Recombinant dna

technology
 introduction
• Used to alter phenotype of organism (host) – introducing genetically
transformed vector & assimilating into genome of organism

• Includes insertion of foreign part of DNA assembly into genome

which comprises concerned gene

• r-DNA technology includes selection of desired gene for

administration into host, selection of perfect vector (gene is
combined)

• r-DNA has to be introduced into host, sustain in host & get carried
forward to off-spring

• Allows probable isolation of one gene or different DNA segment –

helps researchers to regulate nucleotide arrangement, study
transcripts, mutate & re-insert modified sequence into living
• DNA is utilized as molecule of heredity by all organisms

• DNA from diverse organisms can be cut & pasted together to

form r-DNA

• 1972- Paul Berg – 1st r-DNA molecule; combined DNA

fragment from 2 diverse viruses using specific restriction
enzymes & ligase

• Restriction enzymes – molecular scissors

• DNA from different source – cut with same rest. enzyme, cut
ends can be combined together & sealed into constant DNA
strand by enzyme ligase
 steps
1. Selection & isolation of DNA insert – concerned DNA
fragment selected, isolated enzymatically; known as DNA
insert or foreign DNA or target DNA

2. Selection of proper cloning vector – cloning vector is self-

replicating DNA molecule into which DNA insert is
combined; e.g.: bacteriophages & plasmids

3. Introduction of DNA-insert into vector to form r-DNA

molecule – extracted DNA insert (step 1) is cleaved
enzymatically by selective restriction endonuclease enz &
combined to vector DNA (step 2) using enz ligase – produce
r-DNA molecule; known as cloning vector insert DNA
construct
4. Introduction of r-DNA molecule into suitable host –
suitable host cell selected, r-DNA (step 3) introduced into
host cell by transformation (process of entry of r-DNA into
host); e.g.: E.coli, yeast & fungi

5. Selection of transformed host cells – transformed cells (host

cells that have taken up r-DNA molecule) separated form
non-transformed cells – using marker genes

6. Expression & multiplication of DNA insert in host –

confirm that foreign DNA introduced into vector DNA is
expressing preferred character in host cells; transformed cells
multiplied – adequate no. of copies; such gene can be
transmitted & expressed into different organism
 tools
 Enzymes
 DNA ligase
 Exonuclease
 Restriction endonuclease
 DNA polymerase
 Other enzymes

 Cloning vector

 Host organism

 DNA insert or foreign DNA

 Linker & adaptor sequences

 DNA ligase
• Enables linking of DNA strands composed by catalyzing
development of phosphodiester bond

• Important role – repairing single-strand breaks in duplex DNA

• Single strand breaks – repaired by DNA ligase using

complementary strand of double helix as template, DNA ligase
produces final phosphodiester bond to complete repair in DNA

• Purified DNA ligase – link DNA molecules together – form r-

DNA
 Mechanism of DNA ligase
• Forms two covalent phosphodiester bonds among 3’ hydroxyl
ends of one nucleotide (acceptor) with 5’ phosphate end of
another nucleotide (donor)
• ATP is essential for ligation

• Steps:
1. Adenylation (addition of AMP) of residue in active center of
enzyme; pyrophosphate is released

2. Transfer of AMP to 5’ phosphate donor; formation of

pyrophosphate bond

3. Formation of phosphodiester bond among 5’ phosphate of

donor & 3’ hydroxyl of acceptor
 Types of DNA ligase
1. Bacteriophage T4 DNA ligase (ATP) – derived from T4
bacteriophage – extensively used, monomeric polypeptide,
mol wt. 68KDa & encoded by bacteriophage gene30; border
specific & repairs single stranded nicks in duplex DNA, RNA
or DNA:RNA hybrids

2. E.coli DNA ligase – derived from E.coli cells, requires

NAD+ - cofactor, monomeric enzyme, mol wt. 74KDa,
catalyzes development of phosphodiester bond in duplex
DNA comprising cohesive ends, slight substrate specificity,
convenient tool; used for ligation of cohesive ends, catalyze
sticky end ligation, cloning of full length cDNA – high
efficiency cloning
3. Taq DNA ligase (NAD+) – thermostable ligases, derived
from thermophilic bacteria, recognizes mutation, maintains
action after experiencing higher temp; beneficial in DNA
amplification – identify mutation in mammalian DNA

4. T4 RNA ligase – only phage RNA ligase, weekly

categorized, monomeric enzyme with 373 deduced amino
acid residues, product of T4 gene 63; used for phosphodiester
bond formation of RNA molecule by hydrolysis of ATP to
PPI
 Exonuclease
• Eliminates nucleotides from ends of nucleic acid molecule
• Eliminates nucleotide from 5’ or 3’ end of DNA molecule
• Never produces internal cuts in DNA

• Types:
1. Exonuclease Ba131 - DNA fragment with blunt ends shorter
from both its ends

2. λ exonuclease – change 5’ ends of DNA; eliminates

nucleotides from 5’ terminus of linear DNA molecule

3. E.coli exonuclease III – 3’ end modification; capable of

removing nucleotides from 3’ OH end of DNA
 Restriction endonuclease
• Cuts sugar-phosphate support of DNA strands
• Identify particular DNA base sequence & cut both strands of
double stranded DNA molecule at or adjacent to recognition
site
• Classes:
1. Class I – mol wt. 300,000 Daltons, made up of non-identical
subunits, require Mg2+, ATP (adenosine triphosphate) &
SAM (S-adenylyl methionine) – factors

2. Class II – very small mol wt. 20,000-110,000 Daltons, equal

subunits, require only Mg2+ - cofactor

3. Class III – large molecules mol wt. 200,000 Daltons, require

Mg+ & ATP – cofactors; rarest endonuclease
 Mechanism of action:
• Begins with binding of homodimer with recognition site
(specific binding) or nearby (non-specific binding)

• Non-specific binding – if recognition site is near, enzyme

travels along DNA strand until it hits recognition site, links &
hydrolyzes sugar phosphate bonds of DNA, enzyme gets
released & cleaved DNA molecule is left behind

• Specific binding – few enzymes bind to one magnesium

divalent ion, few bind to two magnesium divalent ions
 DNA polymerase
• Synthesize new corresponding DNA strand of existing DNA or
RNA template

• Few significant types – utilized in genetic engineering

• DNA polymerase – organized from E.coli

• Klenow fragment of DNA polymerase I – create protruding

ends double –stranded by extension of shorter strand

• Taq DNA polymerase – used in PCR

• Reverse transcriptase – use RNA as template to synthesize

new DNA strand – cDNA (complementary DNA)
 Other enzymes
1. Polynucleotide kinase enzyme – reverse influence of
alkaline phosphatase; it adds phosphate group to 5’-terminus
of DNA molecule

2. Terminal deoxynucleotidyl transferase enzyme – enhances

single stranded sequence to 3’-terminus of DNA molecule,
one or more deoxyribonucleotides (dATP, dGTP, dCTP)
added onto 3’-end of blunt ended fragments

3. Alkaline phosphatase enzyme – eliminates phosphate group

from 5’-end of DNA molecule
 Cloning vector
• When foreign DNA is combined to DNA molecule – it has
competence to duplicate it within itself to give birth to several
clones of r-DNA

• Phages & plasmids are used

• Gene cloning – important invention

• Cloning vector – vital role in gene cloning

 Types of cloning vectors
1. Bacteriophage –cloning limit 4-5 kbp of donor DNA, size 48502 bp; e.g.:
lambda (λ) genome

2. Plasmid – size 4361 bp, cloning limit 0.1-10 kb, marker gene – ampicillin
& tetracycline resistant gene, isolated from E.coli; e.g.: PBR322

3. Cosmid – size 7900 bp, cloning limit 30-50 kb, characteristics similar to
phage & plasmid; e.g.: super COS1

4. Human artificial chromosome (HAC) – artificial chromosome used to

transfer human gene, no cloning limit, transmit large segment of DNA

5. Yeast artificial chromosome (YAC) – size 11400 bp, cloning limit 100-1000
kb, marker similar to yeast, artificial, yeast centromere isolated from
Saccharomyces cerevisiae

6. Bacterial artificial chromosome (BAC) – size 11827 bp, cloning limit 35-
300 kb, marker gene – lactose metabolizing gene &chloramphenicol,
alteration of f-plasmid & artificially synthesized; e.g.: pUvBBAC
 Host organism
• Good host organism – important for successful genetic
engineering

• E.coli most common host

• Isolation & cloning of DNA inserts – very simple

• Ideal host – easy to transform & simple replication

• No restricting element present during replication of r-DNA in

host cell
 DNA insert or foreign DNA
• The anticipated DNA fragmented to be replicated – DNA
insert or foreign DNA or target DNA

• Selection of suitable target DNA – most important step

• Target DNA – animal, plant, bacterial or viral origin

 Linker & adaptor sequences
• Vital role – modification of cut ends of DNA fragments

• Preferred changes obtained by joining linkers & adapters to

cut ends of DNA molecule

• Linkers - short, chemically produced, double stranded DNA

structures

• Comprise one or more restriction endonuclease sites

• Adaptors have one or both sticky ends

 Linkers – target site for action of several restriction enzymes, ligate to
blunt ends of vector DNA

• They go through treatment with particular restriction endonuclease to

yield consistent ends of DNA fragments
• e.g.: EcoRI-linker

 Adaptors – chemically produced moieties with pre-formed cohesive

ends

• Used for end modification where recognition site for restriction

endonuclease enzyme exists inside foreign DNA

• Foreign DNA is ligated with adaptor on both ends

• Novel moiety produced is phosphorylated at 5’-terminii

• Foreign DNA improved with adaptors is combined into vector DNA to

applications
 Hormone production – bacterial cells of E.coli – somatostatin,
insulin, p-endorphin, somatotropin; concerned genes
integrated in bacterial cells & cloned – commercial
significance

 Transgenic animals production – preferred gene introduced in

animal – transgenic animal; helps animal breeders – speed &
range of selective breeding; production of healthier farm
animals –more profit, manufacture of proteins &
pharmaceutical composites

 Transgenic plants production – yield transgenic or genetically

modified plants; resistance to insects, herbicides, viruses;
enhanced post-harvest characteristics
 Production of antibiotics – active against diverse bacterial,
viral or protozoal diseases; antibiotics – penicillin,
tetracycline, novobiocin, streptomycin, bacitracin

 Vaccine production – biochemical preparation comprising

pathogen in reduced or inactive state, initiates immunity
against infection; useful against serious diseases – malaria,
polio, rabies, hepatitis; DNA vaccine comprises gene encoding
immunogenic protein from concerned pathogen

 Biosynthesis of interferon – glycoproteins, antiviral & anti-

cancer properties; genetic factor of human fibroblasts –
introduced into bacterial plasmid, cloned, cultured & gene
expressed; interferons are created – high amount, extracted &
purified
 Commercially significant chemical production – alcohol &
alcoholic beverages, citric acid, acetic acid, vitamins –
fermentation process

 Application in enzyme engineering – modification of enzyme

structure by inducing alteration in genes that encode specific
enzyme

 Prevention & diagnosis of diseases – resolved difficulty –

conventional approaches for analysis; deliver ways to inhibit
several diseases – AIDS, cholera; monoclonal antibodies – tool
for disease diagnosis, produced by hybridoma technique (fusion
of lymphocyte & single myeloma cell)

 Gene therapy – delivers particular gene into human body to

correct disease; treatment of disease by transfer & expression of
gene into patients’ cells – confirm restoration of normal cellular
activity; in vivo & ex vivo approach
Applications of genetic engineering in
medicine
• DNA vaccines – injection of proteins – vaccination (common),
immune system desires definite properties for effective
immunization; DNA mediated immunization – DNA vaccines;
direct injection of plasmid DNA encoding antigenic protein inside
the organism through needles or skin – less cells are affected,
reaction efficiently immunizes body

• Gene therapy – cloned functional copy of gene introduced in

individual – balance the individual’s defective copy; employed for
SCID (severe combined immunodeficiency) & hemophilia

• Manufacture of pharmaceutical products – pharming (farming &

pharmaceutical); use of genetic engineering to insert genes into host
animals & plants – appearance of useful pharmaceutical products
 fabrication of interferon
• Natural glycoproteins – virus-infected eukaryotic cells; protect
host cells from virus infection

• Interferon – interferes with intracellular development of virus

• Manufactured by living animal cells – in vivo & cultured cells

• Interferon fabrication & function – obstructed by transcription
& translation inhibitors

• Virus-infected cells do not yield interferons in presence of

actinomycin D (since it is eukaryotic RNA polymerase
inhibitor)

• After 2 hrs of infection, interferon production is not inhibited

which indicates that transcription is completed within 2 hrs
• Interferon inducer – responsible for interferon synthesis

• double stranded RNA – e.g.: reovirus

• single stranded RNA virus act as inducers only after

replication by forming double-stranded replicative
intermediates

• DNA virus e.g.: vaccinia virus prompt interferons by

overlapping transcription of viral DNA

• fungal virus – mostly double stranded RNA genome – act as

interferon inducers

• synthetic polymers – riboinosinic acid, ribocytidylic acid,

riboadenylic acid & ribouridylic acid - inducers
Steps:
1. Production starts after initiation of viral maturation &
continues for 20-50 hr.

2. Production stops due to formation of repressor (activated

when interferon conc exceeds threshold conc)

3. Interferon is transported from producing cell to neighboring

cells

4. When interferon conc in generating cell rises beyond

threshold, triggers alternative gene which codes for repressor
protein – feeds back & stops extra interferon production
Steps:
5. Virus-infected cells yield only minor amounts of interferons

6. Interferon molecules leave producing cells & reach

neighboring uninfected host cells; bind to cell membrane or
nuclear membrane receptors

7. Cells start synthesizing antiviral proteins

8. Act as agents – deliver safety to host cells against viral

infection
 fabrication of hepatitis-b vaccine
• Recombinant HB vaccines – linked with adverse reactions (17%
population receiving vaccine)

• Existing HBV spot fabrication method uses baker’s yeast Saccharomyces

cerevisiae – heterologous protein appearance host – comprise entrapped
carbohydrates

• Carbohydrates from yeast – different structure than manufactured in

mammalian cells

• Chemical alterations cause immunological reactions in humans

• Decreases efficacy of vaccine

• Modern method: distinct variety of tropical monocot banana – genus

Musa, family Musaceae – engineered by genetic engineering – production
of hepatitis B vaccine
• Reduced cost of vaccine
 Steps:
1. Isolation of HBs antigen fabricating gene from HB virus –
standard isolation technique (protein denaturation, cell lysis,
centrifugation, drying)

2. Plasmid DNA taken from bacterium E.coli & cut with

restriction enzyme – EcoRI (forms plasmid vector)

3. Insertion & location of isolated HBs antigen constructing

gene into bacterial plasmid vector – form r-DNA

4. r-DNA (with target gene) is hosted in yeast cell – creates

recombinant yeast cell
Steps:
5. This recombinant yeast cell proliferates in fermentation tank
& yields HBs antigens

6. After 2 days, yeast cells are cracked to free HBs Ag

7. Fusion is treated for extraction & HBs antigens are purified

8. HBs Ag joined with preserving agent & extra constituents &

bottled

9. Vaccine ready for injection in humans

 fabrication of insulin
• Recombinant insulin – outcome of effective genetic engineering

• Old method:
1. production of insulin comprised dispersed production of
insulin A &B chains in two bacterial strains

2. Equal insulin A & B chain genes placed under control of lac

promoter – inducible expression by lactose inducer

3. Chains were purified from bacteria & connected chemically –

yield final insulin

• Recombinant insulin – diabetes therapy

 Steps:
1. Human insulin removed from pancreas & insulin-producing gene is
isolated

2. Plasmid DNA extracted from bacterium & cut with restriction

enzyme – forms plasmid vector

3. Human insulin-producing gene inserted into bacterial plasmid vector

– form r-DNA of human insulin-producing gene

4. R-DNA introduced into bacterial cell – form recombinant bacteria

5. Recombinant bacteria reproduce in fermentation tank & yield human

insulin

6. Insulin is extracted, purified & bottled

7. Preparation is ready to be injected in diabetic patients