Genetic engineering/Recombinant DNA technology

• Genetics is a science of the structure, function 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• Chromosomes as circular DNA in

cytoplasm, e.g. Bacteria, viruses

Cellular genetic materialGenes• 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 eukaryotesChromosomes• 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 • 22+ sex chromosomes (allosome) XX in females and XY in males

• Chromosomes duplicate before cell division sister chromatids connected by centromere

• Repetitive nucleotide sequences at each end of a chromatids: Telomeres; protech chromosomes from degradation/fusion

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 string, DNA

wound around the nucleosome; 50 base pairs of DNA separating each pair of nucleosomes

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 thymine (T)

• A always pairs with T• C always pairs with G

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 the ribosome, the sites of protein synthesis (translation) in the cell

• rRNA (Ribosomal RNA): component of the ribosomes and the protein synthetic factories in the cell, decoding mRNA into amino acids and interacts with tRNAs, 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

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

Steps involved

• 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

• Unwinding of the strands: helicase

• 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 built in 5’ to 3’ direction , linked by enzyme ligase

• Source of nucleotides:

• De- novo synthesis (endogenous)• Nucleotide = ribose sugar + phosphate + nitrogen base

Phosphates: readily available in the cytoplasmRibose 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 • 5’to 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 released from the

DNA

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

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

• Termination–elongation 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 geneIsolation

• 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

•  Haemophilus aegyptius bacteria

• 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• the order in which the restriction enzyme was identified and isolated

Types of cuts

• 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 and expression

vectors

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

multiple copies can be obtained

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 mythological beast (chimera) with

body parts from several creatures• a hybrid DNA molecule that has been

constructed in vitro by joining fragments of

separate plasmids and that forms a 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 a "recombinant plasmid”• Plasmid can replicate when placed in a

suitable bacterial host

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 λ: Head and tail• DNA in the head • Tail for attachment to bacterial cell wall

Natural Recombination in Bacteria• Transformation: direct uptake, incorporation and expression of

exogenous DNA (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 from one bacterium to another via bacteriophage

• 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. A selectable marker for antibiotic (e.g. Chloramphenicol) resistance; T7 & Sp6 phage promoters for transcription of inserted genes and a cloning segment

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 the telomeric, centromeric, and replication origin sequences named autonomous replicating sequence needed for replication and preservation in yeast cells

• Built using an initial circular plasmid, which is typically broken into two linear molecules using restriction enzymes; DNA ligase is 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 YAC

• Less stable than BACs, cloned DNA ≈ multiple genomic regions, deletion of segments from a cloned region, and rearrangement of genomic segments

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' NNNNGAATTCNNNN 3' 3’ NNNNCTTAAGNNNN 5'

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' NNNNGAATTCNNNN 3' 3' NNNNCTTAAGNNNN 5’

This results in a recombinant DNA molecule.

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 for DNA melting and enzymatic replication of the DNA

A process conceived by Kary Mullis in 1983

PCR requirements:• DNA template that contains the DNA region (target) to be amplified• Two primers that are complementary to the 3‘ ends of each of the sense

and anti-sense strand of the DNA target• Taq (Thermus  aquaticus) polymerase (stable at the high temperatures

≈95oC or another DNA polymerase with a optimum at around 70 °C• Deoxynucleoside triphosphates (dNTPs; nucleotides containing

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: Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis

• Monovalent cation: potassium ions (primer annealing)

Steps in PCR• Initialization step: Heating the reaction to a temperature of 94–96 °C (or

98 °C for extremely thermostable polymerases), held for 1–9 minutes, only for DNA polymerases requiring heat activation by hot-start PCR

• Denaturation: heating the reaction to 94–98 °C for 20–30 seconds, causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, Templates for synthesis from primers

• Annealing: temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template

• Stable DNA-DNA hydrogen bonds: only in case of complimentarity; • Polymerase binds to primer-template hybrid and begins DNA formation

• Extension/elongation step: Temperature of this step governed by the polymerase; Taq polymerase : optimum activity: 75-80 °C; 72 °C 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 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended

• Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction

Number of specific DNA molecule

copies grows exponentially 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) may be

performed to assist in primer design

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

sample—determine levels of gene 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 leukaemia virus

(MuLV) reverse transcriptases

• Real-time PCR: DNA quantification that measures the accumulation of

DNA product after each round of PCR amplification

• Early diagnosis of malignant diseases such as leukemia and lymphomas

• Identification of non-cultivatable or slow-growing microorganisms such

as mycobacteria, anaerobic bacteria, or viruses

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

Inserting Recombinant Vectors into Living Cells• Bacterial cell takes up the recombinant DNA in the process 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 DNAGel 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 (5–10 kb) small fragments (0.2–1 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 circular DNA molecule

• 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 ( nitrocellulose or 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

Southern Blot

• 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 by capillary action moves 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 to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA

• Non specific binding reduced by blocking the membrane with salmon or herring sperm DNA, deionized formamide, and detergents such as SDS

• X-ray film by autoradiography in the case of a radioactive or fluorescent probe or colour reaction in case of probe labelled with chromogenic dye

• 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

chromatography to isolate only those RNAs with a poly(A) tail• Buffer contains formamide, reduces annealing temperatureof the probe-

RNA interaction, and hence chances of RNA degradation

Northern Blot

• 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

Applications

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 and sucrose

Staining dye• Coomassie Brilliant Blue R-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 beta-mercaptoethanol• 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• As SDS is also anionic, it may interfere with staining process; large volume of

staining solution is recommended, at least ten times the gel volume

• Two-dimensional (2-D) gel which 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 according to their molecular weight in the second dimension

• 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

• 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 nitrocellulose or polyvinylidene difluoride (PVDF) membrane

• Primary method for transferring the proteins is called electroblotting

Western Blot (Immunoblot)

• 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

by capillary 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 transfer checked by staining

membrane with Coomassie Brilliant Blue or Ponceau S dyes, latter preferred due to higher sensitivity and water solubility, making it easier to destain and probe the membrane

Blocking• Prevent interaction of probing antibodies with membrane non-

specifically• 3-5% Bovine serum albumin (BSA) or non-fat dry milk (both are

inexpensive) in Tris-Buffered Saline (TBS), with small percentage of detergent such as Tween 20 or Triton 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

reactionsTwo 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 bindingSecondary antibody• directed at a species-specific portion of the primary antibody• an anti-mouse secondary will bind to almost any mouse-sourced primary

antibody

• due to its targeting properties, tends to be referred to as "anti-mouse," "anti-goat”

• Linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish 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, produces luminescence 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 known protein 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 is normalized to 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

• Extension of western blot• Analyzes protein with post translational modifications (PTM) such as

lipids and glycoconjugates• Probes used may detect lipids, carbohydrate, phosphorylation or any

other protein modification• Probes used are is an aptamer (DNA/RNA/peptide molecule) rather than

an antibody

Eastern Blot

Significance • Most proteins that are translated from mRNA undergo modifications

before becoming functional in cells; post-translational 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 the N-terminus of the amino acid: translocation across biological membranes.

• These include secretory proteins in prokaryotes and eukaryotes and also proteins that are intended to be incorporated in various cellular and organelle membranes eg lysosomes, chloroplast, mitochondria and plasma membrane

• Expression of post-translated proteins is important in several diseases

• After the cloned DNA is isolated; technique that exploits variations in homologous DNA sequences

• Differences between samples of homologous DNA molecules that come from differing locations of restriction 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 in genome mapping, localization of genes for genetic

disorders, determination of risk for 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 a carrier of the mutant genes

Restriction Fragment Length Polymorphism (RFLP)

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 a 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

• Plasmid that is used to introduce a specific gene into a target cell

• Engineered to contain regulatory sequences that act

as enhancer and promoter regions and lead to efficient transcription of

the gene carried on the expression vector

• Goal: production of large amounts of stable messenger RNA, and

therefore proteins

• Basic tools of biotechnology for production of recombinant proteins like

insulin

Expression vectors

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 complexes by bringing the sites into direct contact, may be by formng a loop-ike stucture.

• 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 be histidine (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

• 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.

Prokaryotic systems

• Cloned gene is introduced into an expression vector 3’ to 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 three-dimensional structures• E.g. human interleukin (hIL)-l

Yeast expression vector

• 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 N- and C-termini• proteolytic degradation • addition of tags might result in aggregation and insolubility

Eukaryotic systems

Pichia pastoris

• High growth rate and is able to grow on a simple, inexpensive medium• Shake flasks or a fermentor, suitable for both small and large scale

production• Two alcohol oxidase genes, AOX1 and AOX2, which have a

strongly inducible promoter• Use methanol as a carbon and 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

• 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 Baculovirus expression system

Baculovirus expression system

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

• 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 functional DHFR gene

Mammalian cell lines expression systems

• 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 a

strong cytomegalovirus (CMV) promoter

• Increasing concentrations of the MTX selection agent stepwise (from 50 nM

up to 5 µM) 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 genesAdvantages• Sufficient quantities of higher eukaryotic proteins with post-translational

modificationsDisadvantages• 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 extensively

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

Therapeutic proteins

• 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 obtaining the

product

Recombinant DNA Technique for Humulin

• 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 β-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

• 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

Human Growth Hormone (HGH)

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

• protein, or peptide chain containing 191 amino acids which is injected; broken down or digested by the oral route

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-β• Human fibroblasts produce Interferon-β 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• First commercial e.g. VectorVax FP-N (Zeon Corporation, Japan)

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,

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