Genetic Engineering: Aliyu, Habibu

Genetic Engineering
By
Aliyu, Habibu
oBiotechnology
All inclusive term for several technologies
including but not limited to recombinant DNA.
Refers to the use of technology in applications
for solving fundamental problems in biology.
the application of organisms/Biological
systems/ processes in the manufacturing and
Service industries.
oGenetic Engineering
Use of techniques involving recombinant DNA
technology to produce molecules and/or
organisms with new properties/heritable,
directed alteration of an organisms DNA.
oRecombinant DNA technology

Set of molecular techniques for locating, isolating,
altering, and studying DNA segments. The term
recombinant is used because frequently the goal
is to combine DNA from two distinct sources.
Genes from two different bacteria might be
joined, for example, or a human gene might be
inserted into a viral chromosome.
Used to create a number of commercial products,
including drugs, hormones, enzymes, and crops
oRecombinant DNA
DNA molecule produced artificially and
containing sequences from unrelated organisms.

oHistory
1966 The genetic code is deciphered when biochemical analysis

reveals which codons determine which amino acids.
1970 Hamilton Smith, at Johns Hopkins Medical School, isolates
the first restriction enzyme, an enzyme that cuts DNA at a very
specific nucleotide sequence. Over the next few years, several
more restriction enzymes will be isolated.
1972 Stanley Cohen and Herbert Boyer combine their efforts to
create recombinant DNA. This technology will be the beginning of
the biotechnology industry.
1976 Herbert Boyer cofounds Genentech, the first firm founded
in the United States to apply recombinant DNA technology
1978 Somatostatin, which regulates human growth hormones, is
the first human protein made using recombinant technology.
1983 Kary Mullis does PCR. 1985 Kary Mullis publishes method.
Patents follow.
2000? Gateway cloning
Needles in Haystacks
How to find one gene in large genome?
A gene might be 1/1,000,000 of the
genome. Three basic approaches:
1. cell-based molecular cloning: create
and isolate a bacterial strain that
replicates a copy of gene.
2. Polymerase chain reaction (PCR).
Make many copies of a specific region
of the DNA.
3. hybridization: make DNA single
stranded, allow double strands to reform using a labeled (e.g. radioactive)
Polymerase Chain Reaction

Based on DNA polymerase creating a
second strand of DNA.
Needs template DNA and two primers
that flank the region to be amplified.
Primers are short (generally 18-30
bases) DNA oligonucleotides
complementary to the ends of the
region being amplified.
DNA polymerase adds new bases to the
3' ends of the primers to create the new
second strand.
go from 1 DNA to 2, then 4, 8, etc:
A key element in PCR is a special form of

DNA polymerase from Thermus aquaticus, a
bacterium that lives in nearly boiling water in
the Yellowstone National Park hot springs. This
enzyme, Taq polymerase, can withstand the
temperature cycle of PCR, which would kill
DNA polymerase from E. coli.
advantages:
rapid, sensitive, lots of useful variations, robust
(works even with partly degraded DNA)
disadvantages:
Only short regions (up to 2 kbp) can be
amplified.
limited amount of product made
oRestriction endonucleases
Also called restriction enzymes: digest
DNA at specific sequences
oSequence Recognition -R.E.

Restriction endonucleases -- cut double
stranded DNA at specific sequences,
protection against viruses in bacteria.
Sequences often palindromes: a
sequence which is the same when read
in either direction. A man a plan a
canal: Panama
Cloning Vector Types

For different sizes of DNA:
plasmids: up to 5 kb
phage lambda () vectors: up to 50
kb
BAC (bacterial artificial
chromosome): 300 kb
YAC (yeast artificial chromosome):
2000 kb
Plasmids
Extrachromosomal, circular small (2-3

kb) DNA in a bacterial cell which
can replicate independently but which
cannot integrate into the host
chromosome.
Drug resistance plasmids are not
essential for the cell's growth, but
confer antibiotic resistance.
Plasmids used for molecular cloning
have been artificially created by
recombining fragments of various
Example of a Plasmid
Example of a plasmid + insert (DNA of

interest)
oDNA ligase
DNA ligase joins 5'-phosphate and 3'hydroxyl ends of DNA
Two fragments formed by EcoRI can
be rejoined by ligase.
Similarly, Eco RI fragments from two
different pieces of DNA can be joined
Ligation
Tools of recombinant DNA - cloning
Sources of DNA to Clone

Genomic DNA: cut up whole genome
and clone small pieces. Advantage is,
you get everything. Disadvantage is, a
lot of it is junk.
oTwo general methods:
1. randomly shear DNA into
small pieces, then ligate linkers to
the ends: oligonucleotides that
contain a useful restriction site.
2. partially digest the DNA with a
restriction enzyme that has a 4 base
recognition site. These sites will
cDNA: DNA copy of mRNA, made

with reverse transcriptase. Advantage:
you just get the expressed genes.
Disadvantages: you don't get control
sequences or introns, and frequency
depends on level of expression.
Synthetic DNA: synthesized de novo
(for example multiple cloning sites or
linkers), or made by PCR
Basic Cloning Process
Plasmid is cut open with a restriction

enzyme that leaves an overhang: a
sticky end
Foreign DNA is cut with the same
enzyme.
The two DNAs are mixed. The sticky
ends anneal together, and DNA ligase
joins them into one recombinant
molecule.
The recombinant plasmids are
transformed into E. coli using heat plus
Recombinant DNA molecule
oInserting recombinant DNA into Host

Transformation
cell made competent to take up DNA
competent cells: electroporation
poke holes in membrane and calcium
chloride- make cells more permeable
to DNA
Transfection
when the cloning vector used has
aspects of a virus, the host cell can be
infected (transfected) to insert the
recombinant molecule
Electroporation
the cell is placed in an electric field
such that small pores are temporarily
opened in the membrane. Added DNA
can enter through these pores.
Transformation
oSelection
Antibotic resistance
Plasmid vector contains an ampicillin
resistance gene making the cell
resistant.
Growth of transformed cells (cells
receiving the plasmid) can be identified
on agar medium containing (e.g.)
ampicillin.
Transformation
oFurther selection
The plasmid vector contains another identifiable gene (e.g.,
a second drug resistance or an enzyme activity), with the
coding sequence of this gene containing the restriction site
for insertion.
Insertion of the foreign DNA at this site interrupts the
reading frame of the gene and result in insertional
mutagenesis.
In the following example, the -galactosidase gene is
inactivated. The substrate "X-gal" turns blue if the gene is
intact, ie. makes active enzyme. White colonies in X-gal
imply the presence of recombinant DNA in the plasmid.
X-gal selection
Applications
Gene products- using GMOs ie
microbes to produce chemicals used for
medical or industrial purposes
New phenotypes- using gene
technology to alter the characteristic of
an organism ie farm animals or crops
Gene therapy- using gene technology on
humans to treat disease
Transfer a gene of interest from human

into a host usually microbes
Product produced quickly cheaply
Used in commercially important crops
or farm animals to increase their yield
Disadvantages
Safety of food
Adverse effect on human health or
ecosystem
Toxins
Allergies
Decreased nutritional value
Antibiotic resistant bacteria

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Genetic Engineering

oRecombinant DNA technology

1966 The genetic code is deciphered when biochemical analysis

Polymerase Chain Reaction

A key element in PCR is a special form of

oSequence Recognition -R.E.

Cloning Vector Types

Extrachromosomal, circular small (2-3

Example of a plasmid + insert (DNA of

Tools of recombinant DNA - cloning

Sources of DNA to Clone

cDNA: DNA copy of mRNA, made

Basic Cloning Process

Plasmid is cut open with a restriction

Recombinant DNA molecule

oInserting recombinant DNA into Host

Transfer a gene of interest from human

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