2a-basic of genetic engineering.pdf
TRANSCRIPT
GENETIC
ENGINEERING
by : Dr. Lanny Hartanti, M.Si.
Faculty of Pharmacy
2014
AD Hershey & M Chase experiment
two chains are
complementary:
A binds to T
C binds to G
4 building blocks: A, C, G
and T (“bases”)
•3 bases form a “codon”
a codon encode 1 amino acid
A GENE consists of hundreds to
millions of bases and encodes a protein
5’
5’ 3’
3’
exon exon intron upstream downstream
Initiation codon termination codon
TRANSCRIPTION
DNA RNA
TRANSLATION
mRNA protein
Conclusion:
gene works
through protein
DNA (code: ACTG)
RNA
transcription
mRNA (without introns)
(“messenger”-RNA)
protein
“splicing”
translation
RNA (code: ACUG)
intron
THE GENETIC CODE
How to understand gene functions ?
Certain stimuli receptor
signal transduction
intracellular pathway
nuclear response
certain gene on
RNA
m RNA
Protein (a.a)
transcription
splicing
translation
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 versus prokaryote Eukaryote versus prokaryote
Prokaryote vs eukaryote PROKARYOTE
(Bacteria)
EUKARYOTE
CHROMOSOMAL
DNA
-double helix,
-circular,
-usually single
-double helix
-linear,
-usually multiple
EXTRA
CHROMOSOMAL
DNA
- Plasmid -Mitochondrial
-Chloroplast
Circular shape of microbial DNA
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 GAATTC
CTTAAG 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 AAGCTT
(Hind III)
TTCGAA
Haemophilus aegyptus GGCC
(Hae III) 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.
Template DNA
Denaturation of
the template
DNA : 94°C
Annealing of the
oligonucleotide
primers (50-60°C)
Synthesis of
new DNA : 74°C
Taq DNA
polymerase
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
Target Molecul Separation Base
Agarose gel
Horizontal, submarine
DNA/RNA Size
SDS-Page
Vertical, slab gels proteins apparent molecular
weight
Isoelectric focussing (IEF PAGE)
horizontal (strips), vertical (capillaries)
proteins Isoelectric point
Sequencing Gel
Vertical: slab gel, capillary gel
ss DNA Size
AGAROSE POLYACRYLAMIDE
Gelation of the polysaccharide sol by chilling Chemical polymerisation of acrylamide monomers andNN´-methylenbisacrylamide (Bis)
1% agarose (w/v) ca. 150 nm;0.16 % agarose (w/v) ca. 500 nm.
Total acrylamide concentrationand Crosslinking:
T = 100 [%]; C = 100 [%]a + ba + b
a:g acrylamide; b:g Bis;V: volume in mL
5 % T / 3 % C 5 nm
V
b
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
DNA fragment
On the nylon membrane
Add the radio labeled-
DNA probe
Wild type
Contained
fragment
Autoradiograph
cDNA
synthesis
Genomic DNA library construction
cDNA library construction