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and
artificial
alteration
of
genes
for
specific
Cell
Simple
bacteria
and
Archaea
Membrane and a cell
wall
No
nucleus,
mitochondria
Chromatin
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
James D.
Watson
Francis H.
Crick
Maurice H. F.
Wilkins
Rosalind Franklin
RNA
DNA replication
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
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
DNA Translation
the
the
the
the
the
Restriction Enzymes
Types of cuts
blunt end: cutting at the same position such that all the
nucleotides are paired
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
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
from
one
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
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
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
diagnosis
ofmalignantdiseases
such
asleukemiaandlymphomas
Identification
of
non-cultivatable
or
slow-growing
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
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
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
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
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
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
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
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
primers
(short
pieces
of
DNA
that
are
both
is
transferred
to
nitrocellulose
filter
and
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
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.
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
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
or
other
recombinant
protein)
under
strong
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
Restriction enzymes
used to cut out insulin
gene and to cut a
bacterial
(E. coli)
plasmid at the same
sticky ends
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
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
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
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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