Genetic

engineerin
g? No idea.
But you
can ask me
about
aliens…
 RECOMBINANT
DNA TECHNOLOGY/
GENETIC
ENGINEERING
By: Feliza Maren Israelita R.
Barican
Review on
Genetics
 Deoxyrubonucleic Acid
(DNA)
• Double stranded
– Composed of a sugar
(deoxyribose), a phospate
residue and one of the four
bases: adenine (A), cytosine
(C), guanine (G) and thymine
(T)
• Base Pairing ; Purine – Pyrimidine
• DNA Replication
 Ribonucleic Acid
• Single stranded
– Consist of a ribose sugar, a
phospate residue and one of the
four bases: adenine (A), cytosine
(C), guanine (G) and uracil (U)
• RNA species; rRNA, tRNA, mRNA
• Role of mRNA; intermediate product
b/w amino acid and DNA
• Hybridzation – strands of nucleic acids
binding together forming
Replication

dsDNA
ssDNA
Transcription
dsDNA

AMINO ACID

mRNA
Reverse Transcription

dsDNA
ssDNA
Translation

mRNA

PROTEIN
 Biotechnology

• The industrial application of

microorganisms, cells or cell
components to make a useful
product.
 Recombinant DNA
Technology

• Manufacturing and manipulating

genetic material in vitro.
• Its product is called recombinant
DNA.
 Recombinant/ Chimeric
DNA

• Synthetic DNA that is engineered

through the combination or
insertion of one or more DNA
strands, thereby combining DNA
sequences that would not
normally occur together.
 Chimeric Molecules

• Recombinant DNA that is further

changed to host additional
strands of DNA.
• Regular in occurrence
• Propagation by vectors ensures
the presence of hundreds of
thousands of organismal and
bacterial cells that all contain
copies of the original chimeric
 Recombinant
DNA Technology
Procedures
 DNA Isolation

• Cells in a broth culture are lysed

by using a detergent to isolate
bacterial DNA.
• Burst cells release fragile DNA.
• The fragile DNAs are treated with
specific enzymes to purify the
obtained DNA by removing
unnecessary RNA and proteins.
 Examples of
Enzymes Used in
Recombinant
DNA Research
 ENZYME PRIMARY ACTION PRIMARY USE

Alkaline Dephosphorylates Removal of 5’ PO4

phosphatase 5’ ends of RNA and groups prior to
DNA kinase labeling to
prevent self ligation
BAL 31 nuclease Degrades both the Progressive
3’ and 5’ ends of shortening of DNA
DNA molecules
DNA ligase Catalyzes bonds Joining of DNA
between DNA molecules
molecules
DNA polymerase I Synthesize dsDNA Synthesize dsDNA;
from ss DNA Nick translation;
generation of blunt
ends from sticky
DNAse I Under appropriate Nick translation;
ends
conditions, mapping of
produces ss nicks in hypersensitive
DNA sites; mapping
protein DNA
interaction
Exonuclease III Remove nucleotide DNA sequencing;
from 3’ ends of mapping of DNA
DNA protein interactions
λ exonuclease Removes DNA sequencing
nucleotides from 5’
ends of DNA
Polynucleotide Transfer terminal 32P labeling of DNA
kinase phospate (γ and RNA
position) from ATP
to 5’ OH groups of
Reverse Synthesize
DNA or RNADNA Synthesis of DNA
transcriptase from RNA template from mRNA; RNA
c5’ end mapping
study
SI nuclease Degardes single Removal of hairpin
stranded DNA in synthesis of
cDNA; RNA
mapping
Terminal Adds nucleotides to Homopolymer
transferase the 3’ ends mapping
 Using Restriction Enzymes
to Generate DNA
Fragments

• Purified DNA is cut (or

“digested”) into smaller
fragments by restriction
enzymes.
• Digestion of DNA with restriction
enzymes generates blunt-ended
DNA or sticky ended (or
 Restriction Enzymes
• An enzyme that causes cleavage
of both strands of DNA at highly
specific sites dictated by the
base sequence.
• First isolated in 1970, when
certain bacteriophages were
found to have a restricted host
range.
• Protects a bacterial cell by
hydrolyzing phage DNA.
• Named after the bacterium in
which they are isolated.
• Recognizes palindrome
(repeated, inverted) gene
sequence
Examples of
Restriction
Enzymes
RESTRICTION SEQUENCE
ENDONUCLEA RECOGNIZED BACTERIAL
SE CLEAVAGE SITES SOURCE
Bam HI GGATCC
SHOWN Bacillus
amyloliquefaciens
Bg III AGATCT H
Bacillus glolbigii

Eco RI GAAATTC Escheriecia coli

RY13
Eco RII CCTGG Escheriecia coli
R245
Hind III AAGCTT Haemophilus
influenzae Rd
Hha I GCGC Haemophilus
haemolyticus
Hpa I GTTAAC Haemolyticus
parainfluenzae

Mst II CCTNAGG Microcoleus strain

Not I GCGGCCGC Nocardia otitidis -

caviarum

Pst I CTACAG Providencia stuartii

Sma I CCCGGG Serratia

marcescens

Taq I TCGA Thermus aquaticus

YTI
 Types of
DNA Fragments
 Blunt-Ended DNA

• Two strands of DNA duplex

having ends that are flush with
each other.
• Example; Alu I
Sticky (Cohesive) Ended
DNA

• Complementary single strands of

DNA that protude from opposite
ends of a DNA duplex or from the
ends of different duplex
molecule
– Example; Bam HI
 Using DNA Ligase to Join
Vector and Insert
• VECTOR
– Plasmid or bacteriophage into
which foreign DNA can be
introduced for the purpose of
cloning
• INSERT
– Additional length of a base
pairs in DNA
 Properties of an Ideal
Vector

• Replicator (“ori”)
• Selective markers resistant to
antibiotics
• Multiple cloning sites
 lacZ
gene

CLEAVA
GE
AMP SITES
R

ORI
 Examples of
Cloning Vectors
Bacteriophage Virally infected bacterium
Cosmid A plasmid into which the DNA
sequence from bacteriophage
lambda that are necessary for
packaging of DNA
Plasmid Small, extrachromosomal,
circular molecule of DNA that
replicates indepently of host
DNA; with cos sites
Bacterial Artificial Derivatives of the F plasmid of E.
Chromosome (BAC) coli; contains inserts as large as
300,000 nucleotieds in length
Yeast Artificial Chromosome Stores 1,000,000 nucleotides in
(YAC)
Expression Vector length
Engineered to certain and
appropriate protomer and
ribosmome binding site adjacent
to multiple cloning site
Ti Plasmid Tumor inducing plasmid
(Agrobacterium tumefaciens
• BACTERIOPHAGE T4 DNA
LIGASE
– Glues the DNA fragments
together.
– Forms covalent bonds between
sugar phosphate residue of
adjacent nucleotide .
Introducing Recombinant
DNA into a New Host

• Selecting a suitable host

• Introducing DNA into cells
• Selecting for transformants
Selecting a Suitable Host

• Escheriecia coli
– Most preferred host
• Easily grow in laboratory
• Can’t grow in normal
environmental conditions
found outside the laboratory
 Introducing DNA into
Cells

• DNA MEDIATED
TRANSFORMATION
– Cells take up DNA from
surrounding environment.
• ELECTROPORATION
– Inducing an electrical current
to form microscopic pores in
the membrane of the cells;
DNA enters the cells through
the cells.
• MICROINJECTION
• DNA introduced directly into
an animal cell.
 Genetic Cloning

• GENETIC LIBRARIES
– Collection of genetic clones
containing different DNA
fragments.
• Reverse transcriptase
–An enzyme that is
produced by artificial
genes.
 Selecting for
Transformants/Clones

• Blue white screening

• Colony hybridization
 Blue-White Screening

• Color of bacterial colonies

formed at the end of the
screening process
– (+) – Blue
– ( - ) – White
 Colony Hybridization

• Identification of a colony
containing a desired gene by
using a DNA probe that is
complementary to the gene.
 Applications of
Recombinant
DNA Technology
Production of Recombinant
Vaccines
• RECOMBINANT VACCINE
– Vaccines produced by
recombinant DNA technology
– Contains either a protein or a
gene encoding a protein of a
pathogen origin that is
immunogenic and critical to the
pathogen function
Pharmaceutical
Products of
Genetic
Engineering
 PRODUCT COMMENTS

Alpha-interferon Therapy for leukemia, melanoma and

hepatitis, prduced by E. coli and S.
Antitrypsin cerevisiae (baker’s patients;
Assist emphysema yeast) produced
by genetically modified sheep
Beta-interferon Treatment for multiple sclerosis;
produced by mammalian cell culture
Bone Induces new bone formation; useful in
morphogenic healing fractures and reconstructive
proteins surgery; produced mammalian cell
Colony- culture
Counteracts effects of chemotherapy;
stimulating factor improves resistance to infectious
(CSF) disease such as AIDS; treatment of
leukemia; produced by E. coli and S.
cerevisiae (baker’s yeast)
Epidermal Heals wounds, burns, ulcers; produced
growth factor by E. coli
(EGF)
Erythropoietin Treatment of anemia; produced by
(EPO) mammalian cell culture
Factor VII Treatment of hemophilia; improves
clotting; produced by mammalian cell
Gamma- culture
Treatment of CGD; produced by E. coli
interferon
HBV Produced by S. cerevisae that carries
hepatitis virus gene on a plasmid
Human Growth Corrects growth deficiencies in
Hormone (hGH) children; produced by E. coli
Influenza Trial vaccine made from E. coli or S.
vaccine cerevisiae carrying virus genes
Interleukins Regulates the immune system;
possible treatment for cancer;
produced by E. coli
Monoclonal Possible therapy for cancer and
antibodies transplant rejection; used in
diagnostic tests; produced by
mammalian cell culture (from fusion of
Orthoclone cancer cell and
Monoclonal antibody-producing
antibody used in
cell)
transplant patients to help suppress
the immune system, reducing th
Prourokinase echance of tissue
Anticoagulant; rejection;
theray produced
for heart
by mouse
attacks; cells
produced by E. coli and yeast
Human insulin Therapy for IDDM; better tolerated
than insulin extracted from animals;
produced by E. coli
Pulmozyme Enzyme used to breakdown
(rhDNAse) mucuos secretion in cystic fibrosis
patients; produced by mammalian
Relaxin cell
Usedculture
to ease childbirth; produced
by E.coli
Superoxide Minimizes damage caused by
dismutase oxygen-free radicals when blood is
resupplied to oxygen deprived
tissues; produced by S. cerevisiae
Taxol and
PlantPichia pastoris
produced used for treatment
of ovarian cancer in E. coli
Tissue plasminogen Dissolves the fibrin of clots;
activator (Activase) therapy for heart attacks; produced
by mammalian cell culture
Tumor necrosis Causes disintegration of tumor
factor (TNF) cells; produced by E.coli
 Other
Applications of
Recombinant
DNA Technology
• Normal Gene Variation
– Human Genome Project
• Disease-Causing Gene Variation
• Xenotransplant
• Nucleotide Sequence
 “We cannot help
ourselves from growing
wiser. Those who are
helpless to their search
for knowledge are those
who can’t transcend
Thanks for
listening!
Have a nice
day!