Genetic engineering/Recombinant DNA

Genetics is a science of the structure, function

technology

and

movement of genes whichcode for physiological and

biological, inheritable characteristics
Manipulation of an organisms nucleic acid
Organisms:

artificial

alteration

of

genes

for

specific

purpose: Genetically modified organism (GMO)

Recombinant DNA technology (rDNA): process of cutting a
part of the DNA of one organism or specie and inserting it
into a plasmid (circular DNA, 5,000-25,000 bp) of another
organism
Alteration in genotype and phenotype of organism
Genetic engineering/ gene cloning

Cell

fundamental unit of life

cellular organisms : Eukaryotes and Prokaryotes

Mitochondria, nucleus, plasma

membrane,
cytoplasm
and
other membranes
Chromosomes in nucleus

Simple
bacteria
and
Archaea
Membrane and a cell
wall
No
nucleus,
mitochondria

Cellular genetic material

Genes
Basic units of heredity consisting of DNA
Information for protein synthesis
Growth, physiological and psychological characteristics
Genetic code : sequence of three nucleotide bases called a
codon
Each codon codes for one unique amino acid
21 standard amino acids in eukaryotes
Chromosomes
delicate thread like structures
in the nucleus which
contain condensed DNA
No. depends on the species 23 pairs in humans, 30 pairs
in cattle
When paired: 2n: diploid number
Gamete production: half: haploid (n) number

 Chromosomes duplicate before cell division sister

chromatids connected by centromere
Repetitive nucleotide sequences at each end of a
chromatids: Telomeres; protech chromosomes from

Chromatin

Eukaryotic chromosomes contain an enormous amount of

DNA relative to their condensed length
2 x 108 nucleotide pairs (23 chromosomes 1.87m
condensed into 90 micrometers)
DNA molecule several times longer than the cell diameter
elaborate, multilevel system of DNA packing
Histone: first level of packing
positively charged amino acids bind tightly to negatively
charged DNA
five types of histones: similar amongst different
eukaryotes and even bacteria (H1/H5, H2A, H2B, H3 and
H4),
Two H2A-H2B dimers and a H3-H4 tetramer form the
nucleosome core, H1 linker histone at entry & exit sites of
DNA locking it in place
Unfolded chromatin has the appearance of beads on a

DNA
Deoxyribonucleic acid plus
protein constitute chromatin
shape of a twisted ladder
called a double helix
One strand is labeled 5- 3
and the other is labeled 3- 5
"beginning" of a strand is
defined as 5
5 end: phosphate group
attached to the 5 carbon end
end" of the strand of DNA
molecule is defined as 3
3 end will always have a
hydroxyl (OH) on the 3 carbon

 Repeating
subunits
called nucleotides
phosphate group, a
sugar,
and
a
nitrogenous base
sugar/phosphate
backbone is on the
outside
Inside
four
bases:
adenine (A), guanine
(G), cytosine (C), and

1962: Nobel Prize in Physiology and Medicine

James D.
Watson

Francis H.
Crick

Maurice H. F.
Wilkins

Rosalind Franklin

RNA

 mRNA (Messenger RNA) : copy of DNA encoding a gene.

Carries information from DNA to theribosome, the sites
of protein synthesis (translation) in the cell
rRNA (Ribosomal RNA): component of the ribosomes and
the
protein
synthetic
factories
in
the
cell,
decodingmRNAintoamino
acidsand
interacts
withtRNAs, extremely abundant , 80% of RNA of
eukaryotic cell
tRNA (Transfer RNA): bring the necessary amino acids
corresponding to the appropriate mRNA codon. >20
different tRNA molecules

DNA replication

duplication of DNA prior to cell division

Copying of chromosomes
Regulated by many enzymes including primase, DNA
polymerase, ligase, helicase

Steps involved
1. Identification of the origins of replication
2. Unwinding (denaturation) of dsDNA to provide an
ssDNA template
3. Formation of the replication fork
4. Initiation of DNA synthesis and elongation
5. Formation of replication bubbles with ligation of the
newly synthesized DNA segments
6. Reconstitution of chromatin structure
HIGHLY COORDINATED PROCESS

 DNA could be selectively denatured at sequences

unusually rich in A=T base pairs
replication loops always initiate at a unique point, termed
as origin
Specific origins of replication have been identified and
characterized in bacteria and lower eukaryotes.

when the DNA is unzipped, one strand is oriented in the

opposite direction from 3 to 5. This unzipping takes place
in both directions (from the replication origin) creating a
replication bubble

 DNA is synthesized only in

the 5 to 3 direction
DNA
polymerase
adds
nucleotides
to
each
template strand
One strand is synthesized
continuously:
leading
strand,
proceeds in the
same
direction
as
replication fork movement
The strand oriented in the
opposite direction, from 3
to 5,
is lagging stand
because
the
DNA
polymerase cannot move
continuously
Short
DNA
fragments
(Okazaki fragments), few
hundred to a few thousand
nucleotides in length, are

 Source of nucleotides:
De- novo synthesis (endogenous)
Nucleotide = ribose sugar + phosphate + nitrogen base
Phosphates:
readily
available
in
the
cytoplasm
Ribose is made by utilising the HMP shunt pathway of
glucose
breakdown
Nitrogen bases (purines n pyrimidines) have there own de
novo synthetic pathways from simpler components
Dietary source (exogenous)
Beef (liver, kidney, heart, brain), Pork liver , chicken (liver,
heart), Fresh sea food, sardines, squids, salmon, mackerel,
clams, Dried legumes, split peas, lentils, pinto beans.

 Semi-conservative
Daughter DNA contains one
strand from the original DNA
helix and one new strand
each old strand forms a new
template for a new DNA
strand

DNA Transcription

Also known as Gene expression: Process through which a

messenger RNA (mRNA) strand is copied from template
DNA
mRNA leaves the nucleus to the cytoplasm
RNA polymerase: essential enzyme
helical DNA unwinds near the gene that is to be
transcribed
Only one strand may be transcribed: template
strand/antisense strand
Complimentary DNA strand is called sense strand/coding
strand
5to 3 direction
Prokaryotes: RNA polymerase
Eukaryotes: RNA polymerase
I, II and III

 Initiation: RNA polymerase binds to the promoter gene in

the DNA
promoter dictates finding of the start sequence by RNA
polymerase
Specific sequences on the non coding strand of DNA starts
the unwinding process
Elongation: RNA polymerase reads DNA sequence
Only one strand of DNA is read for the base sequence
RNA synthesized complimentary to this strand
Termination: base sequence at end of the gene
(terminator) which
signals termination of growing RNA chain
RNA chain and the enzyme RNA polymerase are then

DNA Translation

Cytoplasm has special cell structures called ribosomes

(two subunits)
sequences on mRNA are used to make protein
smaller ribosomal subunit binds to mRNA
5 end of mRNA
Initiation: AUG codes for amino acid methionine, initiator
codon
initiator complex: small subunit + mRNA + large ribosomal
subunit
along with another codon initiates reading the information
on the mRNA and protein synthesis

 ElongationtRNA binds mRNA within the context of

ribosome and gets attached to it. The ribosomes align
amino acids correctly according to the information in
mRNA, and peptide bond formation takes place. Then
ribosome moves one codon towards the 3 end of
mRNA which is called translocation.

the
the
the
the
the

 Terminationelongation till the ribosomes reach the

terminator codon, release of polypeptide from the tRNA,
release of tRNA from the ribosome, and breaking up of
the ribosomal subunits from mRNA

Principles of Recombinant DNA Technology

Isolate the gene

Insert it in a host using a vector
Produce as many copies in the host as possible
Separate and purify the product of the gene
Isolation

enzymes are used to recognize a particular nucleotide

sequence and to cut the DNA at that site

Restriction Enzymes

Bacteria contain restriction endonucleases as their defence

mechanism which identify and destroy the invading viral
DNA

 scan the DNA sequence

Recognize and make a cut within specific palindromic
sequences, known as restriction sites, in the DNA
4- or 6 base pair sequence in which the 5 to 3 base pair
sequence is identical on both strands

Types of Restriction endonucleases

Type I- multi-subunit, both endonuclease and methylase
activities, cleaves at random up to 1000 bp from
recognition sequence
Type II- single subunit, cleave DNA within recognition
sequence
Type III- multi-subunit, endonuclease and methylase,
cleaves about 25 bp from recognition sequence
E.g. Hae III

Types of cuts
blunt end: cutting at the same position such that all the
nucleotides are paired

sticky end: cutting at different positions such that each

strand has some unpaired nucleotides to leave staggering
ends
form base pairs with any DNA molecule that has the
complementary sticky end

E.g. EcoRI: name type of bacteria in which the enzyme is

found

 for example EcoRI

R strain of E.coli bacteria
I as it is was the first E. coli restriction enzyme to be
discovered

DNA Ligase
Joins DNA fragments together
Enzymes that cut with staggered cuts result in
complementary ends that can be ligated together
Complimentary sticky ends can be easily ligated, blunt
ends can be ligated together with lower efficiency
covalent bonds between the phosphate and sugar
molecule of the adjacent nucleotide
Ligated DNA usually cannot be re-cut by either original
restriction enzyme

Vectors
Vehicle to carry DNA
to the host cell
plasmids

and

bacterial phages
Cloning
expression vectors

and

Cloning vectors
plasmid that can be modified to carry new genes
If the vector is used only for reproducing the DNA fragment
Essential characteristics of clonng vector:
An origin of replication for autonomous replication
A selectable marker (antibiotic resistance gene, such as
ampr and tetr).
Multiple cloning site (MCS) (site where insertion of foreign
DNA will not disrupt replication or inactivate essential
markers)
Easy to purify away from host DNA
Preferably small in size for easy handling
Relaxed control of replication so that

Plasmids
circular DNA of bacteria
produce genetic products of a foreign DNA segment
carry antibiotic resistance genes, genes for receptors,
toxins or other proteins
Replicate separately from the genome of the organism
can be engineered to form cloning vectors
Plasmid vectors can be designed with a variety of features:
Antibiotic resistance
Colorimetric markers
Strong or weak promoters for driving expression of a
protein

Chimeric DNA
Named

for

(chimera)

mythological

with

body

parts

beast
from

several creatures
a hybrid DNA molecule that has
been constructed in vitro by joining
fragments of separate plasmids and
that

forms

new,

biologically

functional replicon when inserted

into a cell
After cleavage of a plasmid with a
restriction enzyme, a foreign DNA
fragment can be inserted
Ends of the plasmid/fragment are
closed

to

form

"recombinant

Bacterial phages
DNA molecule and a protein coat called capsid
Infect bacteria by attaching to the cell wall and insert the
DNA therein
Bacterio-phage : Head and tail
DNA in the head
Tail for attachment to bacterial cell wall

Natural Recombination in Bacteria

Transformation:
direct
uptake,
incorporation
andexpressionof exogenousDNA (and recombinant
plasmids) taken up from its surroundings through the cell
membrane
Expression of bacterial genes whose proteins carry out the
process

 Transduction: transfer of DNA fragments

bacterium to another via bacteriophage

from

one

 Conjugation: sexual process for transfer of DNA from one

bacterial cell to another

Bacterial Artificial Chromosomes(BACs)

Artificial DNA construct, can hold up to 300 kbs.
The F factor of E.coli is capable of handling large segments
of DNA (150-350kbp)
Recombinant BACs are introduced into E.coli by
electroporation ( a brief high-voltage current). Once in the
cell, the rBAC replicates like an F factor. Example:
pBAC108L
Has a set of regulatory genes, oriS, repE F for plasmid
replication & regulation of copy number; parA and parB for
partitioning F plasmid DNA to daughter cells during
division and stable maintenance of the BAC. Aselectable
marker forantibiotic (e.g. Chloramphenicol) resistance; T7
& Sp6 phage promotersfor transcription of inserted genes

Yeast Artificial Chromosomes(YACs)

Can hold up to 100-3000 kbs.

Designed to replicate as plasmids in bacteria when no foreign
DNA is present. Once a fragment is inserted, YACs are
transferred to cells, they then replicate as eukaryotic
chromosomes.
Contains thetelomeric,centromeric, andreplication origin
sequencesnamedautonomous replicating sequenceneeded
for replication and preservation inyeastcells
Built using an initial circularplasmid, which is typically
broken
into
two
linear
molecules
usingrestriction
enzymes;DNA ligaseis then used to ligate a sequence or
gene of interest between the two linear molecules, forming a
single large linear piece of DNA
For cloning purpose YAC is digested with restriction enzymes
and recombinants are produced by inserting a large fragment
of genomic DNA. This molecule can be maintained in yeast as

Use of restriction endonucleases and DNA

ligases
cut the DNA at different sequences to generate DNA

fragments or cDNA (complimentary DNA) with sticky ends

joined to vectors at complimentary sites
analyzed for correct size by using gel electrophoresis
vectors (plasmids) are first treated with restrictive
enzymes
DNA fragments inserted into vectors with ligases
join DNA by catalyzing formation of covalent bonds
between the 5-phosphate group and 3-sugar group
blunts, large amounts of ligases
Linkers & Adapters: small oligonucleotides for ligation of
blunt end DNA;
add small DNA fragments to the blunt ends sticky ends

Consider a plasmid with a unique

EcoRI site:
5'NNNNGAATTCNNNN3'
3NNNNCTTAAGNNNN5'
An EcoRI restriction fragment of
foreign DNA can be inserted into a
plasmid having an EcoRI cloning site
by:
a) cutting the plasmid at this site with
EcoRI,
b) annealing the linearized plasmid
with the EcoRI foreign DNA fragment,
and,
c) sealing the nicks with DNA ligase.
5'NNNNGAATTCNNNN3'

Cloning: Polymerase Chain Reaction (PCR)

Production of large number of recombinant molecules in a
short time
Amplification, allows for quick and efficient cloning
Thermal cycling, consisting of cycles of repeated heating
and
cooling
of
the
reaction
mixture
forDNA
meltingandenzymatic replicationof the DNA

A process
conceived by Kary
Mullis in 1983

PCR requirements:
DNA templatethat contains the DNA region (target) to be
amplified
Twoprimersthat arecomplementaryto the3ends of
each of thesense and anti-sensestrand of the DNA target
Taq (Thermus aquaticus) polymerase (stable at the high
temperatures 95oC or anotherDNA polymerasewith a
optimum at around 70C
Deoxynucleoside
triphosphates(dNTPs;nucleotidescontaining triphosphate
groups), the building-blocks from which the DNA
polymerase synthesizes a new DNA strand
Buffer solution, providing a suitable chemical environment
for optimum activity and stability of the DNA polymerase
Co-factors:
Divalentcations,magnesiumormanganeseions;
generally Mg2+is used, but Mn2+can be utilized for PCR-

Steps in PCR
Initialization step: Heating the reaction to a temperature of
9496C
(or
98C
for
extremely
thermostable
polymerases), held for 19 minutes, only for DNA
polymerases requiring heat activation byhot-start PCR
Denaturation: heating the reaction to 9498C for 2030
seconds, causesDNA meltingof the DNA template by
disrupting the hydrogen bonds between complementary
bases, Templates for synthesis from primers
Annealing: temperature is lowered to 5065C for 2040
seconds allowing annealing of the primers to the singlestranded DNA template
Stable DNA-DNA hydrogen bonds: only in case of
complimentarity;
Polymerase binds to primer-template hybrid and begins

 Extension/elongation step: Temperature of this step

governed by the polymerase; Taq polymerase: optimum
activity: 75-80C; 72C is used; DNA polymerase
synthesizes a new DNA strand complementary to the DNA
template strand by adding dNTPs that are complementary
to the template in 5' to 3' direction
extension time depends both on the DNA polymerase used
and on the length of the DNA fragment to be amplified;
generally a thousand bases per minute
Final elongation: temperature of 7074C for 515 minutes
after the last PCR cycle to ensure that any remaining
single-stranded DNA is fully extended
Final hold: This step at 415C for an indefinite time may
be employed for short-term storage of the reaction

Number of specific DNA

molecule

copies

exponentially

grows

with

each

PCR cycle.
20-40 cycles: enough DNA
for most applications
Starting with 2 molecules,
after 30 cycles you will
have more than a billion

PCR optimization
Contamination with extraneous DNA: spatial separation of
PCR-setup areas from areas for analysis or purification of
PCR products
Thorough cleaning of the work surface between reaction
setups
Primer-design:

improving

PCR

product

yield

and

in

avoiding the formation of spurious products

Usage of alternate buffer components or polymerase
enzymes can help with amplification of long or otherwise
problematic regions of DNA e.g.formamide, in buffer
systems may increase the specificity and yield of PCR
Computer simulations of theoretical PCR results (Electronic

PCR applications
Isolation of DNA fragments from genomic DNA by selective
amplification of a specific region of DNA
high amounts of pure DNA, enabling analysis of DNA samples
even from very small amounts of starting material
DNA

sequencing:to

determine

unknown

PCR-amplified

sequence
Genetic fingerprinting: a forensic technique used to identify a
person or organism by comparing experimental DNAs
Quantitative PCR: estimation of the amount of a given
sequence present in a sampledetermine levels ofgene
expression; quantitatively measures starting amounts of
DNA, cDNA, or RNA

RT-PCR
thermostable polymerase used in the basic PCR requires a
DNA template
Limited to DNA amplification
Sometimes amplification of RNA is preferred
analyses involving the differential expression of genes in
tissues during development
RNA sample is first reverse-transcribed to cDNA to provide
the

necessary

DNA

template

for

the

thermostable

polymerase
Process is called reverse transcription (RT), hence the
name RT-PCR
Avian myeloblastosis virus (AMV) or Moloney murine

 Real-time PCR: DNA quantification that measures the

accumulation of DNA product after each round of PCR
amplification
Early

diagnosis

ofmalignantdiseases

such

asleukemiaandlymphomas
Identification

of

non-cultivatable

or

slow-growing

microorganisms such asmycobacteria,anaerobic bacteria,

orviruses
Viral DNA can likewise be detected by PCR; detection of
viral diseases

Inserting Recombinant Vectors into Living

Bacterial cell takes up the recombinant DNA in the process
Cells

called Transformation
Rate of uptake of the recombinant plasmids by the
bacterial cells is less
Calcium/electroporation enhances the rate of uptake
Growing the cells in agar medium containing an antibiotic
like tetracyclin

Calcium

 The plasmids have naturally occurring genes for antibiotic

resistance
Bacteria containing plasmids with these genes will grow
on a medium containing the antibiotic- the others die, so
only transformed bacteria survive
Bacterial cells with rDNA are allowed to replicate so that a
very large number of recombinant vectors can be
produced

Selection methods
Phenotypic screening- the protein encoded by the gene
changes the colour of the colony
Using antibodies that recognize the protein produced by a
particular gene

 Detecting the DNA sequence of a cloned gene with a probe

(DNA hybridization)

Analysis of recombinant DNA

Gel Electrophoresis
Separating DNA based on physical properties like size,
electric charge and conformation
Charged molecules in electric field migrate toward either
the positive or negative pole according to their charge
nucleic acids have a consistent negative charge
(phosphate backbone) migrate towards the anode
Gel may be of agarose or polyacrylamide

Agarose gel electrophoresis

Agarose:
polysaccharide
from
seaweed,
typical
concentrations : 0.5 to 2%, non-toxic, can separate DNA
200 to 50,000 bp

Resolution of DNA fragments depends on Gel concentration

large DNA fragments (510 kb) small fragments (0.21 kb)

 DNA sample is added to the gel and electrical charge is

applied through external electrodes
Electric charge causes DNA to migrate to opposite
electrodes
The gel acts as a sieve through which the DNA has to
travel, altering the rate of migration
Buffer: [tris(hydroxymethyl)aminomethane acetate/borate
buffer: pH maintainance, ions to support conductivity
Loading agents: Glycerol & bromophenol blue/Sucrose &
xylene cyanol / bromophenol blue
The DNA is first stained with fluorescent dye (usually EtBr),
then visualized with UV light and the size of the DNA is
analyzed
Comparison against DNA ladder consisting of DNA of
known size

 Chromosomal DNA sample is usually digested with

restriction enzymes to produce a range of fragment sizes
E.g. Three billion base pairs cleaved by a restriction
enzyme recognizing 6 bases results in 750,000 fragments
They appear as a background smear of DNA cleaved into
all possible sizes by the restriction enzyme
If a restriction site is in a repetitive sequence, digestion
will produce a large number of fragments of identical
length
This will appear as a band in the background smear of
fragments

 Plasmid in different supercoiled

forms in the bacteria
Upon
isolation
from
bacterial
culture, they migrate differently on
the gel, major bands and many
minor bands
With a restriction enzyme, the
different forms linearize and unwind,
hence become identical and run at
the same rate, & only one band on
the gel
Supercoiled
forms
and
linear
migrate at different rates
Unknown mol.wt. determined by
comparing distance run by standard
mol.wt. DNA; true for linear DNA
DNA markers cannot be used to
estimate the molecular weight of a

Southern Blot
The technique in which the individual DNA sequences in
agarose gel may be detected by probe hybridization
The DNA within the gel is denatured by exposing it to a
solution of sodium hydroxide
The DNA is then neutralized and transferred out of the gel
onto a membrane (nitrocelluloseor nylon)that binds DNA:
Blotting
It exposes the DNA to the surface so that it may hybridize
to complementary sequences
The membrane bound DNA is then hybridized to a short
specific sequence known as a probe

 Pressure is applied evenly to the gel (either using suction,

or by placing a stack of paper towels and a weight on top
of the membrane and gel), to ensure good and even
contact between gel and membrane.
If transferring by suction,buffer is used to ensure a seal
and prevent drying of the gel.
Buffer transfer bycapillary actionmoves the DNA from the
gel on to the membrane
Ionic interactions bind the DNA to the membrane due to
the negative charge of the DNA and positive charge of the
membrane
Membrane baked in a vacuum or oven at 80 C for 2 hours
or exposed toultraviolet radiation(nylon membrane) to
permanently attach the transferred DNA
Non specific binding reduced by blocking the membrane
with salmon or herring sperm DNA,deionizedformamide,
and detergents such asSDS

Northern Blot
Similar to southern blot, but analyzes RNA instead of DNA
DBM paper: diazobenzyloxymethyl paper
Probes can be DNA, RNA, or oligonucleotides with a
minimum of 25 complementary bases to the target
sequence
Total RNA from a homogenized tissue sample or from cells
is extracted
mRNA can then be isolated through the use of oligo (dT)
cellulose chromatographyto isolate only those RNAs with
apoly(A) tail
Buffer
contains
formamide,
reduces
annealing
temperatureof the probe-RNA interaction, and hence
chances of RNA degradation

Applications
Observe a particular gene's expression pattern between
tissues, organs, developmental stages, environmental
stress levels, pathogen infection, and over the course of
treatment e.g. to show overexpression of oncogenes and
downregulation of tumour-suppressor genes in cancerous
cells when compared to 'normal' tissue, gene expression
in the rejection of transplanted organs
Abundance of mRNA: discovery of newer genes
Expression patterns obtained under given conditions can
provide insight into the function of that gene
Variance in the level of each band on the membrane:
Insight into the size of the product
Variance in size of a gene product can also indicate
deletions or errors in transcript processing

Polyacrylamide gel electrophoresis (PAGE)

Used to separate components of a protein mixture based on
their size
cross-linked polymer of acrylamide
Typical concentration: 3.5 and 20%
Gels poured in between glass plates (or cylinders) as oxygen
inhibits polymerization
Ammonium persulphate-TEMED (tetramethylethyldiamine)
system is conventionally employed, TEMED catalyzes the
formation of free radicals from persulphate and these free
radicals initiate polymerization
Potent neurotoxin, disposable gloves, polyacrylamide is non
toxic, gels may be toxic due to free acrylamide
small range of separation, but very high resolving power
Tracking dyes: bromophenol blue and xylene cyanole;
Loading aids: glycerol andsucrose

Staining dye
Coomassie Brilliant BlueR-250 (CBB): most popular protein
stain
It is an anionic dye, which non-specifically binds to
proteins
used in methanolic solution acidified with acetic acid
Proteins in the gel are fixed by acetic acid and
simultaneously stained
The excess dye incorporated into the gel can be removed
by destaining with the same solution without the dye
The proteins are detected as blue bands on a clear
background

 Stacking gel: larger pore size, does not retard the

migration during the focusing
Resolving gel: smaller pore size, sieving effect,
determines the electrophoretic mobility of the proteins

SDS PAGE
general electrophoresis techniques cannot be used to
determine the molecular weight as mobility depends on both
size and charge
sample of protein, often freshly isolated and unpurified, is
boiled in the detergent sodium dodecyl sulfate and betamercaptoethanol
The mercaptoethanol reduces disulfide bonds
The detergent disrupts secondary, tertiary and quaternary
structures
On the molecular level, proteins are stretched out and coated
with the detergent (which has a negative charge) by this
treatment (charge is proportional to mass)
They will then migrate through a gel towards the positive
pole at a rate proportional to their linear size
Molecular weights with respect to size markers may then be
determined

 Two-dimensional
(2-D)
gelwhich
spreads
the
proteins from a single
sample
out
in
two
dimensions
according to isoelectric
point (pH at which they
have neutral net charge)
in the first dimension, and

 Separation of proteins in two steps, according to two

independent properties
Generally no two proteins will have similarity in two
distinct properties
the first-dimension is isoelectric focusing (IEF), which
separates proteins according to their isoelectric points
(pI);
the second-dimension is SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), which separates proteins
according to their molecular weights (MW)
Placing the sample in gel with a pH gradient, and
applying a potential difference across it
In the electrical field, the protein migrates a long the pH
gradient, until it carries no overall charge
This location of the protein in the gel constitutes the
apparent pI of the protein

Western Blot (Immunoblot)

A technique used to separate and identify proteins
Proteins separated by SDS page (based on molecular
weight) are probed using antibodies
The antibodies are usually labelled
Those that remain bound to their specific targets identify
specific proteins in the cell
The intensity of the signal reflects the level of expression
It can also reveal mutations by anomalies of migration
E.g. A deletion might cause a shorter protein
Proteins are made accessible to antibody detection by
transferring them from within the gel onto a
nitrocelluloseorpolyvinylidene
difluoride(PVDF)
membrane
Primary method for transferring the proteins is
calledelectroblotting

 Proteins transferred as per the same organization as in the

gel
Older method: placing a membrane on top of the gel, and
a stack of filter papers on top of that
Entire stack is placed in a buffer solution which moves up
the paper bycapillary action
Proteins are exposed on a thin surface layer for detection
Membranes chosen for their non-specific protein binding
properties
Binding based upon hydrophobic interactions, charged
interactions
Nitrocellulose membranes are cheaper than PVDF, but are
far more fragile and do not stand up well to repeated
probings
Uniformity and overall effectiveness of transferchecked by
staining
membrane
with
Coomassie
Brilliant

Blocking
Prevent interaction of probing antibodies with membrane
non-specifically
3-5%Bovine serum albumin(BSA) or non-fat dry
milk(both are inexpensive) inTris-Buffered Saline(TBS),
with small percentage of detergent such asTween
20orTriton X-100 (prevent elution of blocking protein and
non-specific interactions with blocking protein)
Protein in the dilute solution binds to membrane in all
places where the target proteins have not attached
Antibodies bind only to target proteins
Reduces "noise" in the final product of the western blot
Clearer results, and eliminates false positives

Detection
modified antibody which is linked to a reporter enzyme
colorimetric reactions
Two steps : Primary antibody
Generated when a host species or immune cell culture is
exposed to the protein of interest (or a part of it)
sensitive and specific detection tools that bind the protein
directly
buffered saline solution with a small percentage of
detergent, and sometimes with powdered milk or BSA
Incubation time 30 min to overnight, at different
temperatures, higher temperature higher specific and
non-specific binding
Secondary antibody
directed at a species-specific portion of the primary
antibody

 due to its targeting properties, tends to be referred to as

"anti-mouse," "anti-goat
Linked tobiotinor to a reporterenzymesuch asalkaline
phosphataseorhorseradish peroxidase
Several secondary antibodies will bind to one primary
antibody and enhance the signal
E.g. horseradish peroxidase-linked secondary antibodies
are used to cleave a chemiluminescent agent,
producesluminescence proportional to amount of protein
One step method
Antibody which both recognizes the protein of interest and
contains a detectable label, probes which are often
available for knownprotein tags

Analysis
unbound probes are washed away
Size approximations are taken by comparing the stained
bands to that of the marker or ladder loaded during
electrophoresis
a structural protein, such as actin or tubulin, that should
not change between samples is analyzed in same way
amount of target protein isnormalizedto the structural
protein to control between groups
Allows for correction in case of errors/incomplete transfers
Detection may be by colorimetric, chemiluminescent,
radioactive and fluorescent methods

Eastern Blot
Extension of western blot
Analyzes
proteinwith
post
translational
modifications(PTM) such as lipids and glycoconjugates
Probes
used
may
detectlipids,
carbohydrate,phosphorylationor
any
other
protein
modification
Probes used are is anaptamer (DNA/RNA/peptide
molecule)rather than an antibody

Significance
Mostproteinsthat are translated frommRNAundergo
modifications before becoming functional in cells; posttranslational modifications(PTMs)
The nascent or folded proteins, which are stable under
physiological conditions, are subjected to a battery of
specific enzyme-catalyzed modifications on the side chains
or backbones
Those occurring at theN-terminusof theamino acid:
translocation across biological membranes.
These include secretory proteins in
prokaryotesandeukaryotesand also proteins that are
intended to be incorporated in various cellular and
organelle membranes
eglysosomes,chloroplast,mitochondriaandplasma

Restriction Fragment Length Polymorphism

(RFLP)
After the cloned DNA is isolated; technique that exploits

variations in homologous DNA sequences

Differencesbetween
samples
ofhomologousDNAmolecules that come from differing
locations ofrestriction enzyme sites
Restriction mapping provides a compilation of the number,
order, and distance between restriction endonuclease
cutting sites along a cloned DNA fragment
Detected by southern blotting
Important tool ingenome mapping, localization of genes
forgenetic disorders, determination ofriskfor disease, and
paternity testing
Analyze the DNA of members of a family afflicted by the
disease, analysis of other families could reveal who was at
risk for the disease, or who was likely to be acarrierof the

Sanger dideoxy DNA method

Manual DNA sequencing, chain termination method
Technique
utilizes
dideoxynucleotide
triphospates
(ddNTPs), molecules that differ from deoxynucleotides by
the having a hydrogen atom attached to the 3' carbon
rather than an OH group

Molecules terminate DNA chain elongation because they

cannot form a phosphodiester bond with the next
deoxynucleotide

 Sanger reaction consists of the following:

a strand to be sequenced (denatured from dsDNA using
NaOH)
DNA

primers

(short

pieces

of

DNA

that

are

both

complementary to the strand which is to be sequenced

and radioactively labelled at the 5' end), a mixture of a
particular ddNTP (such as ddATP) with its normal dNTP
(dATP in this case), and the other three dNTPs (dCTP,
dGTP, and dTTP). The concentration of ddATP should be
1% of the concentration of dATP
If the ddATP is only 1% of the total concentration of dATP,
a whole series of labelled strands will result

 Reaction is performed four times using a different ddNTP

for each reaction
When these reactions are completed, a polyacrylamide gel
electrophoresis (PAGE) is performed
One reaction is loaded into one lane for a total of four
lanes
Gel

is

transferred

to

nitrocellulose

filter

and

autoradiography is performed so that only the bands with

the radioactive label on the 5' end will appear
Shortest fragments will migrate the farthest
The bottom-most band in a particular lane indicates that
its particular dideoxynucleotide was added first to the
labeled primer

 the band that migrated the farthest was in the ddATP

reaction mixture Therefore, ddATP must have been added
first to the primer, and its complementary base, thymine,
must have been the base present on the 3' end of the
sequenced strand
If one reads the bases from the bottom up, one is reading
the 5' to 3' sequence of the strand complementary to the
sequenced strand
Thus sequenced strand can be read 5' to 3' by reading top
to bottom the bases complementary to the those on the
gel

Expression vectors
Plasmidthat is used to introduce a specificgeneinto a
target cell
Engineered to contain regulatory sequences that act
asenhancerandpromoterregions and lead to efficient
transcription of the gene carried on the expression vector
Goal: production of large amounts of stablemessenger
RNA, and therefore proteins
Basic tools ofbiotechnology for production of recombinant
proteins like insulin

The promoters are found in all genes that help in transcription which
eventually will lead to produce proteins, the core promoter. This is a
sequence of dna bases which are located upstream of about -35
bases( ie. the opposite direction of the transcription reation). They help in
aid of the transcription to happen smoothly by having effective
interaction with the transcription factors, thus forming a transcription
complex and it also contains the RNA polymerase binding and regulatory
sites necessary for transcription to happen. They usually have sequences
called consensus sequence like TATA box in eukaryote or prinbow box in
prokaryotes, which initiates the transcription by unwinding the DNA.
Apart from the basal promoter, there are unique upstream promoter
sequences which attract other sequence-specific transcription factors and
help construct the transcription complex. Different genes are thus
regulated by different promoters and combinations of transcription
factors even though transcription factors are shared among genes within
the cell.
Enhancer DNA sequences bind transcription factors with special protein
called enhancer-binding proteins which increase the rate of transcription.
Enhancer sequences may be at a distance of kilobases away from the
gene they influence. An enhancer complex may interact with promoter

 Types:
E. coli expression vector
Yeast expression vector
Mammalian expression vector
Purification of the protein is required; since the vector is
introduced to a host cell, the protein of interest should be
purified from the proteins of the host cell. To simplify, the
cloned gene should have a tag This tag could behistidine
(His) tag (nickel/cobalt)or any other marker peptide
Features
In addition to the origin of replication, selective marker,
multiple cloning site, expression vector has to contain a
promoter and terminator for transcription
The inserted gene should have a start codon and a stop
codon for translation

E. coli expression vector

Prokaryotic systems
E. coli is a popular and well understood system for
heterologous protein expression
Simple, convenient, rapid and cheap

Expression options
Direct expression. E. coli cytoplasm is a reducing
environment - difficult to ensure proper disulphide bonds
formation.
Fusion expression. Ensures good translation initiation. Can
overcome insolubility and/or instability problems with
small peptides. Has purification advantages based on
affinity chromatography.

 Cloned gene is introduced into an expression vector 3to a

sequence, known as carrier sequence, which codes for
amino terminus of a highly expressed protein known as
the carrier protein
The carrier sequence provides the necessary signals for
good expression, and the expressed fusion protein
contains an N-terminal region encoded by the carrier
The carrier sequence can also code for an entire functional
moiety or even for an entire protein that can be exploited
in purifying the protein, either with antibodies or with an
affinity purification specific for that carrier protein
Alternatively unique physical properties of the carrier
protein (e.g., heat stability) can be exploited to allow
selective purification of the fusion protein

 Bacillus subtilis is a better choice for secretion of a

prokaryotic protein than E.coli: Secretes proteins to the
medium, including own proteases : therefore there might
be a problem with proteolysis. Overcome with mutants
which are protease deficient
It is capable of secreting functional extracellular
proteins directly to the culture medium
It is non- pathogenic; it has no significant bias in
codon usage, and a great deal of vital information
concerning its transcription and translation mechanisms
is available,
genetic manipulation
and large-scale
fermentation have now been acquired
Industrial enzymes in large quantities
Medical applications require intact proteins with both
authentic primary sequences and properly folded threedimensional structures

Yeast expression vector

Eukaryotic systems
Yeast
systems
for
heterologous
expression:
Saccharomyces cerevisiae
Eukaryote, unicellular, GRAS (Generally Regarded As Safe),
capable of performing post-translational modifications.
Intracellular expression - higher protein yields, but more
difficult extraction and purification. Additional potential
problem:
co- and post-translational processing of proteins at Nand C-termini
proteolytic degradation
addition of tags might result in aggregation and
insolubility

Pichia pastoris
High growth rate and is able to grow on a simple,
inexpensive medium
Shake flasks or afermentor, suitable for both small and
large scale production
Twoalcohol oxidasegenes, AOX1 and AOX2, which have a
stronglyinduciblepromoter
Use methanol as a carbonand energy source
Gene for the desired protein is introduced under the
control of the AOX1 promoter, protein production by the
addition of methanol
Protein purification is easier as protein is secreted into the
medium

Baculovirus expression system

Insect cells from Spodoptera frugiperda infected with
baculoviruses Autographa californica (multinucleocapsid
nuclear polyhedrosis virus AcMNPV)
The baculovirus genome contains the gene, encoding
polyhedrin, an abundant viral protein. This protein
accumulates in the insect cell towards the end of the
infectious cycle and is the major constituent of a protein
matrix, containing many virions trapped (polyhedron). Many
of these polyhedrons are released into the environment
after cell lysis and the death of a single host organism
The promoter of the polyhedrin gene is very strong,
however the gene is not essential for the viral reproduction
cycle. For these reasons it could be replaced with a
heterologous gene and this is the strategy used in the

Advantages
The polyhedrin gene is not required for the continuous
production of infectious virus in insect cell culture. Its
sequence is replaced with that of the heterologous gene
The polyhedrin gene promoter is very strong. This
determines a very high level of production of recombinant
protein
The polyhedrin promoter is highly active very late in
infection when the lytic virus is already killing the host
cells, giving a reasonable chance for high levels of
expression
This system is capable of post-translational modifications;
proteins unable to be expressed in E. coli have been
successfully expressed in the insect cell system

Disadvantages
Expensive
Glycosylation in insect cells is different (insect cells unable
to produce complex N-linked side chains with penultimate
galactose and terminal sialic acid) from that in vertebrate
cells, therefore, a problem for therapeutic proteins
A large fraction of the RP can be poorly processed and
accumulates as aggregates
Discontinuous expression: baculovirus infection of insect
cells kills the host and hence the need to re-infect fresh
cultures for each round of protein synthesis
Inefficient for production on a commercial scale

Mammalian cell lines expression systems

Two modes of expression - transient and stable
Cell lines used. Three cell types are dominant in transient
expression: human embryonic kidney (HEK), COS
(fibroblast-like from monkey kidney tissue) and baby
hamster kidney (BHK), whilst CHO (Chinese hamster ovary)
cells are used predominantly for stable expression
Mammalian expression vectors: Eukaryotic origin of
replication is from an animal virus: e.g. Simian virus 40
(SV40). Popular markers for selection are the bacterial gene
Neor (encodes neomycin phosphotransferase), which
confers resistance to G418 (Geneticin), and the gene,
encoding dihydrofolate reductase (DHFR). When DHFR is
used, the recipient cells must have a defective DHFR gene,
which makes them unable to grow in the presence of
methotrexate (MTX), unlike transfected cells with a

 DHFR-deficient CHO host cells are transfected with an

expression vector containing the DHFR gene under a weak
sv40 promoter and the gene of interest (for expressing a
MAb

or

other

recombinant

protein)

under

strong

cytomegalovirus (CMV) promoter

Increasing concentrations of the MTX selection agent
stepwise (from 50 nM up to 5 M) leads to several hundred
copies of the DHFR gene
During that amplification process, a proportional increase in
the copy number of a nearby linked gene of interest
dramatically increases the clones specific productivity
levels

 Promoter sequences that drive expression of both marker

and cloned heterologous gene, and the transcription
termination (polyadenylation signals) are usually from
animal viruses (human CMV, SV40, herpes simplex virus) or
mammalian genes (bovine growth hormone, thymidine
kinase)
Strategies for co-expression of two cloned genes
Advantages
Sufficient quantities of higher eukaryotic proteins with posttranslational modifications
Disadvantages
Cultures characterised by lower cell densities and lower
growth rates. Maintenance and growing very expensive
Gene manipulations are very difficult
Mammalian cells might contain oncogenes or viral DNA, so
recombinant protein products must be tested more

Therapeutic proteins
Insulin
Made by pancreatic beta cells
Enables cells to take up glucose from the bloodstream to
use in production of ATP
Insufficient insulin causes diabetes (insulin dependent
diabetes mellitus -IDDM)
Cells cannot take up glucose
Insufficient ATP is made
Glucose spills into urine (excreted by kidneys; kidney tries
to dilute glucose by excreting large amounts of water)
Necessity to inject insulin to avoid physiological
complications

 Complications of diabetes (60 x 106 worldwide cases)

Retinopathy
Kidney failure
Nerve disorders
Circulatory diseases (including gangrene and stroke)
Diabetic coma (pH imbalance caused by fat metabolism
producing ketones)
Insulin is made up of 2 chains = 51 amino acids total
A chain = 21 amino acids; B chain = 30 amino acids
Two disulfide bonds hold A and B together (interchain
disulfide bond)
One disulfide bond within the A chain (intrachain disulfide
bond)
Gene encoding the insulin protein is found on chromosome
11

 Before recombinant insulin was available, insulin was

obtained from cows or pigs pancreas (7-10 lb pancreatic
tissue per patient per year)
Cow (Bovine) = 3 amino acid differences
Pig (Porcine) = 1 amino acid difference
Amino acid differences can stimulate allergic responses
Therefore human insulin is preferred
Patients immune systems do not produce antibodies
against human insulin as they do with bovine or porcine
insulin
Projected decline in the production of animal-derived
insulin
Need for a more reliable and sustainable method of

Recombinant DNA Technique forHumulin

Restriction enzymes
used to cut out insulin
gene and to cut a
bacterial
(E. coli)
plasmid at the same
sticky ends

 Mutant strains of E. coli used to avoid bacteria attacking

foreign genes
Insert insulin gene next to E. Coli -galactosidase gene
which controls transcription of genes
Bacterial cells replicate and make copies of insulin gene
Insulin protein is purified (B-galactosidase removed)
Chains are mixed and disulfide bridges form
Yeast cells provide a sterile growth medium
Final product is Humulin - chemically identical to human
insulin

Human Growth Hormone (HGH)

HGH promotes overall body growth by increasing: amino
acid uptake by cells, protein synthesis and fat utilization
for energy
Dwarfism caused by insufficient production of HGH by
the pituitary gland
Growth retardation
Chubby face
Baby fat around waist
Unusual body properties as an adult
~ 4 feet tall only
IQ = Normal
HGH can treat dwarfism to help undersized children
reach their normal height and size

Old method:
Purification of HGH from cadaver pituitary
glands
8 cadavers/year for 8 10 years per patient
Risky to use brain tissue
Prion disease transmission: Creutzfeldt-Jacob Disease
(CJD)
Muscle wasting
Convulsions
Tremors
Dementia
24 cases reported by 1993 in France from cadaver
HGH
New method
Gene production for HGH synthesis
Protropin Genentech
Humatrope Eli Lilly

Monoclonal antibodies

Hybridoma technology

Interferon

Proteins produced by a cell infected by a virus

Resist the immediate invasion by virus of the neighbouring
healthy cells
Resist abnormal cellular multiplication
rDNA technology: interferons with enhanced specific activity
(antiviral activity)
Yeast cells are the perfect host due to mechanism to carry
out post translational modifications
DNA sequence encoding for human interferon is isolated and
cloned to yeast alcohol dehydrogenase gene in a plasmid
vector
Large industrial fermenters
Hybrid interferons

Production of Interferon-
Human fibroblasts produce Interferon- biomolecules
Gene is isolated and incorporated into a plasmid vector
E.coli cells are used for multiplication in large industrial
fermentors
Purification
Uses
treatment of large number of viral diseases
Cancer
Common cold, influenza

Tissue Plasminogen Activator

Enzyme that helps in dissolving blood clots

Tissue plasminogen activator activates

plasminogen

to

produce plasmin
Removing arterial thrombi: protects heart and brain
Does not compromise blood clotting capability in other
tissues
faster than other thrombolytic agents
i.v. administration, lower side-effects
Recombinant Tissue Plasminogen Activator
cDNA molecule complementary to concerned gene
synthetic plasmid, vector: mammalian cells
Cloning in fermentors, isolated, purified from culture media
first pharmaceutical product of mammalian cell culture
2nd generation: ALTEPLASE and RETEPLASE

Vaccines
live genetically modified organisms
recombinant inactivated (killed) vaccines
genetic vaccines
Advantages: safer, more efficacious, and/or less expensive
Requirements
Antigens critical for inducing protection
Lack of pathogenicity of the disease agent
Disease response of host

Live genetically modified organisms

Viruses or bacteria with one or more genes deleted or
inactivated: attenuate the disease agent
double-knockout to avoid revertation
Genes for pathogenecity should not be the ones governing
viability and ability to induce immune response
Creating infectious clone from genome of disease agent
modified to produce live genetically modified organism
Vaccines carrying a foreign gene from another disease agent
(vaccine vector) : bacteria, viruses, or plants carrying a gene
from another disease agent
Expressed and induces an immune response when the host is
vaccinated
Viral and bacterial vectors induce a protective response
against themselves and the disease agent
Foreign genes must be inserted into the genome in a viable
form

Recombinant Inactivated Vaccines

subunit vaccines containing only part of the whole organism
synthetic peptides representing basic portion of protein
inducing the immune response
whole proteins extracted from disease agent or expressed
from cloned genes
Expression may be cell free or using whole cells
Virus-like particles (VLPs) : one/more cloned genes
representing structural proteins of a virus are expressed
simultaneously and self-assemble into VLPs
VLPs are immunogenic
Structure like virus but cannot replicate because they do not
contain any genetic material
Injected along with adjuvant that stimulates the immune
system to respond to the vaccine

Genetic Vaccines
Also referred to as DNA vaccines; circular pieces of DNA,
called plasmids, which contain a foreign gene from a disease
agent and a promoter to initiate the expression of the protein
from that gene in the host
Intradermal or intramuscular injection of naked DNA after
purification of recombinant plasmids from bacteria
Host cells take up the DNA, and an immune response is
induced to the protein expressed from the foreign gene
Designed to include different immune-stimulatory genes that
trigger different compartments of the immune system
CpG motifs: these unmethylated motifs act as an adjuvant
E.g. Gardasil, made by Merck & Co., Inc.: for the prevention
of genital warts, vaginal and vulvar cancer, cervical cancer
due to human papillomavirus (HPV)
Studies underway for DNA vaccines for influenza, malaria,
HIV, cancers, Hepatitis B virus, emerging infectious diseases
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