Faculty of Medicine

Department of Genetics Universitas Swadaya

Gunung Jati

Genetic Engineering and

Molecular Techniques
dr. Tiar M Pratamawati.,MM.,M.Biomed
Department of Genetics, Faculty of Medicine
Unswagati University
Real or Fake ?
Aku anak siapa??
 Definition

§ Biotechnology
Technic which applies biological system (in organisms, cellular and molecular level) aiming
to obtain the expected result according to human needs.

§ Genetic engineering (Rekayasa genetika)

Biotechnology application by using gene/DNA manipulation.

§ Genetic engineering in medicine

Gene/DNA manipulation to obtain information regarding normal gene function and its
pathology in a disease; and to create a new prototype according to human needs (vaccine,
gene therapy, etc.)
 Biomedical importance
1. Rational approach to understanding the molecular 4. Human proteins can be produced
basis of a number of diseases in abundance for therapy
E.g: muscular dystrophy, cystic fibrosis, cancer, diabetes E.g: Insulin, growth hormone

2. Diagnostic testing & predict the risk of

5. Protein for vaccines
developing a given disease and individual response
E.g: Hepatitis B vaccine
to pharmacological therapeutics
E.g: Ebola test, familial cancer
6. Forensic medicine
E.g: Method for identifying criminal
3. Gene therapy for potentially curing diseases
suspect
caused by a single gene deficiency
E.g: thalassemia, adenosine dealinase deficiency
Murray RK, et al. 2009. A LANGE medical book: Harper’s illustrated
biochemistry. 28th ed. New York: The McGraw-Hill Companies, Inc
 1856 1956
Gregor Mendel Tjio & Levan 1990-2003
Mendelian inheritance; Mendel Law I & II The right number of chromosome HGP (Human Genome
Project)
1877
1839 Flemming
Schleiden & Chromosome
Schwan identification 1977 1985
Cell à basic in the nucleus Sanger K. Mullis
unit of life Sequencing PCR

MILESTONES OF GENETICS & MOLECULAR GENETICS

1983
1859 1903
1953 JB Martin
Charles Darwin Sutton & Boveri
Watson & Crick 1st gene identification
Evolution theory; Chromosome contains
double helix responsible for genetic
The Origin of genes/blue print of life
disease: Huntington
DNA
Species
Disease
1860-1868
1959
Haeckel
Leujeun dkk.
Heredity is transmitted via sperm & ovum
Human chromosomal disease(trisomy 21)
 Human
genome

Garland. 2008. Molecular basis of the cell.

 Recombinant DNA technology
§ DNA isolation & manipulation
§ Makes chimeric molecules
§ Involves several unique techniques and reagents
Several important reagents:
üRestriction enzymes
üDNA ligase
üDNA polymerase
 Important reagents: Restriction enzymes
§ Endonuclease
§ Cut DNA chains at specific locations (palindromic 4-7 bp)
§ Result: unique DNA fragment in a sequence-specific manner
§ Isolated from bacteria
§ Protect the host bacterial DNA from the genome of foreign organism
§ Named after the bacterium from which they are isolated
§ EcoRI à isolated from Escherichia coli strain RY13 which is 1st discovered
§ BamHI à isolated from Bacillus amyloliquefaciens strain H which is 1st discovered

§ DNA cut ends result:

§ Sticky ends (overlapping/cohesive)
§ Blunt ends
Murray RK, et al. 2009. A LANGE medical book: Harper’s illustrated
biochemistry. 28th ed. New York: The McGraw-Hill Companies, Inc
Blunt ends

Sticky ends
Restriction enzymes
Important reagents: DNA ligase & DNA polymerase
DNA ligase
§ Isolated from cell culture or bacteria
§ Ligates 2 fragments of DNA by catalyzing bonds between DNA molecules

DNA polymerase I
§ Isolated from bacteria
§ Incorporates nucleotides to complementary template DNA to synthesizing
new dsDNA; generates blunt ends from sticky ends.
Murray RK, et al. 2009. A LANGE medical book: Harper’s illustrated biochemistry. 28th ed. New York: The McGraw-Hill Companies, Inc
 Genetic engineering techniques

1. Gene identification
§ Polymerase chain reaction (PCR) & electrophoresis
§ Restriction fragment length polymorphism (RFLP)
§ Hybridization: Western, southern, northern blot
§ DNA sequencing

2. Gene cloning

3. Transgenic animal
 Gene identification: Polymerase chain reaction
(PCR)
§ Gene identification by amplifying DNA
sequence using primers compatible to DNA
target.
§ Principle: amplification of target DNA by
using thermal cycler

Target DNA
 Gene identification: Polymerase
chain reaction (PCR) – cont.

§ Requires:
§ DNA template
§ A pair of primers
§ DNA polymerase (thermostable; obtained from
thermophilic bacteria) à Thermus aquaticus
§ Taq polymerase

§ dNTP
§ Thermal cycler (PCR machine)
 Step 1 (94oC)
Denaturation
dsDNA to ssDNA

Step 2 (56-60oC)
Annealing
Primers onto template

Step 3 (72oC)
Extension
dNTPs extend the 2nd strand

extension products in one cycle serve as template in the next cycle

PCR Animation

Process

Denature

Anneal Primer

Replicate
DNA

1st cycle 2nd cycle 3rd cycle

 Gene identification:
§ To separate charged cells/molecules component Electrophoresis
(protein, nucleotide).
§ Principle: charged molecule in electric field will
migrate to opposite electrode. DNA is negatively
charged, will migrate to anode.
§ Samples are loaded into well in one of the plate
site
§ Velocity of the migration depends on:
§ Charged level
large moderate small
§ Molecule’s size
§ Molecule’s shape
§ Supporting media: Agarose/polyacrylamide gel &
Buffer
Electrophoresis

Loading the sample

Amplification result of Y chromosome fragment with PCR technic

N O N O N O N O M
1 2 3 4 5 6 7 8 9

800 bp 1000 bp
472 bp
500 bp
326 bp
311 bp

Y chromosome fragment amplification: sY14, sY84, sY143, and RBM1 in

Indonesian males with normozoospermia (N) and oligozoospermia (O).
Well no. 1-2 = sY14 3-4 = sY84
5-6 = sY143 7-8 = RBM1
9 = Marker 100 bp
 Gene identification: RFLP (Restriction fragment
length polymorphism)
difference in homologous DNA sequences that can be
detected by the presence of fragments of different
lengths after digestion of the DNA samples in question
with specific restriction endonucleases

§ Detection of gene polymorphism (variant in a gene) by using restriction

enzyme

§ E.g:
Polymorphism in exon 10 of FSH receptor gene in position 680th
nucleotide à resulting in 2 different amino acids: Ser & Asn.
AACC CGGGAG AACC AGGGAG
TTGGGC CCTC TTGGGT CCTC
 Gene identification: RFLP (Restriction fragment
length polymorphism) – cont.

§ PCR product of FSHR gene exon 10 in position 680

AACC CGGGAG AACC AGGGAG
TTGGGC CCTC TTGGGT CCTC

§ Cut by BsrI restriction enzyme

5’CC CGGG 3’
3’GGGC CC 5’
§ RFLP Electrophoresis result:
homozygous serin/serin : one band (300 bp)
homozygous asparagin/asparagin: 2 bands (200 bp & 100 bp)
heterozygous serin/asparagin : 3 bands (100bp, 200bp & 300 bp)
a b c d

1 kb
a. Marker
b. Ser/Ser
500bp c. Asn/Asn
d. Ser/Asn
100bp
 Application in forensic medicine

§ Variation in DNA for identification of an

individual: criminals or biological relationship.

§ Use of microsatellite markers (Alec Jeffreys,

1984) à DNA fingerprinting.

§ Variations are visualized as band patterns after

electrophoresis.

§ Each person has a unique pattern, except

identical twins.
Application in paternity test

Who are their parents/sons/daughters?

 Gene identification: Hybridization

§ Detect certain sequence/fragment (DNA, RNA, or protein) by

creating its hybrid (couple between two complementary strands)
§ The detected fragment will be marked by a probe
§ Types:
1. DNA hybridization (southern blot)
2. RNA hybridization (northern blot)
3. Protein hybridization (western blot)
Southern blot
hybridization

Hybridization
 Gene identification: DNA
sequencing

§ Principle: amplify DNA using DNA

template identified by DNA polymerase.
Incorporation of nucleotides in new
strand is terminated by ddNTP.
Gene identification: DNA sequencing (cont’)
 Gene cloning

§ Clone
A large population of identical molecules, bacteria, or cells that arise from a common
ancestor.
§ Chimeric/hybrid DNA molecules can be constructed in cloning vectors (gene porter, e.g:
bacterial plasmids, phages, cosmids) inserted to host cell.
§ Amplify the chimeric DNA àthe gene can replicate in host cell (e.g. bacteria, yeast).
§ Aims :
1. Produce gene library to be used for many technics
2. Produce certain gene product in vitro

Murray RK, et al. 2009. A LANGE medical book: Harper’s illustrated biochemistry. 28th ed. New York: The McGraw-Hill Companies, Inc
 Gene cloning: required
components
§ Gene/DNA template
§ Vector/ gene porter, should have:
1. gene for replication
2. restriction site
3. selectable marker to antibiotic
§ Host cell, should be able to:
1. Adopt foreign DNA
2. Replicate the adopted DNA
3. Express the adopted foreign
DNA
 Transgenic animal

§ Animal carrying other organism gene with different genetic

composition

Gene recipient animal/ transgenic animal

express the foreign gene

 FM UNSWAGATI

Making of
transgenic animal
How Dolly was created
 Somatic or body
cells has 54
Chromosomes

Egg or ovum has

cells has 27
Chromosomes

Fused cells has 54 tricking

the cell into thinking it got
fertilized by a sperm
 Transgenic animal: The benefit

§ Transgenic can transcript as endogen gene

E.g: Human lacto ferine gene can be expressed in transgenic cow and
produce cow milk similar to breast milk

§ Transgenic can produce mutagenesis (thus the cell does not

express the gene/ knockout animal)
E.g: Create animal model with class II MHC deficient.
Ethical aspect of Genetic engineering

The benefit VS ethical dilemma

§ Law
§ Culture & stigma
§ Religion & beliefs
§ Socioeconomics
Ethical aspect of Genetic engineering – cont.

§ Diagnostic
§ Prenatal (before birth) à “designed baby”
§ Postnatal (after birth) à having disease later in life; patient may
feel sick but does not yet suffer from the disease

§ Management & therapy

§ Trial with germ cell or early embryo is highly controversial &
currently banned by many countries
§ Abortion (termination of pregnancy) is illegal in many countries
THANK YOU