2a-basic of genetic engineering.pdf

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

Splicing

Eukaryote Prokaryote

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