acids)
are usually termed proteins.

Amino Acids (contd.)

R group varies, thus, can be classified
based on R-group.
Glycine is the simplest amino acid. Side
chain R=H.
Unique because Gly -carbon is achiral.
H
H2N-C-COOH
H
Glycine, Gly, G
Chiral: when a molecule is not superimposable on its mirror image

Amino Acids: Classification based on

polarity of side chain (R group)
Non-polar side chain
Alanine, Ala, A
Valine, Val, V
Leucine, Leu, L
Isoleucine, Ile, I Proline, Pro, P Methionine, Met, M
Phenylalanine, Phe, F Tryptophan, Trp, W
Cysteine, Cys, C

Uncharged Polar side chain

Serine, Ser, S Threonine, Thr, T
Asparagine, Asn, N
Glutamine, Gln, Q
Tyrosine, Tyr, Y
Glycine, Gly, G

Polar, Acidic side chain (negatively charged)

Aspartic acid, Asp, D Glutamic acid, Glu, E
Polar, basic side chain (positively charged)
Lysine, Lys, K

Arginine, Arg, R

Histidine, His, H

Amino Acids: Molar Mass and Chemical Formula

Amino Acids: Structural Classification

Amino Acids: Classification based

on R group

Basic amino acids

Acidic amino acids
Aliphatic amino acids
Aromatic amino acids
Hydroxyl containing amino acids

Sulfur containing amino acids

Secondary amino acids

Natural amino acids: pKa

Zwitterionic character, pK and pI

At the pH under physiological conditions (pH 67), the amino group (pK 8.7~10.7) is ionized to
NH3+ and the carboxyl group (pK 1.8~2.5) is
ionized to COO-. So, at physiological pH,.
amino acids are zwitterionic

Zwitterionic character, pK and pI

(contd.)
pK is the dissociation constant for H+.
pI (isoelectric point) - It is a specific pH
value at which aa exhibits no net charge.
It can be estimated via the HendersonHasselbalch equation pI = (pKNH3++pK ),
where pKi and pKj are the dissociation
constants of the ionization groups involved.
At its isoelectric point, amino acid remains stationary
under an applied electric field.
COOH

Acid-Base Properties

Peptide Bond Formation

Condensation reaction.
Between NH2 of n residue and COOH of
n+1 residue.
Rigid, inflexible.
Loss of 1 water molecule.

The peptide bond presents a significant barrier to rotation.

The resonance structure on the right (see figure) is invoked
to explain this, and bond length comparisons are consistent
with partial double bond character. As a consequence, the
atoms shown in the figure are all constrained to lie in the
same plane. (Note that in polypeptide chains, R and R'
repesent C atoms and their attached groups.)

The peptide bond as we've seen is planar. But the planar

conformation can be accommodated in two alternate forms
denoted as trans and cis. Shown above is the more stable
trans form. The cis form is less stable because of its greater
steric repulsion between the C atoms and their attached
groups. Therefore, trans peptide bonds predominate in
proteins.

Peptides and Proteins

Peptides and protein are macromolecule
made up from long chains of amino acids via
peptide bonds.
In general, there are two types of protein
structures: fibrous (elongated proteins not
soluble in water and providing structural
support), e.g., extracellular matrix proteins
and globular (spherical proteins soluble in
water and have specific function in the
immune system and metabolism).
e.g., growth

factors

The structural proteins

Primary
Secondary
Tertiary
Quarternary

Primary Structure
The sequence of amino acids
e.g: MSNKLVLVLNCGSSSLKFAV
e.g: MCNTPTYCDLGKAAKDVFNK
The peptide bond is rigid and can not move due
to its partial double bond character of C-N bond.
To write peptide and protein always from
N-terminal to C-terminal.

Secondary Structure
Regular elements such as -helices and sheets, which are formed between
relatively small parts of the protein
sequence.
They are determined by the local
conformation of the polypeptide backbone.

-helix
Most abundant; ~35% of
residues in a protein
Repetitive secondary
structure
1.5 rise in 100 rotation
C=O of i forms H bonds
with N-H of residue i+4
Intra-strand H bonding
C=O groups are parallel to
the axis; side chains point
away from the axis
Hence, polar ends present
at surfaces
Amphipathic
All N-H and CO are H-bonded, except
first N-H and last CO

-sheet

Other major structural element

Basic unit is a -strand
Usually 5-10 residues
Can be parallel or anti-parallel based on the
relative directions of interacting -strands
Pleated appearance

-sheet (with primary structure)

PG-1 -sheet arrangements. Topological diagrams for the PG-1 dimers in the (a)
antiparallel and (b) parallel -sheet arrangements. Blue and white beads represent the
positively charged (Arg) and hydrophobic residues, respectively, and the polar
residue (Tyr) and Gly residues are denoted by green beads. Solid lines indicate the
disulfide bonds between Cys residues, and dotted lines indicate the backbone
hydrogen bond (H-bond). The first and the last residues for each monomer are
indicated by the residue number.

-pleated sheet
In a beta-pleated sheet, the chains are folded so that they lie
alongside each other. The next diagram shows what is
known as an "anti-parallel" sheet. All that means is that
next-door chains are heading in opposite directions. Given
the way this particular folding happens, that would seem to
be inevitable.

Nucleoplasmin (PDB 1K5J)

Nucleoplasmins are also known as chromatin decondensation proteins. They

bind to core histones and transfer DNA to them in a reaction that requires
ATP. This is thought to play a role in the assembly of regular nucleosomal
arrays.

Tertiary Structure
Describe the complete three-dimensional
structure of whole polypeptide chain.
Include the relationship of different domains
formed by the proteins secondary structure and
the interactions of the amino acid substituent R
group.
The specific folding of a protein is only
thermodynamically stable within a restricted
range of environmental parameters, e.g.,
Temperature, pH, ionic strength

Phosphofructokinase (PDB 4PFK)

Phosphofructokinase (PFK) is a glycolytic enzyme that

catalyzes the irreversible transfer of a phosphate from ATP to
fructose-6-phosphate.

Quaternary Structure
Quaternary structure is the 3-Dimensional
arrangement of multiple folded protein or
coiling protein molecules in a multi-subunit
complex by hydrogen bond, electrostatic
attraction and sulfide bridge.

Hemoglobin (PDB 1A3B)

Hemoglobin: an iron-containing respiratory pigment of vertebrate red

blood cells that consists of a globin composed of four subunits each of
which is linked to a heme molecule.
Hemoglobin functions in oxygen transport to the tissues after conversion to
oxygenated form in the gills or lungs, and that assists in carbon dioxide
transport back to the gills or lungs after surrender of its oxygen

HEME Molecule

In the 1950s, Christian Anfinsen performed a classic

experiment that demonstrated that the linear sequence
of amino acids in a protein determines the folded
structure of that protein.
Using a chemical called urea, he gently unfolded (denatured) a
protein called Ribonuclease-A, and then reduced its internal disulfide
bonds with mercaptoethanol. (Disulfide bonds act like little molecular
staples that stabilize the folded protein in its original shape.)

Urea

Denaturation
(8M urea, mercaptoethanol)

mercaptoethanol

When Anfinsen then gently and slowly removed the urea

and mercaptoethanol (by dialysis), the ribonuclease
renatured back to its original shape, and regained full
enzymatic activity.
Based on experiments like this, we conclude that the information
for the complete, correct folding of a protein is contained in the
linear amino acid sequence of that protein.

Renaturation
(Remove urea, mercaptoethanol)

(Christian Anfinsen, Nobel laureate.

(After graduating medical school, he worked for a while with Linderstrm-Lang before coming to work at the 141
NIH; he loved sailing and (playing the viola) and was a modest and quiet man.)

Protein Characterizations

Circular Polarization of Light

Electromagnetic radiation consists of an electric (E) and magnetic
(B) field that oscillate perpendicular to one another and to the
propagating direction, a transverse wave.
While linearly polarized light occurs when the electric field vector
oscillates only in one plane, circularly polarized light occurs when the
direction of the electric field vector rotates about its propagation
direction while the vector retains constant magnitude.
In electrodynamics, circular polarization of an electromagnetic wave
is a polarization in which the electric field of the passing wave does
not change strength but only changes direction in a rotary manner.
At a single point in space, the circularly polarized-vector will trace
out a circle over one period of the wave frequency, hence the name.

 The two diagrams below show the

electric vectors of linearly and
circularly polarized light, at one
moment of time, for a range of
positions; the plot of the circularly
polarized electric vector forms a
helix along the direction of
propagation (k).
For left circularly polarized light
(LCP) with propagation towards
the observer, the electric vector
rotates counterclockwise.
For right circularly polarized light
(RCP), the electric vector rotates
clockwise.

http://en.wikipedia.org/wiki/File:Circular.Po
larization.Circularly.Polarized.Light_Right.
Handed.Animation.305x190.255Colors.gif

 Simply put, since circularly polarized light itself is "chiral", it

interacts differently with chiral molecules.
That is, the two types of circularly polarized light are absorbed to
different extents.
In a CD experiment, equal amounts of left and right circularly
polarized light of a selected wavelength are alternately radiated into a
(chiral) sample.
One of the two polarizations is absorbed more than the other one,
and this wavelength-dependent difference of absorption is measured,
yielding the CD spectrum of the sample.
It is important that the chirality of the molecule can be
conformational rather than structural. That is, for instance, a protein
molecule with a helical secondary structure can have a CD that
changes with changes in the conformation.

Circular Dichroism (CD)

Circular dichroism (CD) is dichroism involving
circularly polarized light, i.e., the differential absorption of
left- and right-handed light.
Left-hand circular (LHC) and right-hand circular (RHC)
polarized light represent two possible spin angular
momentum states for a photon, and so circular dichroism is
also referred to as dichroism for spin angular momentum.
It is exhibited in the absorption bands of optically active
chiral molecules.
CD spectroscopy has a wide range of applications in many
different fields.

Circular Dichroism (CD)

Most notably, UV CD is used to investigate the
secondary structure of proteins.
UV/Vis CD is used to investigate charge-transfer
transitions.
Near-infrared CD is used to investigate geometric and
electronic structure by probing metal dd transitions.
Vibrational circular dichroism, which uses light from the
infrared energy region, is used for structural studies of
small organic molecules, and most recently proteins and
DNA.

Delta Absorbance in CD
By definition,

where A (Delta Absorbance) is the difference between

absorbance of left circularly polarized (LCP) and right
circularly polarized (RCP) light (this is what is usually
measured).
A is a function of wavelength, so for a measurement to be
meaningful the wavelength at which it was performed must
be known.

Molar circular dichroism

It can also be expressed, by applying Beer's law, as:

where:
L and R are the molar extinction coefficients for LCP
and RCP light
C is the molar concentration
l is the path length in centimeters (cm)

is the molar circular dichroism. This intrinsic property is what is usually meant by
the circular dichroism of the substance. Since is a function of wavelength, a molar
circular dichroism value ( ) must specify the wavelength at which it is valid.

Synthesis of Proteins
CENTRAL DOGMA
DNA
replication

DNA

RNA synthesis
(transcription)

RNA

Protein

Protein synthesis
(translation)

Transcription
Translation
Post-translational modification such as phosphorylation,
acetylation, methylation, glysosilation, etc..

Degradation of Proteins
Proteins are hold together by hydrogen
bonding, electrostatic attraction and sulfide
bridges, which are very sensitive to its
chemical and physical environment.
The change of temperature, pH or ionic
strength disrupts these interactions, causing
protein denaturation

Protein loses its activity once its normal

shape is lost.

Instrumentation Platform for our

Biomolecular Targets

Electrophoresis
Electrophoresis is a bioanalytical tool used in fundamental
research and diagnostic settings for the isolation and
identification of high molecular weight biomolecules.
The separation is based upon the mobility of charged
macromolecules under the influence of an electric field.
Mobility is a fundamental property of a macromolecule, and its
value depends on the magnitude of its charge, its molecular
weight, and its tertiary or quaternary structure (i.e., its shape),
and its isoelectric point.
Because most biopolymers, such as proteins and nucleic
acids, are charged, they can be separated and quantitated by
electrophoretic methods.
Awarded Nobel Prize in 1948

The Nobel Prize in Chemistry 1948

Prior to the speech, Gustaf
Hellstrm, member of the Royal
Academy of Sciences, addressed
the laureate:

Professor Arne Wilhelm Kaurin Tiselius

Sweden
Uppsala University
Uppsala, Sweden
b. 1902
d. 1971
"for his research on electrophoresis and
adsorption analysis, especially for his
discoveries concerning the complex
nature of the serum proteins"

Your work within molecular

research is more significant than it
may appear to the layman.
According to the experts, proteins
and other macromolecules are the
building blocks of the living
organism,
As life itself can be described
chemically as an exchange between
matter and energy in the proteins of
the cell.

Different modes of electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
Isoelectric focusing (IEF)
2D-gel electrophoresis (2D-EP)
Capillary zone electrophoresis (CZE)
Capillary isoelectric electrophoresis (CIEF)
Capillary gel electrophoresis (CGE)
Micellar electrokinetic chromatography (MEKC)

Principle and Theory of Electrophoresis

An electrophoretic separation occurs in an intervening
medium that separates two electrodes.
At one end of the medium is the positively charged
anode, and at the other is the negatively charged
cathode.
The intervening (support) medium may be as short as 10
cm or as long as 1 m.
Throughout this medium, positively charged species will
migrate toward the cathode and negatively charged
species will move toward the anode.
Differences in charge and size lead to different mobilities and
thus separation of different sample components.

Principle and Theory of Electrophoresis

(contd.)
Electrophoretic separations can be performed in free
solution or in a solution containing a non-conductive
matrix such as agarose or polyacrylamide gel.
For free solution, the separation of ions occurs due to
differences in mobility (different in charge to size ratio)
The separation of analytes in a gel is also based on
differences in mobility, additionally, the gel has sieving
effect. Large compounds are retarded more than smaller
compounds. So two compounds with same charge to
size ratio can be separated as long as they are different
in size.
The efficiency of an electrophoretic separation is
governed by two main factors:
The electrophoretic mobility (ep) of the analytes and the
electroosmotic flow (EOF) of bulk solution.

Electrophoretic Mobility
Electrophoresis is the process in which sample ions move under the
influence of an applied voltage. The ion undergoes a force that is
equal to the product of the net charge and the electric field strength.
It is also affected by a drag force that is equal to the product of f, the
translational friction coefficient, and the velocity. This leads to the
expression for electrophoretic mobility:
EP = q / f = q / (6r)
Electrostatic Force: Fef = qE
Drag Force: Fef = Ffr = fep= 6rep

where f for a spherical particle is given by the Stokes law; is the

viscosity of the solvent, and r is the radius of the molecule.

Electrical Force equal to Frictional Force

The rate at which these ions (charged biomolecules)
migrate is dictated by the charge to mass (which is
proportional to r) ratio. The actual velocity of the
ions is directly proportional to E, the magnitude of
the electrical field and can be determined by the
following equation:
ep= EP * E = (q /f) * E
Once force equilibrium is reached, the velocity of the ion is
constant. The electric field will dictate the velocity. During
the turn-on of electric field, the ions accelerate from zero to
v at acceleration of Force/Mass.

Electroosmotic Flow (EOF)

Many of materials used for
electrophoretic separation
exhibit surface charges.
At the surface of capillary,
an electric double layer is
formed.
The negative surface charges
are compensated by positive
ions from the buffer solution
to form the Stern layer.
A diffuse layer of mobile
cations is formed next to the
Stern layer (zeta-potential).
The potential drop inside the
diffuse layer is exponential.

Actual Flow Pattern

Electrical Double Layer

Electrical double layer consists of a region near an interface in which the net charge
density is nonzero. As compared to the bulk solution, the counterions (ions with
charge opposite the wall) are present at higher concentration, while the coions (ions
with charge of same sign as the wall) are present at lower concentration.

Electroosmotic Flow (EOF) Equation

Upon application of an electric field, the
cations in the diffuse layer (+ charge) move
towards the cathode and drag the bulk
solution with them.
This movement of the bulk solution is called
electroosmotic flow (EOF).

E/(4)
= /E

EOF=

EOF

EOF

For most biomolecular separations, the analyte ions are

negatively charged and will be dragged to the anode,
whereas the EOF is directed to the cathode.

Electroosmotic Flow (EOF) (contd.)

In capillary, the EOF can be controlled.
1. low pH (<4)surface charges are
neutralized by protonating the silanol group.
2. Chemical surface modification. (eg. coating
of the capillary walls with polymer layer)
3. Using additives to change the viscosity
and the zeta-potential .
E.g., Organic solvent methanol and acetonitrile used
to reduce or increase the viscosity

Separation Efficiency and Resolution

Efficiency and resolution of an
electrophoretic separation are influenced
by the electrophoretic motion as well as
the EOF.
The apparent mobility is the sum of
electrophoretic mobility and the
electroosmotic mobility
In simplified terms,

app = ep + EOF

Separation Efficiency and Resolution

Separation Efficiency and Resolution

 The electrophoretic mobility of the analyte and the

electro-osmotic mobility may act in the same direction or
in opposite directions, depending on the charge of the
solute.
In normal capillary electrophoresis, anions (-) will
migrate in the opposite direction to the electro-osmotic
flow and their velocities will be smaller than the electroosmotic velocity.
Cations (+) will migrate in the same direction as the
electro-osmotic flow and their velocities will be greater
than the electro-osmotic velocity.
Under conditions in which there is a fast electro-osmotic
velocity with respect to the electrophoretic velocity of the
solutes, both cations and anions can be separated in the
same run.

Addition of EOF and Electrophoretic Motion

eof
2+

ep
eof

n
Anode
+
ep

ep

2-

total

ep

ep=0

total

eof
total
eof

total
eof
total

ep = q / (6r)

Cathode
-

Capillary Electrophoresis

Resolution

Theoretical Plate

Efficiency
It is important to remember that the plates do not really
exist; they are a figment of the imagination that helps us
understand the processes at work in the column.
They also serve as a way of measuring column
efficiency, either by stating the number of theoretical
plates in a column, N (the more plates the better).
In practice, other phenomena such as heat dissipation,
sample adsorption onto the capillary wall, mismatched
conductivity between sample and buffer, length of the
injection plug, detector cell size and unlevelled buffer
reservoirs can also significantly contribute to band
dispersion (N becomes smaller).

Resolution
Separation between 2 bands, a and b (expressed
as the resolution, Rs) can be obtained by modifying
the electrophoretic mobility of the analytes, the
electro-osmotic mobility induced in the capillary
and by increasing the efficiency for the band of
each analyte, according to the equation:

Rs is higher the better.

Gel Electrophoresis (GE)

Separation takes place in an electrically nonconductive hydrogel medium such as agarose or
polyacrylamide, containing an electrolyte buffer.
The pores of gel serve as a molecular sieve and
retard the migrating molecules according to their
size.
The gels act as anti-convective support medium,
reducing the band broadening.
NO ELECTROOSMOTIC FLOW
Only analytes with a net charge can be separated.
Neutral compounds can not.

Instrumentation for GE
Power supply
Electrophoresis chamber with buffer
reservoirs
Thermostat for temperature control

Instrumentation for GE (contd.)

Power supply
Plastic Cell with
reservoir

Different Orientations
Separation can be
performed:
vertically or horizontally.

Instrumentation for GE (contd.)

Applied potential typically 200-500V
Buffer pH chosen such that the analytes are
negatively charged, so loading sample at the
cathode.
After gel electrophoresis, the analyte bands
are visualized, usually by staining
E.g., Ethidium bromide

Choice Gel media

Agarose or polyacrylamide gels
The gel pore size is an important parameter
for electrophoresis separation.
In restrictive gels, pore are small enough to
act as molecular sieves.
In non-restrictive gels, the pores are too
large to impede the sample movement:
migration time only depends on the mobility of the sample.

Agarose
Agarose was used for the first time as an electrophoresis
medium in the early 1970s.
It is a linear polymer of D-galactose and 3,6anhydrogalactose that is isolated from seaweed.
Agarose contains 0.04% sulfate, may be dissolved in
boiling water and forms a continuous gel when cooled to
<38 C; the gel structure is maintained by hydrogen
bonding.
The concentration of agarose in the gel determines the
average pore size (pore size 150nm at 1% concentration
and 500nm at 0.16% concentration).
Pore sizes are much larger with agarose than with
polyacrylamide gels.
So that nucleic acids and proteins too large to be separated
on polyacrylamide gels can be separated and quantitated
using agarose gels.

Agarose is a chain of sugar molecules, and is

extracted from seaweed.

Agarose (contd.)

Agarose slab gels are used in both vertical and horizontal modes. In
the vertical mode, agarose concentrations as low as 0.8% can be
made, allowing the separation of proteins and nucleic acids up to
molecular weights of ~ 5x 107 Da.
In the horizontal mode, agarose gels can be made from as little as
0.2% agarose, allowing molecular weights of ~ 1.5 x 108 to be
determined.
Agarose gels tend to be relatively fragile (especially at very low
agarose concentrations), and gels are either examined directly or
carefully dried to a thin film prior to staining.
Agarose gels contain charged groupsmainly sulfate and some
carboxylate groups.

These charged groups interact with charged groups on proteins, and

lead to ion-exchange effects; they may also lead to significant
electroosmotic flow.

Agarose (contd.)
The pretreatment of agarose in alkaline solution
leads to the hydrolysis of these groups, and improves
the sieving characteristics of the gels.
The physical properties of agarose gels, especially
the viscosity, are very sensitive to temperature
fluctuations, so that strict control of temperature during
electrophoresis is essential.
Because of the large pores in agarose gels, their
major area of application is in DNA separation and
analysis.

Polyacrylamide gel
Polyacrylamide gels are prepared by the reaction of acrylamide
(monomer) with N,N-methylenebis(acrylamide) (cross-linker) in the
presence of a catalyst and initiator.
Initiators include ammonium persulfate and potassium persulfate,
where the S2O82-- dianion decomposes into two SO4-. radicals, while
the commonly used catalyst is tetramethylethylenediamine [TEMED,
(CH3)2N(CH2)2N(CH3)2], which reacts with the sulfate radical anion
to produce a longer lived radical species.

Polyacrylamide gel (contd.)

The pore size of a polyacrylamide gel controls the
mobility and resolution of components because of
the sieving effect of the pores on macromolecular
species.
The pore size may be controlled by varying the
total concentrations of monomer and cross-linker,
and by varying their ratio.
Gel compositions are defined by two parameters,
their %T and %C values, that represent the total and
crosslinker contents respectively.

 These parameters are defined by following Eqs.

%T= (weight in grams of monomer plus crosslinker)/100mL
%C= 100 x (grams cross-linker)/(grams monomer plus
cross-linker)
The higher the %T, the more restrictive the gel. E.g. at
5%T and 3%C, the pore size is about 5 nm, whereas at
20%T and 3%C the pore size decreases to 3 nm.
Polyacrylamide gels are generally restrictive and acts as
molecular sieves. High molecular compounds (MW>800
kDa) can not be run on it.

Sample preparation and Buffer systems

Sample should not contain any solid particle.
Salt concentration in sample should be low, typically <50
mM.
The buffer must be chosen such that the analyte molecules
are charged, stable and soluble. Typically high pH buffer
used such as pH 9.1 Tris-glycine and pH 8.3 Tris-borate
with concentration 50 mM.
Additives used to increase solubility such as Triton X-100.
Proteins are treated with the denaturing detergent SDS
(sodium dodecyl sulfate) which coats the protein with
negative charges, hence SDS-PAGE.
Disulfide bonds in proteins are also reduced by adding
b-mercaptoethanol.

Visualization and detection

Bands are visualized by staining.

Different staining methods have different
sensitivity ranging from 100 ng to 1 g.
silver staining is more sensitive with
detection limit < 1ng.
DNA/RNA are stained with ethidium
bromide which fluoresces under UV light.
Protein stained with Coomassie Blue, Sypro
Ruby or Silver, colloidal gold.
Quantification can be achieved by
densitomertry.

pMJ1 from
pMJ1 from Transformed 1kb plus
Germany E.coli DH5 DNA ladder

ethidium bromide

Practical
http://www.youtube.com/watch?v=pnBZeL8nFEo

Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE)
The separation principle of SDSPAGE is solely based on the
difference in protein size/molecular
weight.
The protein is denatured in the
presence of anionic detergent SDS
with binding ratio of 1.4 g SDS to 1
g protein.
SDS-protein complex is rod shape
with the large negative charges of
SDS masking the intrinsic charge of
the protein.
Separation totally depends on the
molecular sieving effect of the gels.
The larger the molecular weight, the slower the protein migrates.

SDS-PAGE: Experimental

Loading

Stained Gel

Joule Heating
When an current passes through the conductive
buffer, this causes ohmic heating, also referred to
as Joule heating.
Due to heat transfer, a temperature gradient is
formed across the capillary diameter or gel cross
section, resulting in band broadening and loss of
separation resolution.
There are several ways to minimize Joule Heating.
--Applying a low electric field and decreasing the
conductivity of the buffer.
-- Improving the dissipation of heat by using small
diameter capillary or thin gel.
-- Using a thermostatically controlled environment.

Isoelectric Focussing (IEF)

Separates proteins on basis of isoelectric
point (pI)
If the pH equals to the pI of protein, the protein
is not charged and hence, it does not move in
the electric field anymore.
Basic (positively) charged proteins have a
high pI, acidic (negatively) charged proteins
have a low pI.
Isoelectric focusing has a high resolution.
Bands as narrow as 0.001 pH units can be obtained.

Isoelectric Focusing (IEF) (contd.)

Definition of Isoelectric Point

An amino acid carries simultaneously: a carboxylic acid
function -COOH, which is a weak acid (2 < pKa< 2.5)
An amine function -NH2, which is a weak base (9< pKa<
9.5).
In solution as well as in the solid state, the proton of the
carboxylic acid group is transferred onto the amine to give
a neutral entity, called zwitterion.
Under these form, amino acids can be considered as being
salts of the weak acid -COO- and the weak base NH3+
and therefore they behave as amphoteric particles.

Definition of Isoelectric Point

Amino acids, under their zwitterionic
form behave as amphoteric particles; the
pH of their solutions is given by:

This pH is called isoelectric pH (pI) because

the zwitterion is overall neutral.

Protein at Isoelectric Point

Acidic

Basic

The Charge of Side Chain will be critical

pH > pI

pH < pI

IEF: Instrument

Ampholytes in IEF
A stable pH gradient with constant conductivity is very
important and is achieved by carrier ampholytes or
immobilized pH gradients.
Carrier ampholytes are species that are amphoteric, so that they
reach an equilibrium position along the separation medium,
and are possessing both ionic conductivity, to carry current,
and buffering capacity, to carry pH.
Ampholytes are used to generate stable pH gradients in the presence
of the electric field.

Ampholytes in IEF (Cont.)

Net charge on a protein as a

function of pH. In this example
the protein has a net charge of +2
at pH 5.5, 0 at pH 7.5 (the
isoelectric point), and -1 at pH
8.5.

Creating a carrier ampholyte pH

gradient. (A) No voltage applied.
(B) Ampholytes and proteins move
by electrophoresis when charged.
(C) At isoelectric pH, ampholytes
and proteins are focused.

Principle of Ampholytes
One method for generating pH gradients in IEF gels relies on
carrier ampholytes.
Carrier ampholytes are small, soluble, amphoteric molecules with
a high buffering capacity near their pI.
Commercial carrier ampholyte mixtures comprise hundreds of
individual polymeric species with pIs spanning a specific pH range.
When a voltage is applied across a carrier ampholyte mixture, the
carrier ampholytes with the lowest pI (and the most negative charge)
move towards the anode (+).
The carrier ampholytes with the highest pI (and the most positive
charge) move toward the cathode (-).
The other carrier ampholytes align themselves between the
extremes, according to their pIs, and buffer their environment to the
corresponding pH.

The result is a continuous pH gradient.

 IEF can be run in either a native or a denaturing mode.

Native IEF is the more convenient option, as precast native IEF gels
are available in a variety of pH gradient ranges.
This method is also preferred when native protein is required, as when
activity staining is to be employed.
The use of native IEF, however, is often limited by the fact that many
proteins are not soluble at low ionic strength or have low solubility close
to their isoelectric point.
In these cases, denaturing IEF is employed. Urea is the denaturant of
choice, as this uncharged compound can solubilize many proteins not
otherwise soluble under IEF conditions.
Detergents and reducing agents are often used in conjunction with
urea for more-complete unfolding and solubilization. Urea is not stable
in aqueous solution, so precast IEF gels are not manufactured with urea.
Dried precast gels are a convenient alternative;
they have been cast, rinsed, and dried and can be rehydrated with urea,
carrier ampholytes, and other additives before use.

 Although useful, carrier ampholytes have some limitations.

Because the carrier ampholytegenerated gradient is dependent on
the electric field, it breaks down when the field is removed.
The pH gradients are also susceptible to gradient drift (or cathodic
drift), a phenomenon in which there is a gradual decrease in pH at
the cathodic () end of the gel and a flattening out of the pH at the
anodic (+) end.
For this reason it is important to not over-focus the protein,
because cathodic drift will increase over time.
There can be significant batch-to-batch and company-to-company
variations in the properties of carrier ampholytes, which limits the
reproducibility of focusing experiments.
Another problem encountered with carrier ampholytes is their
tendency to bind to the sample proteins, which may alter

the migration of the protein and render the separation of

carrier ampholytes from the focused protein difficult

IEF with carrier ampholytes

Immobilized pH gradients (IPG)

IPG are produced by incorporating substances
called immobilines in the gel polymerization
process.
Immobilines are not zwitterionic; they are either
acidic or basic.
The pH depends on the ration of these immobilines.
Controlled mixing during gel casting is required to
obtain a good pH gradient.
Once cast, these gels can be prepared in quantity,
dried, and stored, so that a rehydration step just
prior to use with

reproducible gradients.

Properties of IPG
Acrylamido buffers are an alternative means to form pH
gradients that circumvent most of the limitations of carrier
ampholytes.
Chemically, they are acrylamide derivatives of simple
buffers and do not exhibit amphoteric behavior.
The acrylic function of an acrylamido buffer copolymerizes with the gel matrix.
By pouring a gel that incorporates an appropriate gradient
of acrylamido buffers,
an immobilized pH gradient (IPG) is formed.

Creating an immobilized pH
gradient.
(A, B, C) A gradient of
acrylamido buffers in an acrylamide
solution is cast into a slab gel that is
crosslinked to a plastic support
film.
(D) The gel is washed to remove
polymerization byproducts.
(E) The gel is dried for storage.
(F) The pH at any point in the gel
is determined by
the mixture of buffers crosslinked into the gel at that site.

Advantages of IPG
The protein sample can be applied immediately (no
prefocusing is needed).
The pH gradient is stable and does not drift in an
electric field.
Additionally, the gels are not susceptible to cathodic
drift, because the buffers that form the pH gradient
are immobilized within the gel matrix.
Individual Immobiline species with a specific pK
value (or optimum pH buffering range) are available
in biotech supplier (e.g., BioRad, Amersham),
suitable for casting gradients from pH 310.

Advantages of IPG
Because reproducible linear gradients with a slope as low as
0.01 pH units/cm can separate proteins with pI differences of
0.001 pH units.
The resolution possible with immobilized pH gradient gels is
10100 times greater than that obtained with carrier
ampholytebased IEF.
IEF is best performed in a flatbed electrophoresis apparatus.
This type of apparatus allows very effective cooling, which is
necessary due to the high voltages employed for IEF.
Biotech supplier offers a variety of precast gels for IEF,
including ready to use carrier ampholyte gels, dried IPG gels,
and dried acrylamide gels.
These gels are ready for reswelling in a mixture of carrier
ampholytes and any other additives desired,

such as detergent and denaturants.

Immobilized pH gradients (IPG)

(contd.)

Commercial IPG strip

IEF

IEF

http://www.youtube.com/watch?v=45G63IOAS-U

2D Gel Electrophoresis
Two modes of electrophoresis are combined
on a single gel.
Usually, proteins are separated by isoelectric
focusing (IEF) in 1 dimension, based on pI.
Then followed by SDS-PAGE in a
perpendicular direction, based on size.
Mixtures of thousands of proteins can be
separated with high resolution.
The result can be compared to electronic
databases.
Important for Proteomics!

2D electrophoresis with IPG

SDS-PAGE

IEF

SDS-PAGE

IEF

2D electrophoresis with IPG

SDS-PAGE

IEF

Capillary Electrophoresis (CE)

Separation method carried out in a buffer-filled capillary
tube that is typically 20 to 100 m in internal diameter and
10 to 100 cm in length.
The tube extends between two buffer reservoirs that also
hold Pt electrodes.
The sample is introduced into one end of the tubing, and a
dc potential in the 10 to 30 kV range is applied between the
two electrodes throughout the separation.
The separated analytes are observed by a detector at the
end of the capillary opposite the end where the sample was
introduced.
Charged analytes migrate in the presence of an electric
field. Separation is based on
differential rates of migration.

Capillary Electrophoresis (CE) Setup

Hydrodynamic injection

h
Hydrodynamic injection can be performed in following ways: Pressure
injection or vacuum injection, gravity flow injection (siphoning injection). The
anodic end is removed from the buffer reservoir and placed in the sample
solution. The capillary end is then raised so that the liquid level in the sample
vial is at a height h above the level of the cathodic buffer, and is held in this
position for a fixed time t.

Electrokinetic injection
Electrokinetic injection involves drawing sample ions
into the capillary interior with an applied potential. A
high voltage is applied over the capillary between the
sample vial and the destination vial for a given time.
This causes the sample to move into the capillary
according to its apparent mobility, app.
Problem: discrimination occurs between different
components in the sample.

Capillary Electrophoresis
Separation based on electrophoretic mobility
Simple instrumentation
Primary applications in bioanalysis
DNA sequencing (with linear polyacrylamide)
DNA fragment analysis

Multiple modes for improved selectivity of

neutrals
-- Capillary Zone electrophoresis (CZE)
-- Micellar electrokinetic chromatography
(MEKC)

DNA sequencer

DNA Capillary Electrophoresis (Mechanism)

Is there Electroosmotic Flow?

Fluorescently
labeled DNA
fragments move
through a capillary

DNA fragments pass through a laser beam and optical detector

During capillary electrophoresis, the products of the cycle sequencing

reaction are injected electrokinetically into capillaries filled with
polymer. High voltage is applied so that the negatively charged DNA
fragments move through the polymer in the capillaries toward the
positive electrode.

Detection Options
UV-absorption detection
Most common mode
M limit of detection
Peptide =210nm, protein and DNA at 260 or
280nm

Laser-induced Fluorescence
Very sensitive
Limited to fluorescent species
- Mass Spectrometry

Modes of CE
Capillary Zone Electrophoresis (CZE)
Micellar Electrokinetic Chromatography
(MEKC)
Separates compounds with micelles
Capillary Gel Electrophoresis
Size exclusion using sieving gels
Capillary Isoelectric Focusing

Peak #1

Peak #12

L-phenylalanine

Acesulfame

Principles of MKEC
In micellar electrokinetic chromatography, separation takes
place in an electrolyte solution which contains a surfactant at a
concentration above the critical micellar concentration (cmc).
The solute molecules are distributed between the aqueous
buffer and the pseudo-stationary phase composed of micelles,
according to the partition coefficient of the solute.
The technique can therefore be considered as a hybrid of
electrophoresis and chromatography.
It is a technique that can be used for the separation of both
neutral and charged solutes, maintaining the efficiency, speed
and instrumental suitability of capillary electrophoresis.
One of the most widely used surfactants in MEKC is the
anionic surfactant sodium dodecyl sulphate, although other
surfactants, for example cationic surfactants such as
cetyltrimethylammonium salts, are also used.

Mechanism
At neutral and alkaline pH, a strong electro-osmotic flow is
generated and moves the separation buffer ions in the
direction of the cathode.
If sodium dodecyl sulphate is employed as the surfactant,
the electrophoretic migration of the anionic micelle is in the
opposite direction, towards the anode.
As a result, the overall micelle migration velocity is slowed
down compared to the bulk flow of the electrolytic solution.
In the case of neutral solutes, since the analyte can
partition between the micelle and the aqueous buffer, and
has no electrophoretic mobility.
The analyte migration velocity will depend only on the
partition coefficient between the micelle and the aqueous
buffer.

Result
In the electropherogram, the peaks corresponding
to each uncharged solute are always between that
of the electro-osmotic flow marker and that of the
micelle (the time elapsed between these two peaks
is called the separation window).
For electrically charged solutes, the migration
velocity depends on both the partition coefficient
of the solute between the micelle and the aqueous
buffer, and on the electrophoretic mobility of the
solute in the absence of micelle.

Learning Outcomes
Re-establish a solid knowhow in the physiochemical
properties of biological macromolecules such as DNA,
protein, carbohydrates, etc.
Develop a thorough understanding in the targeted
applications of several bio-instrumental techniques
Gain knowledge on the pros and cons in each
technique which is being applied to yield useful
information (i.e. what is measured using the instrument,
how sensitive is the technique, etc.)

Modes of Detection
Fluorescence
Radioactivity

Fluorescence

Excitation Light Source

Arc and Incandescent Xenon lamp
Pulsed Xenon lamp
Low-pressure Hg and Hg-Ar lamps
Ion Lasers and solid state lasers

Xenon arc lamp

Xenon lamp is an electric light that produces light by
passing electricity through ionized xenon gas at
high pressure.
It produces a bright white light that closely mimics
natural sunlight.

Argon Ion Lasers

Fluorescein
A common dye for labeling DNA and protein.

Fluorescein for
labeling actin (green)

Cyanine
Cyanine is a non-systematic name of a synthetic dye family
belonging to polymethine group.
Cy3

Cy5

Applications of Cy3/Cy5 Pair in DNA Detection

Cy3/Cy5
Overlay
Image

Cy3 Image

Cy5 Image

How to capture the fluorescence emission?

Photomultiplier Tube (PMT)
Charge Coupled Device (CCD) Camera
Photodiode

Photomultiplier Tube (PMT)

Photomultipliers are constructed from a glass envelope
with a high vacuum inside, which houses a photocathode,
several dynodes, and an anode.
Incident photons strike the photocathode material, which
is present as a thin deposit on the entry window of the
device, with electrons being produced as a consequence of
the photoelectric effect.

Photomultiplier Tube (PMT)

These electrons are directed by the focusing electrode towards the
electron multiplier, where electrons are multiplied by the process of
secondary emission.
The electron multiplier consists of a number of electrodes called
dynodes. Each dynode is held at a more positive voltage than the
previous one.
The electrons leave the photocathode, having the energy of the
incoming photon.
Upon striking the first dynode, more low energy electrons are emitted,
and these electrons in turn are accelerated toward the second
dynode.

CCD Camera
CCDs are sensors used in digital cameras and video
cameras to record still and moving images.
The CCD captures light and converts it to digital
data that is recorded by the camera. For this reason,
a CCD is often considered the digital version of film.

CCD Camera (light detection)

Photodiode
A photodiode is a type of photodetector capable of
converting light into either current or voltage, depending
upon the mode of operation.
The common, traditional solar cell used to generate electric
solar power is a large area photodiode.
When a photon of sufficient energy strikes the diode, it
excites an electron, thereby creating a free electron (and a
positively charged electron hole).

Electric Current

Radioactive Isotope
The principle behind the use of radioactive tracers is that an
atom in a chemical compound is replaced by another atom,
of the same chemical element.
The substituting atom, however, is a radioactive isotope.
The power of the technique is due to the fact that
radioactive decay is much more energetic than chemical
reactions.
Therefore, the radioactive isotope can be present in low
concentration and its presence detected by sensitive
radiation detectors such as Geiger counters and scintillation
counters.
A radioactive isotope is introduced into DNA or Protein for
quantization purposes through the radioactive decay.

Radioisotopes Are Commonly Used to Detect

Very Small Amounts of DNA/RNA/Protein
Allows for detection of amounts in the femtogram
(1x10-15 gram) to picogram (1x10-12 g) levels
Different radioisotopes used, here are most
common:
32P (DNA & RNA detection)
35S (DNA sequencing)
3H (DNA & RNA detection)
125I (protein detection)

Detect radioisotopes by:

Exposing to x-ray film (autoradiography)
Liquid scintillation Counting (accurate quantification)
determines counts per minute (cpm)

Autoradiography and Quantification by Densitometry

Expose gel to film
Develop film - dark bands indicate radioactive signal
darker band = more signal
fainter band = less signal

Scan x-ray film with light and detector to determine

intensity of band (amount of signal) = densitometry

Autoradiography and Quantification by Densitometry

Gel electrophoresis of DNA fragments in
three parallel lanes on a gel. At this point the
DNA bands are invisible, but their positions
are indicated here with dotted lines.
Place a piece of x-ray film in contact with the
gel and leave it for several hours, or even
days if the DNA fragments are only weakly
radioactive.

Develop the film to see where the

radioactivity has exposed the film.

Southern Blotting
A technique was developed by Professor
Edward M. Southern in 1975.
It is used to detect specific genes in cellular
DNA.
DNA is digested with restriction
endonuclease and DNA fragments are
separated by gel electrophoresis
Then blotted and hybridized with DNA probe.
Can be used to determine (approximately)

the copy number of a gene.

General Scheme for Southern Blotting

Restriction Digest
Gel Electrophoresis
DNA Preparation:
Denaturation
Transfer to filter due to
wicking action: Blotting
Detecting DNA: Probing

Steps of Southern Blotting

Restriction Digest of DNA

Gel Electrophoresis
Denaturation
Blotting Step (over 12 hrs)
Detection of DNA

Genomic DNA

Stain for total DNA (ethidium bromide)

Total fungal
DNA

Phage lambda
DNA (size standards)

23 kb

0.56 kb

Southern Blotting

Experimental Setup

Southern Blotting
Invisible bands
on filter
Hybridize with
labeled probe
(usually radioactive)
Expose to film

Patterns From Southern Blots (genomic DNA)

Enzyme:

EcoRI HpaII BamHI PstI

EcoRI HpaII BamHI PstI

smear

(single copy probe:

detects one part of genome)
A way to visualize a single gene within an entire genome!
(DNA stain)

Southern Blotting In Forensics

DNA analysis and forensics

Marker A - pattern only occurs in 1/100 individuals
Marker B - pattern only occurs in 1/100 individuals
Marker C - pattern only occurs in 1/100 individuals
Combination of A B C only found in 1/106

Each marker examined decreases the probability that

the suspect could match the evidence by chance

Northern Blotting
A variation of the Southern blotting used for
detection of RNA instead of DNA
Total cellular RNA is separated by size, transferred
to a membrane (blotted) and detected by a
complementary radioactive-labelled probe that
hybridises to a specific species of RNA.
Used in studies of gene expression e.g. to determine
whether specific mRNA are present in different types
of cells
To reveal information about RNA identity, size and
abundance.
Northern blottings do not measure transcription rates
or RNA stability,

only steady state mRNA accumulation levels.

Northern Blotting
5 ~10g isolated RNA
260/280nm ~ 2.0

Heat in formamide to denature

agarose gel electrophoresis

Blot/transfer the RNA

onto a membrane using
salt solution

Northern Blotting
RNA transferred to nylon
or nitrocellulose
membrane/filter

Hybridization buffer is important

as is stringency of washing (Rapid
Hybridization ~ 1hr)

Probe (cDNA fragment/antisense

RNA) can be radiolabelled or
chemiluminescent. Random priming
is the usual method for generating a
labelled probe

Procedures for Northern Blotting

mRNA is extracted from the cells and purified.
The mRNA is loaded onto a gel for
electrophoresis. Lane 1 has size standards (a mix
of known RNA fragments) Lane 2 has the RNA.
An electric current is passed through the gel and
the RNA moves away from the negative
electrode.
The distance moved depends on the size of the
RNA fragment.
Since genes are different sizes, the size of the
mRNAs varies. This results in a smear on a gel.
Standards are used to quantitate the size.
The RNA can be visualized by staining first
with a fluorescent dye and then lighting with UV.

Procedures for Northern Blotting

RNA is single-stranded, so it can be
transferred out of the gel and onto a
membrane without any further treatment.
The transfer can be done electrically or by
capillary action with a high salt solution.
A labelled probe specific for the RNA fragment
in question is incubated with the blot.
The blot is washed to remove non-specifically
bound probe.
A development step allows visualization of
the probe that is bound.

How probe works

In this extremely simplified
cartoon, two different mRNAs
are bound to a membrane.
The membrane is incubated with
a solution containing a probe
(another single stranded DNA or
RNA, or oligonucleotide) which
is homologous to one of the 2
mRNAs on the membrane.
The probe specifically binds to
RNA A.
The probe has an label attached to
it so that it may be detected later on.

How probe works

The binding is very specific and requires the base
pairs of the probe and the bound mRNA to be
perfectly complementary, i.e. A to U and C to G.
A wash step removes any probe which is not
tightly bound to the mRNA on the membrane.
Only probe that matches exactly will remain
bound.
In this example, mRNA "A" is the specific mRNA
homologous to the probe.
Therefore a band will develop where this mRNA
is bound to the membrane.
No band will show where "B" is bound.

Northern Blotting
Northern blotting is not used very often for diagnostic
purposes; they are used mainly in research.
A northern blotting is very similar to a Southern blotting
except that it is RNA rather than DNA which is extracted,
run on a gel and transferred to a filter membrane.
There are 3 types of RNA: tRNA (transfer RNA - active
in assembly of polypeptide chains), rRNA (ribosomal
RNA - part of the structure of ribosomes) and mRNA
(messenger RNA - the product of DNA transcription and
used for translation of a gene into a protein).
It is mRNA which is isolated and hybridized in northern
blotting.

Western Blotting
(immunoblotting)
A technique for detecting specific proteins
separated by electrophoresis by use of
labeled antibodies. So called since it has
some similarity to Southern blotting.
We can use this technique to identify a target
protein in a complex mixture.
We can also use it to measure its expression
level.

Protein-Protein Recognition

Western Blotting
use gel electrophoresis to
separate proteins by size
transfer the proteins in
the gel onto a membrane
use the use the specific
antibody to identify the
target protein
visualization or color
development

Visualization
1) colorimetric detection

2) chemiluminescence detection

3) fluorescence detection

Western Blotting
Soluble
fraction

Membrane
fraction

INPNC-OPH
fusion
85 kD

Whole cell
OPH
INPNC-OPH
Other proteins
Membrane

Total Lysate

Total
Soluble Membrane
cell lysate fraction fraction

Summary of blotting techniques

Southern -- Restricted DNA on gel denature and

transfer to filter hybridize with probe = labeled DNA

Northern -- RNA on gel transfer to filter hybridize

with probe = labeled DNA

Western -- protein on gel transfer to filter react

with probe = antibody

"SN0W DR0P":
Match up the 1st word letter with 2nd word letter:
Southern=DNA
Northern=RNA
Western=Protein

Summary of blotting techniques

(contd.)
Use gel electrophoresis to separate the
target
Use sequence-specific or shape-specific
molecular recognition between probe-target
Label the probe and detect the target

Molecular Recognition-Bioassay
Bioassay- most importantly immunoassay, is an
analytical method which uses antibody as
reagents to quantitate specific antigen.
rely on the highly specific reaction between
antibody and antigen.
Very sensitive (fmol)
Widely used in bioanalytical chemistry, especially
for diagnosis and management of diseases

Examples
Pregnancy test

Anthrax

Bioassays
Antibody (Ab) and antigen (Ag) have recognition sites, called
paratope and epitope respectively.
Epitope - a molecular region on the surface of an antigen
capable of eliciting an immune response and of combining with
the specific antibody produced by such a response -- called
also determinant, antigenic determinant
When paratope and epitope match with each other, Ab-Ag
complex is formed.
This kind of binding has very high affinity.
That explains the high sensitivity and low limits of detection
obtained with bioassays.
To detect the assay product, it usually labels either the
antibody or antigen with fluorescent, luminescent, radioactive,
an enzyme or an electrochemically active group.
It can be performed in a large variety of formats,
in solution or on a solid support, with limited reagent or an
excess of reagent.

Antibody
Antibody is a protein complex used by the
immune system to identify and neutralize
foreign objects like bacteria and viruses.
Each antibody recognizes a specific antigen
unique to its target.
It is produced in living organisms via immune
response, in response to immunogen.
Antibodies, also referred to as
immunoglobulins (Ig), consist of four subunits:
two identical light chains (~25KDa) and two
identical heavy chains (~50KDa)

 These subunits are associated via disulfide bonds

and non-covalent interactions to form a Y-shaped
symmetric dimer.
Five types of Ig determined by five types of heavy
chains: IgA, IgD, IgE, IgG (most common antibody
with an abundance of ~70%), IgM

Antibody (contd.)
Two types of antibodies can be distinguished:
monoclonal antibody and polyclonal antibody.
Polyclonal antibodies are isolated directly from
serum.
The resulting antiserum will contain a mixture of
antibodies that bind to different epitopes of
antigens. This may result in significant crossreactivities, or interferences, when employed in
immunoassays.
Monoclonal antibodies are a homogeneous
population of identical antibody molecules,
having identical paratopes and affinity for a single
antigenic epitope.

Structure of antibody

All antibodies have the same basic structure.

Four polypeptide chains linked by disulfide bonds.
Two light chains
Two heavy chains
Antibodies have two antigen binding regions.
A hinge region which confers flexibility on the
molecule

Structure of antibody
A model of an immunoglobulin
molecule.
The heavy chains are coloured
dark red and dark blue
The corresponding light chains
are light red and light blue.
C means crystallizable and ab
means antigen binding.

Useful characteristics of antibodies

They are specific to the substances used to
generate (raise) them.
They are immunogenic themselves (i.e. it is
possible to raise antibodies to antibodies).
The FC portion can be modified without
affecting the specificity or affinity of binding.

They bind the antigen with high affinity

(makes the assay more sensitive).

Antigen
An antigen is a molecule capable of inducing an immune
response when entering the body.
Two classes of antigens can be distinguished: complete
and incomplete antigen.
Complete antigen can induces an immune response by
themselves.
Incomplete antigen , also called hapten, has to attach to
protein carries to trigger the production of antibodies.
The binding site of the antigen, the epitope, makes up a
small area (< 18 amino acids) of the total antigen
structure. Epitopes can be continuous or discontinous.
Disulfide bridge

Antibody-Antigen Complex

The complex formation is reversible and depends on the interplay of

several forces.
Electrostatic interaction between the positively charged amino group
and negatively charged carboxyl group.
Hydrogen bonds between hydroxyl, amino and carboxyl groups
Van der Waals-forces
The binding strength between a single epitope and paratope is
referred to as their affinity, which can be quantified by the
equilibrium constant (Keq)
Keq=[Ab-Ag]/[Ab][Ag]

Immunoassays markets

Agricultural
Environmental
Food
Industrial

Medical
Pharmaceutical
Veterinary
Water Quality

Immunoassay Formats
Limited (competitive) or excess reagents
(non-competitive)
Homogenous or heterogeneous

Labelled or unlabelled

Limited reagent Immunoassay

(Competitive Immunoassay)
+

+
50% bound

Solid phase antibody

Antigen

Bound antigen

Free antigen

+
25% bound

The antigen molecules with the sample compete with a fixed amount of labelled
antigen
for the limited amount of antibody binding sites.
Only a fraction of the antibody binding sites are bound with labeled antigen.

Signal intensity

Response curve for a competitive

assay format
The signal density is
inversely proportional to the
analyte concentration.
C-S

Signal is reduced when the

concentration of non-labeled
antigen increases.

Concentration of antigen

Excess reagent Immunoassay (Noncompetitive Immunoassay)

+
Antigen
Solid phase antibody

Labelled
antibody
Bound antigen

+
Antigen

Primary
Antibody

Secondary
Antibody

The antigen sample is added to an excess of antibody reagent leading to

fractional occupancy of antibody binding sites. A secondary antibody with a
label is then added and a sandwich complex is formed allowing detection.

Signal intensity

Response curve for a noncompetitive assay format

The signal density is
proportional to the
analyte concentration.
C S
The larger the antigen
concentration, the larger
the signal generated.

Concentration of antigen

Labelled and unlabelled assay

Radioisotope (125I, 3H, 32P etc)
Fluorescence label
Enzyme
Luminescence label
Nano/Micro particle (gold)
Ellipsometry

Home Pregnancy Test

Detect the Glycoprotein hormone human chorionic
gonadotropin (hCG)
A few days after conception, hCG appears in the urine
and its concentration increase rapidly during the first

week of pregnancy

Home pregnancy test

The strip component is composed of an adsorbent material.
Once the urine sample is applied, the liquid moves along the
strip by capillary action and the assay-reactions are carried
out in flow.

General Model
Apply sample solution

Negative: no antigen

Immobilised
Immobilised
Control area
Tracer Antibody Capture Antibody

Positive: antigen present

Enzyme-linked immunosorbent assays

(ELISA)
Enzymes are labels Reaction of enzyme with
colorless substrate produces colored product

Y Y Y

Y Y Y
2nd antibody with enzyme

Y Y Y

Antibody/Antigen

Y Y
Y

Y Y Y

Y Y Y

Immobilised Antibody

Y Y Y
enzyme produces colour product

Enzyme-linked immunosorbent assays

(ELISA)
Indirect ELISA detects antibodies in serum

Sandwich ELISA used to detect antigen

ELISA for HIV test

Substrate:
Salicylate
(colorless)

Product:
Oxidized
Salicylate
(brown)

Anti-IgG with
horseradish
peroxidase enzyme
Anti-HIV in serum
of infected person

No brown product
formed

Anti-IgG with enzyme

does not bind
No Anti-HIV in serum
of uninfected person

HIV antigen
Infected Person

Result

HIV antigen
Uninfected Person

Result

Molecular Recognition-Biosensor
Biosensor- is an analytical device that combines a
biological sensing element (such as an antibody,
enzyme or whole cell) with a transducer to produce a
signal proportional to the analyte concentration.
1. Analyte / sampling

2. Biological receptor
5. Processor/ Readout

4. Signal Transducer
3. Immobilization on surface

Molecular Recognition- Biosensor

Biosensors signal is originated from a change
in proton concentration, release or uptake of gases,
light emission, absorption and so forth,
The response is brought about by the reaction of
biomolecule and target compound.

The transducer converts this signal into a

measurable response such as current, potential or
absorption of light through electrochemical or
optical means.
This signal can be further amplified, processed and stored
for later analysis.

Examples
Mining bird (Carbon monoxide)

Glucose biosensor

What do they have in common?

Biosensor

Analyte / bioreceptor / transducer / processor

Nose

Small molecules / olfactory membrane proteins / nerve cells / brain

Eye
Visible light / rods and cones (proteins) / nerve cells / brain

Application areas
Medical
glucose, alcohol, DNA, RNA, proteins, hormones,
aspirin, penicillin
Industrial bio-processes
amino acids, yeast, lactic acid, ethanol, etc.
Environmental
pesticides,fertilizers, CO, CO2
Defense/Forensic
anthrax, small pox, ricin, nerve agents, TNT,
cocaine

The first and still the most important

commercial biosensors: glucose
Diabetes type 1: inability to make the enzyme insulin
Glucose cannot be converted to glucogen to be
stored in body
First glucose biosensor by Clark in 1960
50% of current biosensor research is on glucose
sensors

Glucose-test market is largest for biosensors

Clarks Glucose Sensor 1960

Glucose

Gluconic acid

Glucose Oxidase
(immobilized)

Oxygen
2H+, 2e-

ELECTRODE

5.4 mA

Hydrogen Peroxide

The enzyme is immobilized on a platinum electrode, and

covered with a thin polyurethane (outer) membrane to protect
the enzyme layer, and reduce the dependence of the sensor on
blood oxygen levels. Glucose oxidase, in its oxidized form,
oxidises glucose entering the sensor to gluconic acid; resulting
in the conversion of the enzyme to its reduced form. The
enzyme does not remain in this form for long.

Glucose Sensing

Biosensor components
1. Analyte / sampling
2. Biological receptor
5. Processor/ Readout

4. Signal Transducer
3. Immobilization on surface

Definitions
Biological receptor
A macromolecule / cell / tissue that
recognizes the target analyte
Transducer
Device that converts the biological
recognition event into a measurable signal
Processor
Converts the measured signal into a signal
that can be interpreted by the user, e.g.,
a number, a colour, a meter readout, etc.

1. Analytes
Microbes
anthrax, E. coli, etc.
Small molecules
glucose, alcohol, CO, CO2, nerve
agents, urea, pesticides, aspirin, penicillin,
TNT, cholesterol, amino acids
Bio-macromolecules
DNA, RNA, enzymes, proteins, hormones, viruses

1. Analytes -- Ways of obtaining an

analyte sample
1. Non-Contacting:
Electromagnetic radiation (IR/UV/Visible light, usually requires a probe)
Taking a gas sample near surface
Does not interfere with the subject

2. Contacting:
Invasive/Non-invasive
Can evoke a response/ change analyte

3. Removing:
More or less traumatic (blood versus urine)
Toxic probe molecules or other additives (e.g. heparin) can be added
Most commonly used

2. Biological receptors
Biological interactions
Weak, non-covalent interactions
Spontaneous (self-assembly)
Highly specific (single atom differences)
Complementary (lock-key)

Enzyme

Antibody

Nucleic Acid

2. Biological receptors: proteins

Enzymes/ substrates
Most commonly used biological receptors. They are
catalysts; I.e. a chemical reaction takes place that can be
measured.
Coupled enzyme reactions e.g. with peroxidase,
luciferase to give coloured reaction product or emitted
photons.
Antibodies/ antigens
Highly selective interactions and very tight binding.
Antibodies can be raised against almost any antigen.
Usually needs to be linked to other probe for detection.

2. Biological Receptors
Receptor proteins
Many receptor proteins on surface of cells and embedded into
membranes.
Applications mainly expected in detection of neuro-transmitters,
hormones, neuro-active drugs (e.g. nicotine).
Highly selective but often difficult to isolate. Use of intact
biomembranes or cells.
Nucleic acids
DNA, RNA diagnostic sensors in chip format; used to detect
genetic disorders and expression levels of proteins in parallel.
DNA and RNA can be synthesised in the lab.
Microorganisms
E.g. genetically modified bacterial cells that light up when toxins are presented.

3. Immobilization and surfaces

Bio-receptor molecule has to be
immobilized on or near the surface of the
transducer.
The immobilization is done either by
physical entrapment (e.g. using a
membrane) or chemical attachment.
Biological recognition capability should not be
lost!!

3. Immobilization and surfaces

Chemical methods of bioreceptor
immobilization involve the formation of
covalent bonds, including covalent
binding, e.g., cross-linking.
Physical methods of bioreceptor
immobilization, such as adsorption and
entrapment.

3. Immobilization and surfaces

4. Signal transducers
The transducer converts the
recognition event into a measurable
signal.
The transducer can take many forms
depending upon the parameters
being measured.
Electrochemical, optical, mass and thermal
changes are the most common.

4. Signal transducers
Glucose

Gluconic acid

Glucose Oxidase
Oxygen

Hydrogen Peroxide

Oxygen sensor:

[O2]

e current

pH sensor (gluconic acid):

pH

voltage

Peroxide sensor:

[H2O2]

e current

Coupled to peroxidase rxn:

[Dye]

colour change

Thermal:

enthalpy

4. Signal transducers

Electrochemical
Potentiometric -- detect changes in potential at constant
current (usually zero).
Amperometric -- detect changes in current at constant
potential. (currents generated when
electrons are exchanged between a
biological system and an electrode)
Conductometric-- detect changes in conductivity between
two electrodes.

Piezoelectric crystals
Piezoelectric--These devices detect changes in mass.
Micromechanical systems

Cantilever transducers

Common Transducers
Optical
UV/visible absorption, fluorescence, luminescence
Optical -- correlate changes in concentration, mass, or
number of molecules to direct changes in the characteristics
of light.
For this method to work, one of the reactants or products of
the biorecognition reaction has to be linked to colorimetric,
fluorescent or luminescent indicator molecules.
Usually, an optical fiber is used for guiding the light signals
from the source to the detector.
Thermal

Thermal -- These devices measure changes in temperature.

Electrochemical Transducers -Potentiometric

Measuring cell potential at (near) zero current. The cell potential is
proportional to the ion concentration
Mainly used to quantify inorganic ions, including H+.
In biosensors usually enzyme reactions that involve a significant pH
change: penicillinase, urease, esterase.
Need a reference electrode (e.g. Ag/AgCl)
V

Measuring electrode
Sample to be tested

Salt Bridge

Reference electrode

Enzyme is often immobilised on

electrode surface

Electrochemical Transducers -Amperometric

A constant potential applied between a working and
a reference electrode; the current through the cell is
measured continuously.
When the oxidation potential of a molecule is
reached, it is oxidised and electrons are produced:
measured as a current through the cell.
Current is linearly related to the concentration of the
oxidised molecule.
Most commonly used in reactions involving REDOX
enzymes, e.g., Glucose oxidase, cholesterol oxidase, etc.

Arthrobacter sp. JS443-based

Amperometric Biosensor for PNP
CPE
Pt

RE

1.023nA

Solution

Detector
Recorder
Stir bar

Polycarbonate membrane

CPE
S

O-ring
P

O2

S: substrate
P: product
CPE: carbon paste electrode
RE: Ag/AgCl reference electrode
Lei et al., Electroanalysis, 2004

The Glucose Amperometric Biosensor

Why is needed?

Diabetes
A chronic medical condition associated with abnormally
high levels of sugar (glucose) in the blood known as
hyperglycemia.
Normally, blood glucose levels are tightly controlled by
insulin, a hormone produced by the pancreas which
promotes cellular uptake.
Type I (IDDM) Pancreas undergoes
autoimmune attack by the body itself.
Destruction of beta cells thus renders one
incapable of making insulin. (10%)
Type II (NIDDM) Cells exhibit a lack of
sensitivity to insulin, thus the pancreas
inadequately produces larger than normal
quantities in an attempt to increase cellular
recognition. (90%)

Diabetes
Affects 12 million people (6% of the population)
in the United States and is the third leading cause
of death after heart disease and cancer.
Potentially leads to blindness, kidney failure, and
nerve damage.
Diabetes is also an important factor in
accelerating the hardening and narrowing of the
arteries (atherosclerosis).
Leading to strokes, coronary heart diseases, and
other blood vessel diseases in the body.
The population is at risk now more than ever due
to the growing epidemic of the obesity.

Electrochemical Transducers -Amperometric

Glucose

Gluconic acid

, 2e -

2e

Glucose Oxidase

Hydrogen Peroxide

Ox.
2e

Red.
-

2e

Reduced mediator

Oxidised mediator
2e

Working electrode
with immobilised enzyme
(or mediator)
Auxiliary electrode

ELECTRODE

Contact

Conductive Carbon Track

Reference electrode (Ag/AgCl)

Medisense glucose biosensor

The Glucose Biosensor

How is the disease managed?
Home blood sugar (glucose) testing is an important part
of controlling blood sugar. One important goal of
diabetes treatment is to keep the blood glucose levels
near the normal range of 70 to 120 mg/dl before meals
and under 140 mg/dl at 2 hours after eating.

Medisense
Precision Test Strip Monitor
Requires successive testing throughout each day

Medtronic Minimed
Continuous Glucose Monitoring System
Must be worn three days
Does not operate in real time

Electrochemical Transducers -Conductometric

Measuring conductance/resistance in a
solution.
Useful when charges are produced during
enzymatic conversion (e.g. amidase, esterase,
kinase).
Example: detection of urea (in urine) using
urease:
Urea + 2H2O

urease

2NH4+ + 2HCO3-

Advantages/Disadvantages of
electrochemical transducers
Advantages:
Easy to use
Direct interface with electronic displays
Possibility to miniaturize (faster response times)

Disadvantages:
Limited selectivity

Only works when biological reaction involves electron

transfer

Optical transducers
Photometric behaviour that can be exploited in biosensors:
UV/visible absorption
Fluorescence (and phosphorescence) emission
Bio-luminiscence
Chemi-luminiscence
Internal Reflection Spectroscopy (IRS)
Light scattering methods

Devices: photomultipliers (convert photons to current),

spectrophotomers, optic fibres, etc.

Optical transducers
Advantages:
Easy to use
Can respond simultaneously to different reactants
Can be very accurate, especially when more wavelengths are
used
Can use optical fibres for efficient photon transport
Very high sensitivity (bio-luminiscence)

Disadvantages:
Depend on availability of reactant that changes optical
properties
Dynamic range only around 102 (where Beer/Lambert law
applies)
Difficult to miniaturize
Response time may be slow: analyte diffusion
Background light interference

Thermal transducers
Making/breaking chemical bonds in enzymatic reactions
results in enthalpy changes.
In addition heats of solution change especially with formation
of charged species (e.g. protons).
Typically 10-3 K of temperature change, can be detected.
Reaction of interest may be coupled to reaction that
produces more heat (e.g. glucose oxidase coupled to catalase).
Advantages
They work for most reactions
Disadvantages
Non-selective
Low sensitivity
Difficult to miniaturize

Piezo-electric transducers (Mass)

antibody

piezo electric crystal

antigen

The frequency (f) of crystal

oscillation depends on the mass
(m) of the crystal and that of any
material adsorbed to its surface
(A).
Natural oscillation frequency
decreases with adsorbed mass
according to the Sauerbrey
equation:
f = -2.3 x 106 f2m/A

Piezo electric materials:

Quartz (SiO2 crystals), ceramic materials, organic polymers.

Quartz crystal microbalance

A quartz crystal microbalance (QCM) measures a mass per unit area
by measuring the change in frequency of a quartz crystal resonator.
The resonance is disturbed by the addition or removal of a small mass
due to oxide growth/decay or film deposition at the surface of the
acoustic resonator.

Piezoelectric Transducers
Advantages:
Highly sensitive: detecting antigens in the picogram
range.
Detects antigens in the gas phase as well as in the
liquid phase.
Portable, so the immunological tests could be
performed virtually anywhere.
Quick response times.
No need for labelling.

Disadvantages:
Non-specific adsorption of proteins to the surface can cause false
results!

Cantilever biosensors
Each cantilever is functionalized on
one side with a different
oligonucleotide.
(a) differential deflection signal is
set to zero.
(b) after injection of the first
complementary oligonucleotide
(green), hybridization occurs on
the cantilever providing the
matching sequence (red).

The hybridization increases the deflection of cantilever.

Performance requirements for biosensors

1. Accuracy
2. Sensitivity and specificity no environmental
interference, eg temperature/ contaminations
3. Measurement range
4. Speed of response e.g. nerve agents
5. Self-testing/ calibration
6. Physical robustness
7. Cost capital, running
8. Ease of use
9. Product safety
glucose biosensor
10. Stability
11. Re-usability antibody based often not re-usable due
to strong interactions

Frontier in Biosensor
Nanotechnology in
Biosensor
Due to recent development in
nanotechnology, Si nanowire
and carbon nanotube have
revolutionized our ability to
provide a real-time, sensitive
and selective detection of a
wide range of chemical and
biological species.
Modification with specific
biomolecules can further
enhance the selectivity of
nanosensors.

Cui et al., Science 2001

 Modification with
specific
biomolecules can
further enhance the
selectivity of
bionanosensors.
Silicon nanowire
functionalized with
influenza virus
antibody has been
developed to detect
single influenza virus.

Patolsky et al., PNAS 2005

 Carbon nanotube
functionalized with
human autoantigen
(U1A) has been
developed for rapid,
selective and
sensitive detection of

anti-U1A antibody.

Dai et al., PNAS 2003

Recombinant DNA technology in Biosensor

Neurotoxic organophosphates (OPs) are

widely used as pesticides, insecticides and
chemical warfare agents.
In 2001, WHO estimated 1.1 million US children eat food
containing unsafe levels of organophosphate pesticides.

Organophosphorus Hydrolase (OPH)

OPH

OPs with

p-nitrophenyl

PNP

substituent

PNP Degrader Arthrobacter sp. JS443

NO2

NO2
O2
Enzyme

OH
p-nitrophenol
(PNP)

O2

- OH
NO2

COOCOO-

Enzyme

Enzyme

OH

OH

TCA
Enzyme Cycle

OH
OH
O
4-nitrocatechol 1,2,4-benzenetriol maleylactate
Proposed PNP degradation pathway from J.C Spain, 1994, AEM

An Idea
O2

OPs with

p-nitrophenyl
substituent

OPH PNP

3-methyl-4nitrophenol

Arthrobacter sp NO2
JS443
Electrochemcally
active Intermediates

TCA
cycle

Strategies
Expressing OPH (enzyme) in PNP-degrader
bacteria Pseudomonas putida JS 444.
Then all organophosphates in the environment will be
converted into PNP which will be degraded by the bacteria.

How to Express OPH in Bacteria

Intracellular-expressed OPH

Surface-expressed OPH

OPH
Enzymes involved
in PNP-degradation
Ice nucleation protein anchor

Ice-Nucleation Protein
Isolated from Pseudomonas syrungae.
Membrane targeted protein ideal for cell surface
expression
Phenotypic role to nucleate surrounding water
into ice
Three distinct domains: N and C terminal and an
internally repeating domain
N-Terminal domain
500bp

Internally repeating domain

C-terminal domain
300bp

A molecular anchor (Protein Sequence)

Surface Anchor Systems

Full length INP-OPH fusion (Recombinant DNA Technology)
N-Terminal domain
500bp

Internal repeating domain

C-terminal domain
300bp

OPH 1.0kb

Truncated INP-OPH fusion

800bp

OPH 1.0kb

The use of restriction enzyme and ligase lead to the fusion

of DNA encoding OPH with that encoding INP.
Shimazu et al. Biotech & Bioeng. 2001

Shuttle Vector
EcoRI
BamHI

OHP

InpNC

HindIII

pPNCO33
12 kb

KmR

Replication
gene

Shimazu et al. Biotech & Bioeng. 2001

Surface-expressed OPH

OPH
OP compounds
Enzymes involved
p-nitrophenol
in PNP-degradation
product
Ice nucleation protein anchor
Lei et al., Biotech. Progress, 2005

Microbial Biosensor for Organophosphates

Using Engineered P.p JS444
CPE
Pt

RE

1.023nA

Solution

Detector
Recorder
Stir bar
PC membrane

S: substrate

CPE
S

O-ring
P

O2

P: product
CPE: carbon paste electrode
RE: Ag/AgCl reference electrode
Lei et al., Environ. Sci.&Technol., 2005

Nucleic Acids Preparations,

Amplification and Sequencing

Isolation of Plasmid DNA

Consist of three steps: cell lysis; Isolation; DNA/RNA
concentrated
Cell lysis: cell treated with hypotonic solution (sucrose) or
alkaline detergent (SDS), then cellular wall is ruptured.
RNase to destroy RNA and proteinase K to destroy protein
To isolate plasmid DNA: Rapid neutralization by the high
salt potassium acetate allows the chromosomal DNA to
base-pair in an intrastrand manner, leading to the formation
of an insoluble aggregate that falls out of solution.
The purification of plasmid DNA by the silica-based resin
column is based on the selective adsorption of plasmid
DNA to the resin in the presence of high concentrations of
chaotropic salts.
After efficient removal of contaminants by alcohol-based
washes, DNA is eluted from the column with low ionic
strength solutions or water.

 "Chaotropic" means chaos-forming, a term which in

biochemistry, usually refers to a compound's ability to
disrupt the regular hydrogen bond structures in water.
Hydrogen bonding profoundly affects the secondary
structure of bio-polymers such as DNA, RNA and proteins
as well as how water soluble molecules.
Chaotropic salts increase the solubility of nonpolar
substances in water. They denature proteins because they
have the ability to disrupt hydrophobic interactions. They
do not denature DNA or RNA.
Their function in the Nucleospin Extraction Kit is to
denature cellular proteins (such as DNAse and RNAse).
The high concentration of salt also facilitates binding of
the nucleic acids DNA and RNA to the silica membrane in
the column.

Total cellular DNA Isolation

After cell lysis, cell extract treated with
RNase and Proteinase K.
Ethanol precipitation: only long nucleic acid
chains precipitate
Single nucleotides and products from RNA
digestion remain in solution

RNA Isolation
Proteinase K methodcommon method
The cells are lysed by incubation in a hypotonic
solution followed by centrifugation to remove
DNA and cell debris.
Treatment with the proteolytic enzyme
proteinase K leads to the dissociation of RNAprotein complexes and the digestion of
proteins.
The digestion products are removed by phenol
and chloroform extraction
The RNA in the remaining aqueous solution is
precipitated using ethanol.

Polymerase Chain Reaction (PCR)

The Polymerase Chain Reaction (PCR) provides an extremely
sensitive means of amplifying relatively large quantities of
DNA
First described in 1985, Nobel Prize for Kary Mullis in 1993
The technique was made possible by the discovery of Taq
polymerase, the DNA polymerase that is used by the bacterium
Thermus aquaticus that was discovered in hot springs
The primary materials, or reagents, used in PCR are:
- DNA nucleotides, the building blocks for the new DNA
- Template DNA, the DNA sequence that you want to amplify
- Primers, single-stranded DNAs between 20 and 50
nucleotides long (oligonucleotides) that are complementary to
a short region on either side of the DNA template

- DNA polymerase, a thermal stable enzyme that drives, or

catalyzes, the synthesis of new DNA

Thermal Cycler for PCR

Basically a temperature controlling system which allows

rapid change of sample temperature

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)

There are three major steps in a PCR, which are repeated
for 20 to 40 cycles. This is done on an automated Thermo
Cycler, which can heat and cool the reaction tubes in a very
short time.
Denaturation at around 94C : During the denaturation, the
double strand melts open to single stranded DNA, all
enzymatic reactions stop (for example the extension from a
previous cycle).
Annealing at around 54C : Hydrogen bonds are constantly
formed and broken between the single stranded primer and
the single stranded template. If the primers exactly fit the
template, the hydrogen bonds are so strong that the primer
stays attached
Extension at around 72C : The bases (complementary to
the template) are coupled to the primer on the 3' side (
The polymerase adds dNTP's from 5' to 3', reading the template
from 3' to 5' side, bases are added complementary to the template)

Exponential increase of the number

of copies during PCR

Exponential increase of the number of DNA

Every cycle results in a doubling of the number of
strands DNA present
After the first few cycles, most of the product DNA
strands made are the same length as the distance
between the primers
The result is a dramatic amplification of the DNA that
exists between the primers.
The amount of amplification is 2 raised to the n
power; n represents the number of cycles that are
performed.
After 20 cycles, this would give approximately 1 million
fold amplification. After 40 cycles the amplification would
reach 1 x 1012 (copies).

Verification of PCR product

PCR and Contamination

The most important consideration in PCR is contamination
Even the smallest contamination with DNA could affect
amplification
For example, if a technician in a crime lab set up a test
reaction (with blood from the crime scene) after setting up
a positive control reaction (with blood from the suspect)
cross contamination between the samples could result in
an erroneous incrimination.
Even if the technician changed pipette tips between
samples. A few blood cells could stick to the plastic of the
pipette, and then get ejected into the test sample.
Modern labs take account of this fact and devote
tremendous effort to avoid cross-contamination.

Primer design
Perhaps the most critical parameter for successful
PCR is the design of primers
Primer selection
Critical variables are:
- primer length
- melting temperature (Tm)
- specificity
- complementary primer sequences
- 3-end sequence

- G/C content

Primer length
- specificity and the temperature of annealing
are at least partly dependent on primer length
- oligonucleotides between 20 and 30 (50)
bases are highly sequence specific
- primer length is proportional to annealing
efficiency: in general, the longer the primer, the
more efficient the annealing
- the primers should not be too short as
specificity decreases

Primer design
Specificity
Primer specificity is at least partly dependent on primer
length: there are much more unique 24 base oligos than there
are 15 base pair oligos
Probability that a sequence of length n will occur randomly in
a sequence of length m is: P = (m n +1) x ()n
m= ATCGGGCCTATC..CATGCATTG (20000 bases)
n= ATCTA or ATTAAGGCCT
Example: the genome has about 20,000 bases, the
probability of randomly finding sequences of length n is:
n

Pn

19.52

10

1.91 x 10-2

15

1.86 x 10-5

20

1.82 x 10-8

Complementary primer sequences

- primers need to be designed with absolutely no intra-primer homology beyond 3
base pairs. If a primer has such a region of self-homology, snap back can occur
- another related danger is inter-primer homology: partial homology in the middle
regions of two primers can interfere with hybridization. If the homology should occur
at the 3' end of either primer, primer dimer formation will occur.

intra-primer homology

inter-primer homology

G/C content
- ideally a primer should have a near random mix of
nucleotides, a 50~60% GC content
- there should be no PolyG or PolyC stretches that can
promote non-specific annealing

3- end sequence
- the 3' terminal position in PCR primers is essential for
the control of mis-priming
- inclusion of a G or C residue at the 3' end of primers
helps to ensure correct binding (stronger hydrogen
bonding of G/C residues)

5 ACCTC..CATC 3

(or G, CG, GC)

Polymerase Chain Reaction (PCR)

Denaturing Temperature and time

If the nucleic acid is heated in buffers of ionic
strength lower than 150mM NaCl.
The melting temperature is generally less than
100 C - that is why PCR works with
denaturing temperatures of 91-97 C.
Taq DNA polymerase is given as having a
half-life of 30 min at 95 C, which is partly why
one should not do more than about 30
amplification cycles.
However, it is possible to reduce the denaturation
temperature after about 10 rounds of amplification.

 As the mean length of target DNA is decreased: for

templates of 300bp or less, denaturation
temperature may be reduced to as low as 88 C for
50% (G+C) templates.
It means that one may do as many as 40 cycles
without much decrease in enzyme efficiency.
One will increase the number of cycles that are
possible, whether the temperature is reduced or
not.
Normally the denaturation time is 1 min at 94C: it
is possible, for short template sequences, to reduce
this to 30 sec or less.
Increase in denaturation temperature and decrease in time
may also work: eg. 96 C for 15 sec.

Melting temperature (Tm)

This is the temperature at which 50% of the primer and its
complementary sequence are present in a duplex DNA
molecule. The goal should be to design a primer with an
annealing temperature of at least 50C.
The relationship between annealing temperature and
melting temperature is one of the Black Boxes of PCR.
A general rule-of-thumb is to use an annealing
temperature that is 5C lower than the melting
temperature.
The melting temperatures of oligos are most accurately
calculated using nearest neighbor thermodynamic
calculations with the formula:
Tm = H [S+ R ln (c/4)] 273.15 C + 16.6 log 10 [K+]
(H is the enthalpy, S is the entropy for helix formation, R
is the molar gas constant and c is the concentration of
primer)

 A good working approximation of this value

can be calculated using the Wallace formula:
Tm = 4x (#C+#G) + 2x (#A+#T) C
Both of the primers should be designed such
that they have similar melting temperatures. If
primers are mismatched in terms of Tm.
Amplification will be less efficient or may not
work: the primer with the higher Tm will misprime at lower temperatures
The primer with the lower Tm may not work at
higher temperatures.

Annealing Temperature (Ta)

A consequence of too high Ta is that too little
product will be made, as the likelihood of primer
annealing is reduced.
Another and important consideration is that a pair of
primers with very different Ta may never give
appreciable yields of a unique product.
It may also result in inadvertent "asymmetric" or
single-strand amplification of the most efficiently
primed product strand.
Annealing does not take long: unless the Ta is too
close to the Tm, or unless they are unusually long,
most primers will anneal efficiently in 30 sec or less,

Polymerase Chain Reaction (PCR)

Elongation Temperature and Time

At around 70 C, primer extension occurs at
up to 100 bases/sec. About 1 min is sufficient
for reliable amplification of 2kb sequences.
Longer products require longer times: 3 min is a
good bet for 3kb and longer products.
Longer times may also be helpful in later cycles
when product concentration exceeds enzyme
concentration (>1nM), and when dNTP and/or
primer depletion may become limiting.

Summary for primer sequence design

primers should be 17-28 bases in length;
base composition should be 50-60% (G+C);
primers should end (3') in a G or C, or CG or GC:
increases efficiency of priming;
Tm between 55-80 C are preferred;
runs of three or more Cs or Gs at the 3'-ends of
primers may promote mispriming at G or C-rich
sequences (because of stability of annealing), and
should be avoided;
3'-ends of primers should not be complementary (ie.
base pair), as otherwise primer dimers will be
synthesized preferentially to any other product.
Primers self-complementarity (ability to form 2
structures such as hairpins) should be avoided.

Real-time PCR
Quantitative PCR (QT-PCR)
As opposed to the endpoint detection, the
formation of the reaction products in PCR
can be monitored (in real time) as the
reaction proceeds because of the
fluorescent reporter technology.

Revisit Fluorescence

What is it?
Where does it come from?
Advantages
Disadvantages

Fluorescence
Chromophores are components of molecules
which absorb light
e.g. from protein most fluorescence results from
the indole ring of tryptophan residue
They are generally aromatic rings

Fluorescent Microscope
Arc Lamp

EPI-Illumination

Excitation Diaphragm
Excitation Filter
Ocular

Dichroic Filter
Objective
Emission Filter

Fluorescence Microscope
upright

inverted

Cameras and emission

filters
Camera
goes here

Color CCD camera does not need optical filters to collect all wavelengths
but if you want to collect each emission wavelength optimally, you need a
monochrome camera with separate emission filters shown on the right.
Alternatives include AOTF or liquid crystal filters.

Excitation Sources
Excitation Sources
Lamps
Xenon
Xenon/Mercury
Lasers
Argon Ion (Ar)
Krypton (Kr)
Violet 405nm, 380 nm
Helium-Neon (He-Ne)
Helium-Cadmium (He-Cd)
Krypton-Argon (Kr-Ar)
Laser Diodes
400nm - NIR

2004 sales of approximately 733

million diode laser; 131,000 of
other types of lasers

How many Photons?

Consider 1 mW of power at 488 nm focused to a
Gaussian spot whose radius at 1/e2 intensity is 0.25m
via a 1.25 NA objective
The peak intensity at the center will be 10-3W /[.(0.25 x
10-4 cm)2]= 5.1 x 105 W/cm2 or 1.25 x 1024
photons/(cm2 sec-1)
At this power, FITC would have 63% of its molecules in
an excited state and 37% in ground state at any one
time

C21H11NO5S
Material Source:
Pawley: Handbook of Confocal Microscopy

Absorbance
ln (Io/I) = nd (Beer Lambert law)
Io = light intensity entering cuvet
I=light intensity leaving cuvet
absorption cross section
n molecules
d = cross section (cm)
or

ln (Io/I) = C d (beer Lambert law)

=absorption coefficient
C = concentration

Converting to decimal logs and standardizing quantities we get

Log (I0/I) = cd = A

Now is the decadic molar extinction coefficient

A = absorbance or optical density (OD) a dimensionless quantity

n molecules
absorption cross section
d

Relative absorbance of phycobiliproteins

Phycobiliproteins are stable and highly soluble proteins derived from
cyanobacteria and eukaryotic algae with quantum yields up to 0.98 and molar
extinction coefficients of up to 2.4 106

Protein

B-phycoerytherin

R-phycoerytherin

allophycocyanin

488nm
568nm
633nm
% absorbance % absorbance % absorbance

33
63
0.5

97
92
20
Data from Molecular Probes Website

0
0
56

Fluorescence
Stokes Shift

Fluorescence Intensity

is the energy difference between the

lowest energy peak of absorbance and the
highest energy of emission
Fluorescein
molecule

Stokes Shift is 25 nm
495 nm

Wavelength

520 nm

Raman Scatter
A molecule may undergo a vibrational transition
(not an electronic shift) at exactly the same time
as scattering occurs
This results in a photon emission of a photon
differing in energy from the energy of the
incident photon by the amount of the above
energy - this is Raman scattering.
The dominant effect in flow cytometry is the
stretch of the O-H bonds of water. At 488 nm
excitation this would give emission at 575-595
nm

Rayleigh Scatter
Molecules and very small
particles do not absorb, but
scatter light in the visible
region (same freq as
excitation)
Rayleigh scattering is directly
proportional to the electric
dipole and inversely
proportional to the 4th power of
the wavelength of the incident
light
The sky looks blue because the gas molecules scatter more
light at shorter (blue) rather than longer wavelengths (red)

Photobleaching
Defined as the irreversible destruction of an
excited fluorophore (discussed in later lecture)
Methods for countering photobleaching

Scan for shorter times

Use high magnification, high NA objective
Use wide emission filters
Reduce excitation intensity
Use antifade reagents (not compatible with viable
cells)

Quenching
Not a chemical process
Dynamic quenching =- Collisional process usually
controlled by mutual diffusion
Typical quenchers oxygen
Aliphatic and aromatic amines (IK, NO2, CHCl3)
Static Quenching
Formation of ground state complex between the
fluorophores and quencher with a non-fluorescent
complex, temperature dependent if you have higher
quencher ground state complex is less likely and therefore
less quenching

Antifade Agents
Many quenchers act by reducing oxygen
concentration to prevent formation of singlet
oxygen
Satisfactory for fixed samples but not live cells!
Antioxidents such as propyl gallate,
hydroquinone, p-phenylenediamine are used
Reduce O2 concentration or use singlet oxygen
quenchers such as carotenoids (50 mM crocetin
or etretinate in cell cultures); ascorbate,
imidazole, histidine, cysteamine, reduced
glutathione, uric acid, trolox (vitamin E
analogue)

Photobleaching example
FITC - at 4.4 x 1023 photons cm-2 sec-1
FITC bleaches with a quantum
efficiency Qb of 3 x 10-5
Therefore FITC would be bleaching with
a rate constant of 4.2 x 103 sec-1 so
37% of the molecules would remain
after 240 sec of irradiation.
In a single plane, 16 scans would cause
6-50% bleaching

Probes for Proteins

Probe
FITC
PE
APC
PerCP
Cascade Blue
450
Coumerin-phalloidin
Texas Red
Tetramethylrhodamine-amines

CY3 (indotrimethinecyanines)
CY5 (indopentamethinecyanines)

Excitation
488
488
630
488

Emissi
525
575
650
680

360
350
610
550
540
640

450
630
575
575
670

Probes for Nucleic Acids

Hoechst 33342 (AT rich) (uv)

DAPI (uv)
POPO-1
YOYO-1
Acridine Orange (RNA)
Acridine Orange (DNA)
Thiazole Orange (vis)
525
TOTO-1
Ethidium Bromide
PI (uv/vis)
7-Aminoactinomycin D (7AAD)

346
359
434
491
460
502

460
461
456
509
650
536
509

514
526
536
555

533
604
620
655

DNA Probes
AO
Metachromatic dye
concentration dependent emission
double stranded NA - Green
single stranded NA - Red

AT/GC binding dyes

AT rich: DAPI, Hoechst, quinacrine
GC rich: antibiotics bleomycin, chromamycin
A3,
mithramycin, olivomycin, rhodamine
800

Other Probes of Interest

GFP - Green Fluorescent Protein
GFP is from the chemiluminescent jellyfish
Aequorea victoria
excitation maxima at 395 and 470 nm (quantum
efficiency is 0.8) Peak emission at 509 nm
contains a p-hydroxybenzylidene-imidazolone
chromophore generated by oxidation of the
Ser-Tyr-Gly at positions 65-67 of the primary
sequence
Major application is as a reporter gene for
assay of promoter activity
requires no added substrates

Multiple Emissions
Many possibilities for using multiple probes
with a single excitation
Multiple excitation lines are possible
Combination of multiple excitation lines or
probes that have same excitation and quite
different emissions
e.g. Calcein AM and Ethidium (ex 488
nm)
emissions 530 nm and 617 nm

Filter combinations

The band width of the filter will change the intensity of the measurement

Fluorescence Overlap
Band pass filter
Fluorescence Intensity

525 nm

575 nm
PE
Molecule
(Emission)

Fluorescein
Molecule
(Emission)

450

500

550
Wavelength (nm)

600

Overlap of FITC fluorescence in PE PMT

Overlap of PE fluorescence in FITC PMT

650

Resonance Energy Transfer

Resonance energy transfer can occur when the
donor and acceptor molecules are less than 100
of one another (preferable 20-50 )
Energy transfer is non-radiative which means the
donor is not emitting a photon which is absorbed
by the acceptor
Fluorescence RET (FRET) can be used to
spectrally shift the fluorescence emission of a
molecular combination.
3rd Ed. Shapiro p 90
4th Ed. Shapiro p 115

Energy Transfer
Non radiative energy transfer a quantum mechanical
process of resonance between transition dipoles
Effective between 10-100 only
Emission and excitation spectrum must
significantly overlap
Donor transfers non-radiatively to the
acceptor
PE-Texas Red
Carboxyfluorescein-Sulforhodamine B

FRET properties
Isolated donor
Donor distance too great

Donor distance correct

Resonance Energy Transfer

Molecule2 2
Molecule

Molecule 11
Molecule

Fluorescence
Fluorescence

Fluorescence
Fluorescence
ACCEPTOR

DONOR

Acceptor

Donor
Absorbance
Absorbance

Wavelength

Fluorescence
The longer the wavelength the lower the energy
The shorter the wavelength the higher the energy
eg. UV light from sun - this causes the sunburn, not the red visible
light

The spectrum is independent of precise excitation line but

the intensity of emission is not

Normal PCR--Endpoint detection

Whats Wrong With Agarose Gels?

*
*
*
*
*
*
*

Poor precision
Low sensitivity
Short dynamic range < 2 logs
Low resolution
Non-automated
Size-based discrimination only
Results are not expressed as numbers

Ethidium bromide staining is not very quantitative

Real-Time PCR
By using an additional probe with a fluorescence
label, amplification of DNA can be measured online.
High reproducibility in the beginning of exponential
phase.

Real-Time PCR
Advantages:
Reliable and exact quantification
High reproducibility in the beginning of
exponential phase
High specificity - Usage of an additional third
probe
High sensitivity - lower detection limit = 100
bacteria
High objectivity - fully automated process

Real-Time PCR
Online detection of
amplification.
Reproducibility at end point
of amplification is very low.
Real-Time PCR allows
measurement of the
amplification, there is no
need to determine the end
point of the reaction any
longer (as is the case with
conventional PCR).
High reproducibility at the
beginning of the
exponential phase allows
exact and reliable
quantification.

Principle of quantitation
The number of cycles it takes to reach a certain amount of
fluorescence is proportional to the amount of cDNA present at the start

This can be plotted as cycle no vs. log concentration to give a straight

line

Generating a standard curve using known

amounts of target DNA and measuring the CT for
each reaction.

Increasing Copy

Principles of Real-Time PCR techniques

(a) SYBR Green technique: SYBR Green fluorescence is enormously increased upon
binding to double-stranded DNA. During the extension phase, more and more
SYBR Green I will bind to the PCR product, resulting in an increased fluorescence.
Consequently, during each subsequent PCR cycle more fluorescence signal will be
detected.

dsDNA binding dye assays

SYBR Green

(1)At the beginning of amplification, the reaction

mixture contains the denatured DNA, the primers,
and the dye. The unbound dye molecules weakly
fluoresce, producing a minimal background
fluorescence signal which is subtracted during
computer analysis.
(2) After annealing of the primers, a few dye molecules
can bind to the double strand. DNA binding results in
a dramatic increase of the SYBR Green I molecules
to emit light upon excitation.
(3) During elongation, more and more dye molecules
bind to the newly synthesized DNA. If the reaction is
monitored continuously, an increase in fluorescence
is viewed in real-time. Upon denaturation of the DNA
for the next heating cycle, the dye molecules are
released and the fluorescence signal falls.

Principles of Real-Time PCR techniques

Hydrolysis probe technique: The hydrolysis probe is
conjugated with a quencher fluorochrome, which
absorbs the fluorescence of the reporter fluorochrome
as long as the probe is intact.
However, upon amplification of the target sequence,
the hydrolysis probe is displaced and subsequently
hydrolyzed by the Taq DNA polymerase.
This results in the separation of the reporter and
quencher fluorochrome and consequently the
fluorescence of the reporter fluorochrome becomes
detectable.
During each consecutive PCR cycle this fluorescence
will further increase because of the progressive and
exponential accumulation of free reporter
fluorochromes.

DNA Polymerase 5' exonuclease

activity

Probe is hydrolyzed

Design of Hydrolyzed Probe

Additional species-specific
probe used in the PCR.
The probe is labelled: R =
Reporter = releases a
fluorescence signal
Q = Quencher = neutralizes
the signal, when both
molecules reporter and
quencher are in a short
distance from each other.
As long as the probe is intact, there is no/weakly
fluorescence.

Hydrolysis probe technique: Mechanism

The labelled probe binds to
the single stranded DNA
template. The probe is intact,
no signal is detected.
Next, the primers bind to the
single strands and the
amplification starts.
The fluorescence-labelled
probe is cleaved from the
strand by the activity of the
polymerase and destroyed.

Fluorescence after DNA Amplification

By destruction of the
probe, the connection
between reporter and
quencher is destroyed
as well.
The fluorescence signal
is released and
measured online by the
computer.
The signal remains
active after the double
stranded DNA are
synthesized at the end of PCR Cycle.

Principles of Real-Time PCR

(c) Hybridization probes technique
The Hybridization Probe format is used for DNA detection
and quantitation, providing maximum specificity for product
identification.
Two specifically designed, sequence-specific
oligonucleotide probes, labeled with different dyes, are used.
The sequences of the probes are selected so that they can
hybridize to the target sequences on the amplified DNA
fragment in a head-to-tail orientation, thus bringing the two
dyes into close proximity.
The donor dye (fluorescein) is excited by the blue light
source and emits green fluorescent light at a slightly longer
wavelength.

Hybridization probes
At close proximity, the energy emitted from 1st Probe
excites the acceptor dye attached to the second
Hybridization Probe (15 nucleotides apart).
The acceptor dye emits fluorescent light at a different
wavelength.
This fluorescence signal of acceptor dye is
subsequently detected during the annealing phase and
first part of the extension phase of the PCR reaction.
After each subsequent PCR cycle more hybridization
probes can anneal, resulting in higher fluorescence
signals.

The fluorescence signal is directly proportional to the

amount of target DNA generated during the PCR reaction.

SYBR Green

Hydrolyzed Probe

Hybridization probes

Reverse transcription (RT)-PCR

Convert the RNA to cDNA using reverse transcriptase
Oligo dT

Random primers (pdN6)

Gene-specific primer

3
AAAAAAA
TTTTTTT

3
NNNNNN

NNNNNN

AAAAAAA

3
AGCGA

AAAAAAA

Note the RT and PCR can be done in the same

tube in a one-step reaction.

RT-PCR-- Reverse Transcription + PCR

PCR
mRNA
cDNA
(cDNA)n
TTTTT

Primer with known

sequence

You need to know at least partial sequence of the cDNA you

want to amplify!
RNase H (Ribonuclease H ) is an endoribonuclease that specifically hydrolyzes
the phosphodiester bonds of RNA which is hybridized to DNA

Summary
RT-PCR (reverse transcription-polymerase chain reaction)
RNA is reversely transcribed to DNA.
PCR procedures can be used amplify DNA at exponential
rate.
Gel quantification for the amplified product.
an semi-quantitative method.
Real-Time PCR
The PCR amplification can be monitored by fluorescence
in real time.
The fluorescence values recorded in each cycle represent
the amount of amplified product.
an quantitative method. The current most advanced and
accurate analysis for mRNA abundance. Usually used to
validate microarray result.

Nested primer PCR

Nested primer PCR: PCR amplification is
performed with one set of primers, then some
product is taken - with or without removal of
reagents - for re-amplification with an internallysituated, "nested" set of primers. This process
adds another level of specificity, meaning that all
products non-specifically amplified in the first round
will not be amplified in the second.

This gel photo shows the effect of nested PCR amplification on

the detectability of Chicken anaemia virus (CAV) DNA in a
dilution series: the PCR1 just detects 1000 template molecules;
PCR2 detects 1 template molecule

DNA Sequencing
- The process of determining the order of the nucleotide bases
along a DNA strand is called DNA sequencing
- In 1977 two separate methods for sequencing DNA were
developed: the chain termination method or cycle
sequencing (Sanger) and the chemical degradation method
or Maxam-Gilbert sequencing (Maxam and Gilbert)
- Both methods were equally popular to begin with, but, for many
reasons, the cycle sequencing method is the method more
commonly used today
- This method is based on the principle that single-stranded DNA
molecules that differ in length by just a single nucleotide can be
separated from one another using polyacrylamide gel
electrophoresis

Nobel prize, 1980

Sequencing
Sequencing is the process by which you determine
the exact order of the nucleotides in a given region of
DNA.
Dideoxynucleotide sequencing is done through
complementary chain synthesis and early
termination.
The synthesized chains are visualized by methods
using:
Radioactive labels.
Nonradioactive labels.

DNA Sequencing by Maxam and Gilbert

Synthesize new copies with template, primer (oligo), dNTPs,
and polymerase.
Cleave the strands chemically: Treatment in several steps with
DMS (dimethyl sulfate) or hydrazine, piperidine and various
pH values removes a base and cleaves the two adjacent
phosphoester bonds.
Thus, the DNA ends at G, G+A, C+T, or C.
The DNA sample is treated with dimethyl sulfate (DMS), which
results in methylation of the G residues at the N7 position.
The glycoside bond of the methylated G residue can then be
hydrolysed and the G residue is eliminated.
In the next step, piperidine is added, which reacts with the
hydrolysed sugar residue. This leads to the cleavage of the
backbone.

Overall Cleavage Reaction

Aliquot 1 Cleavage at G only

 Aliquot 2 Cleavage at G and A

DMS also methylates A residues at their N3
position.
Hence, treatment with piperidine also leads to
cleaving of A residues. The rate of this
reaction is only one fifth of that for the
cleavage of G residues.
However, if an acid is added to the reaction
mixture instead of DMS, then both A and G
residues are cleaved at a comparable rate.
The positions of the A residues is determined
by comparing the positions of the G and G +
A residues.

Aliquot 3 Cleavage at C and T

Treatment of DNA with hydrazine and
subsequent reaction with piperidine
releases both C and T residues.

 Aliquot 4 Cleavage at C only

If the reaction is carried out in a 1.5M NaCI
solution, then DNA is only cleaved before
the C residues. Again comparison of the C +
T and C positions

This reaction reveals the positions of the T

residues.

DNA Sequencing Method from Sanger

Synthesize new copies with DNA template, primer (oligo),
dNTPs and ddNTPs, and polymerase.
Dideoxy nucleotides (ddNTP) stop DNA synthesis at
specific nucleotides.
For example, if the ddCTP to the right is incorportated
into a growing strand of DNA, the lack of a free 3 OH
group would prevent the next nucleotide from being
added, and the chain is terminated.
By labeling each ddNTP with a different fluorescent label
the strands with ddA at the end fluorescence different than
those with ddT at the end (and ddG and ddC).

All four reaction is now carried in one tube!

Special Nucleotides in Sanger Reaction

Dideoxynucleotides
Here is an example comparing dATP and ddATP:

dATP

ddATP
NH2

NH2
N

O
-O

P
O-

P
O-

O
H

O
-O

O-

OH

P
O-

P
O-

O
O

O-

The 3 hydroxyl has been changed to a hydrogen in

ddNTPs, which terminates a DNA chain because a
phosphodiester bond cannot form at this 3 location

Mechanism of DNA polymerization

5

O
-O

O-

-O

Base
O

O-

OBase

O-

O
H

H
O

O-

P
O-

OBase

O-

O
H

OBase

:
3
P

H
O

OBase

O
O

H
H

O-

O
O

OH

O
H

-O

Base

DNA polymerase
catalyzed
nucleophilic attack
of the 3-OH on a
phospho-anhydride

H
O

Base

O-

O
Base

O
H

O-

H
H

P
O-

H
OH

-O

P
O-

OH

OH

** Since the 3 OH is changed to a H in ddNTPs, it is unable to form

a phosphodiester bond by nucleophilic attack on the phosphate, and it
will cause a termination in the DNA chain

Sanger DNA Sequencing

(One reaction per tube)

Requirements for Sanger-Coulson Sequencing

DNA to be sequenced must be in single strand
form.
The region to be sequenced must be 3 flanked
by known sequence.
Reagents needed are:
A primer complementary to the known region
to direct chain synthesis.
DNA polymerase.
4 deoxynucleotide triphosphates (dNTPs).
4 dideoxynucleotide triphosphates (ddNTPs).

Sequencing Visualization Methods

Two forms of labeling:
Radioactive
Primer labeled (32P or 33P)
dNTP labeled (35S)
Nonradioactive
Primer labeled
ddNTP labeled (big dye terminator)

Radioactive Primer Labeled Sequencing

1. Unknown fragment

6. One type of ddNTP per reaction

2. with region of known sequence

7. DNA polymerase

3. Complementary primer, 5endlabeled with 32P or 33P

8. ddNTP incorporation
stops chain synthesis

4. dNTPs
dGTP, dCTP, and dTTP

Remember each reaction has many

molecules each one incorporating
its respective ddNTP and stopping
at a different length.

dATP,

5. Four separate reactions

ddATP
5

ddGTP
3

5
Reaction
1

ddCTP
5

5
Reaction
2

ddTTP
3 5

5
Reaction
3

5
Reaction
4

Radioactive Deoxynucleotide Labeled Sequencing

1. Unknown fragment

6. One type of ddNTP per reaction

2. with region of known sequence

7. DNA Polymerase

3. Complementary primer

8. ddNTP incorporation
- stops chain synthesis

4. dNTPs
labeled dATP or dCTP

35S

What is different about

this method? (hint: look at
the colors)

5. Four separate reactions

ddATP
5

ddGTP
3

5
5

Reaction 1

ddCTP

5
5

Reaction 2

ddTTP
3

Reaction 3

Reaction 4

Fluorescent Primer Labeled Sequencing

1. Unknown fragment

6. One type of ddNTP per reaction

2. with region of known sequence

7. DNA Polymerase

3. Four separate reactions

8. ddNTP incorporation
- stops chain synthesis

4. Fluorescent labeled primer.

Different fluorescent dye per
reaction
5. dNTPs: Non-labeled

Whats the big

advantage here?

(dATP, dGTP, dCTP, and

dTTP)
ddATP
5

ddGTP
3

5
5

Reaction 1

ddCTP

5
5

Reaction 2

ddTTP
3

Reaction 3

Reaction 4

Fluorescent Dideoxynucleotide Labeled Sequencing

1. Here we have one reaction vessel, with four copies
of our Unknown fragment.
Dont forget that this and the all the previous reaction
vessels have millions of our unknown fragment. Why
do you think were only showing 4 representatives?
2. A region of known sequence
3. Complementary primer
4. dNTPs
(dATP, dGTP, dCTP, and dTTP)
5. Fluorescent labeled ddNTPs. Each labeled
with a different fluorescent dye
6. DNA Polymerase
7. Again ddNTP incorporation stops
chain synthesis

ddGT
P

5
3 ddATP

3
5
3

3 ddCT
P
5

3
5
3

3
5

ddTT
P

One reaction
vessel
Now we run our products on gel

Gel Separation
The reaction mixtures are separated on a denaturing
polyacrylamide gel.
Denaturing to prevent the DNA from folding up on
itself while it travels through.
Polyacrylamide to separate the strands which differ in
length by only one nucleotide.
Each band corresponds to a sequence of DNA which was
terminated by a particular ddNTP.
This ddNTP is identified by lane in the radioactive method
and by color in the fluorescent method.
The lowest band on the gel is the shortest. The shorter the
strand, the earlier in the synthetic reaction the ddNTP was
incorporated.
The lowest band on the gel is at the 5 end of our
synthesized strand and is complementary to the 3 end of
our unknown fragment.

Gel Visualization
Radioactive method which requires four gel lanes,
one for each reaction vessel.
Readout is done by hand or with a
densitometric scanner.

Nonradioactive fluorescence sequencing

requires only one gel lane because each
nucleotide has a distinct color.
The readout process is done by laser scanner
and recorded by computer.

Gel Electrophoresis and Readout of Reaction Products

ddGTP

ddATP

Nonradioactive

vs.

Radioactive
ddTTP

ddCTP

Sequence of unknown fragment

Sequence of unknown fragment

Longest synthesized band =

3 end of synthesized strand

Shortest synthesized band = 5 end of synthesized strand

Relative Template Quantities needed for Sequencing

Most

Less

Least

Double stranded
plasmid (must
denature) &
standard
sequencing
reaction
Each molecule
of template used
only once

Single stranded
construct such as
phagemid or
M13 & standard
sequencing
Each molecule
of template used
only once

Either double
stranded or
single stranded
construct types
or fragments &
cycle sequencing
Each molecule
is used as
template up to
30 times

DNA Sequencer

Laser-based Detector

Modern DNA Sequencing

4 Labeled ddNTP in one tube

DNA Sequencing

In automated sequencing,
dideoxy nucleotide contain
laser-excitable fluorescent
tags
A scanner controlled by a
PC automatically reads the
gel (four dideoxy rxns run in
the same lane; each dideoxy
emits a unique color)

The lowest band is the

smallest DNA fragment
and provides the first letter
of the sequence.

Capillary Electrophoresis DNA Sequencing

Protein Sequencing
Objective-- determine proteins primary structure (the sequence of the
amnio acids in the polypeptide chain)

Strategy
Determine the number of distinct polypeptide chains
(subunits).
Disulfide bonds must be cleaved.
The amino acid composition of each polypeptide chain can
then be established.
If subunit is too long, they must be fragmented into sets of
smaller peptides by specific cleavage reactions.
The sequence of each fragment is uncovered by
employing Edman degradation.
Put all sequence together by comparing overlaps of the
different sets of fragments.

Nobel prize in 1958

Impact of Protein Sequencing

British biochemist and molecular biologist Dr. Frederick
Sanger is a two time Nobel Prize winner.
Sanger won the 1958 Nobel Prize in chemistry for his
research on the structure of proteins.
The work that won Sanger his second Nobel Prize also led
to his development of the Sanger Sequencing Method
which is the major DNA decoding technique used in the
International Human Genome Project, which has major
health and antiaging implications.
In 1980 he shared the Nobel Prize in chemistry with
American biochemists Paul Berg and Walter Gilbert for
their work on determining the base sequences in nucleic
acids.

Dr. Frederick Sanger

1958

1980

Two Nobel Prizes in Chemistry

Identification of N-terminal amino acid

The N-terminal amino acid can be identified by
Sangers method.
This method involves modification of the N-terminal
residue by flurodinitrobenzene followed by complete
hydrolysis of the peptide.
More recently, fluorescent compounds such as
dansyl chloride or dabsyl chloride are used because
of their higher sensitivity.
The N-terminal aa is the only modified aa and it is
identified by chromatography.
The peptide is completely hydrolyzed and cannot be
reused.

Amino acid sequencing

Peptides are sequenced by Edman Degradation method.
Amino acids are removed and identified sequentially one
residue at a time from the N-terminus.
The N-terminal amino acid is modified by phenyl
isothiocyanate (PITC).
Mild hydrolysis releases the tagged amino acid as a cyclic
derivative phenylthiohydantion-aa (PTH-aa) which is
identified by HPLC ion-exchange chromatography.
The rest of the peptide remains intact, just one aa short
The next cycle releases residue 2.
It is possible to identify ~50 aa from each sample by this
method.

Enzymatic Reactions

Specific Cleavage of Polypeptides

Proteins larger than 50 aa are first hydrolyzed
into shorter peptides
Chemical or enzymatic methods hydrolyze
proteins at specific sites
Peptides are separated by chromatography
Peptides generated by 2 or more cleavage
methods are each sequenced separately.
Sequences of individual peptides are
overlapped together to deduce the entire
protein sequence

Protein Sequencing Example

Method 1 (Trypsin):
ser-glu-phe-his-lys
ala-ile-cys-asp-tyr-thr-ala
gly-leu-pro-arg
Method 2 (staphylococcal protease):
gly-leu-pro-arg-ser-glu
phe-his-lys-ala-ile-cys-asp
tyr-thr-ala
Overall protein sequence:
Gly-leu-pro-arg-ser-glu-phe-his-lys-ala-ile-cys-asp-tyr-thr-ala

N-terminal

C-terminal

Proteins with disulfide linkages

Disulfides are reduced using 2-mercaptoethanol or dithiothreitol (DTT).

-SH groups are blocked by treatment with iodoacetic acid to prevent the
reformation of disulfide bonds.
Position of disulfide linkage is determined by diagonal electrophoresis.
Initially, peptide mixture with intact disulfides are resolved in one direction
by paper electrophoresis.
The paper support is treated with performic acid which oxidizes the
disulfides to charged sulfites and also cleaves the disulfide bond.
Electrophoresis (under the same conditions as in first electrophoresis) in
perpendicular direction (2D-electrophoresis).
Fragments that did not contain any S-S bridges, are positioned along the
diagonal of the matrix as their rate of migration is the same in both
dimensions.
Fragments linked by S-S bond are oxidized by the performic acid and
cleaved, there are two spots for the two fragments, positioned off the
diagonal axis.
The disulfide-linked fragments can be isolated from gel and identified by
sequencing. Then compared to the sequence of whole protein and the
location of a disulfide bond can thus be established.

1st Electrophoresis

4
3

2
1

2nd Electrophoresis