GENETIC

ENGINEERING

by : Dr. Lanny Hartanti, M.Si.

Faculty of Pharmacy
2014

AD Hershey & M Chase experiment

two chains are

complementary:
3 bases form a codon
a codon encode 1 amino acid

A binds to T
C binds to G

A GENE consists of hundreds to

4 building blocks: A, C, G
millions of bases and encodes a protein and T (bases)

upstream

exon intron exon

Initiation codon

downstream

termination codon

TRANSCRIPTION
DNA RNA

TRANSLATION
mRNA protein

Conclusion:
gene works
through protein

intron

DNA (code: ACTG)

transcription
RNA (code: ACUG)

splicing
mRNA (without introns)
(messenger-RNA)

translation
protein

THE GENETIC CODE

How to understand gene functions ?

Certain stimuli
receptor
signal transduction
intracellular pathway
nuclear response
certain gene on
transcription
RNA
splicing
m RNA
translation
Protein (a.a)

How much DNA do we have?

humans have 2 x 23
chromosomes
EACH cells contains
6 billion bases DNA
that is 1 meter of
DNA
a human being has
>100.000.000.000.000
cells
that is 100 billion km
of DNA

How much information is in

our genome?
6 billion bases = 6 Gigabyte
in every cell, including a readout and copying
system

30.000 - 50.000 genes

a lot of junk-DNA contains no code bus
has a different function

DNA technology:
applications
Genetic manipulation
food crops
animals
clones

Inherited disorders / susceptibilities

Diagnostics
Gene therapy

Cancer
origins of cancer
gene therapy
Forensic Test

DNA techniques
microscopes are only for chromosomes
important tools:
enzymes
bacteria
viruses

Eukaryote
Eukaryoteversus
versusprokaryote
prokaryote

Prokaryote vs eukaryote
PROKARYOTE
(Bacteria)

EUKARYOTE

CHROMOSOMAL -double helix,

DNA
-circular,
-usually single

-double helix
-linear,
-usually multiple

EXTRA
- Plasmid
CHROMOSOMAL
DNA

-Mitochondrial
-Chloroplast

Circular shape of microbial DNA

Eukaryote

Prokaryote

Splicing

Gene cloning

paste random pieces of human DNA in

vector such as plasmid, virus, phage
select clones with selectable marker
(antibiotic resistance; X-gal)
grow clones and test with probe
grow specific clones to large volume
sequence inserts

Stages of basic techniques in

cloning gene:
1. DNA / RNA isolation + DNA plasmid isolation
2. Restriction, ligation of DNA/RNA insert.
3. Observation of DNA/RNA restriction or

ligation .
4. Transformation into host cell (E. coli).
5. Isolation of recombinant DNA from host.
6. Analysis of recombinant DNA.

DNA cut and paste

with enzymes

restriction-enzymes:
cut DNA at specific
sequences

ligases:
paste DNA

polymerases:
copy DNA

CLONING STRATEGY

The result of Restriction Enzyme cutting:

Sticky end:

AAATTC
TTTAAG

Blunt / flush end:

GAATTC

CTTAAG

GAATTC

CTTAAG

RESTRICTION ENZYME
Enzyme that is used to cut DNA molecule.

Escherichia coli R
G A *A T T C
(ECO RI)
C T T A* A G
Haemophilus Influenzae d
(Hind III)
TTCGAA
Haemophilus aegyptus
(Hae III)

AAGCTT

GGCC
CCGG

Sel bacteri mampu mengambil plasmid rekombinan yang

terdapat pada media/larutan disekitarnya (dimana bakteri
dikultur), sehingga diperoleh transformasi (masuknya gen
asing ke dalam bakteri).
Setelah bakteri dikultur pada medium bakteri akan tumbuh,
plasmid akan mengalami replikasi bersama sama dengan
replikasi DNA bakteri, sehingga diperoleh klon atau kopi
dari rekombinan plasmid.
Keberhasilan rekombinan ini dapat diketahui dengan tumbuhnya
bakteri pada medium yang ditambah ampisilin sebagai media
seleksi (karena rekombinan disertai gen resistan ampilin).
Sedangkan sel bakteri yang tidak berhasil mengambil rekombinan
akan mati dalam medium ampisilin.

The example of DNA cutting with

Restriction Enzyme

VECTOR

DNA
Cloning

Polymerase chain
reaction

breakthrough technique in DNA

research
make millions of copies of single copy
gene
uses enzymes from hot-water bacteria

Some applications of PCR

Isolation of equivalent gene, ex: genes from

rat to design primer for isolation of human
genes
PCR of human globin genes to test for
the presence of mutations that might cause
thalassaemia
The use of primers specific for the DNA of a
disease-causing virus the PCR is
tremendously sensitive only need 1 molc.

Denaturation of
the template
DNA : 94C

Template DNA

Annealing of the
oligonucleotide
primers (50-60C)

Taq DNA
polymerase

Synthesis of
new DNA : 74C
Taq DNA
polymerase

P C R Instrument

Polymerase Chain Reaction (PCR)

M 1 2 3

Conventional electrophoresis techniques separate biomolecules by their

size, charge or isoelectric point
Gel Media

Electrophoresis
Type
Horizontal,
submarine
Vertical, slab gels

Agarose gel
SDS-Page

Target Molecul

Separation Base

DNA/RNA

Size

proteins

Isoelectric focussing horizontal (strips),

(IEF PAGE)
vertical (capillaries)

proteins

apparent molecular
weight
Isoelectric point

Sequencing Gel

ss DNA

Size

Vertical: slab gel,

capillary gel

AGAROSE
Gelation of the polysaccharide sol by chilling

1% agarose (w/v)
0.16 % agarose (w/v)

ca. 150 nm;

ca. 500 nm.

POLYACRYLAMIDE
Chemical polymerisation of acrylamide monomers and
NN-methylenbisacrylamide (Bis)

Total acrylamide concentrationand Crosslinking:

b
T = a + b 100 [%]; C = a + b 100 [%]
V
a:g acrylamide; b:g Bis;V: volume in mL
5 % T / 3 % C 5 nm

Gel Electrophoresis

DNA hybridization
The attachment by base-pairing of two complementary
polynucleotide. Make use of a strong binding radio
labeled DNA probe whose sequence is in the perfect
complementary to the wild type DNA sequence. Mutant
allele will not able to hybridize to the DNA probe

Wild type
Contained
fragment

Add the radio labeledDNA probe

DNA fragment
On the nylon membrane

Autoradiograph

cDNA
synthesis

Genomic DNA library construction

cDNA library construction