Pedigree of Hemophilia

in One Family
female
normal male
hemophilic male
Hemophilia
Hemophilia - A sex linked genetic disorder in
which blood clotting is deficient

Hemophilia A - lack of antihemophilic globulin
Most common type (80% of cases).
Hemophilia B - defect in thromboplastic
component - a milder form of the disease.
Sex linked - trait found on X chromosome.

Chromosomes
X Chromosome
p
q
22.3
22.2
22.1
21.3
21.2
21.1
11.4
11.3
11.23
11.22
11.21
11.1
11.1
11.2
12
13
21.1
21.2
21.3
22.1
22.2
22.3
23
24
25
26
27
28
coagualation factor IX, hemophilia B
blue-monochr. color blindness
coagulation factor VIIIc,hemophiliaA
homosexuality, male
cleft palate
growth control factor, X-linked
Xg blood roup
ocular albinism
sensorineural deafness
anemia, sideroblastic, with
Spinocerebellar ataxia
lymphoproliferative syndrome
Simpson dysmorphia syndrome
Tahun 50-an : DNA double stranded yang
membentuk Helix (Watson and Crick),DNA
Polymerase (Kornberg)
Tahun 60-an : DNA extrachromosome
(Plasmid), fungsi mRNA, Codon (Triplet
Nucleotide)
Tahun 70-an : Reverse Transcriptase,
Restriction Endonuclease, DNA Ligase,
Recombinant DNA (Berg), Cloning DNA
(BIOTEKNOLOGI)
Tahun 80-an : Transgenic mouse,
Penerapan rekayasa genetika dalam bidang
kedokteran, pertanian dan industri.
Tahun 90-an : Gen therapy, Cloning dan
Sequencing DNA (HGP), Diagnostic dll.
Tahun 2000-an : HGP selesai,
Pathogenese penyakit diketahui dari fungsi
molekul.
BODY PROTEIN
Enzyme
Receptor
Hormone
Growth Factor
Immunoglobulin
Interferon, Interleukin
Adhesions molecules
HLA/MHC
-1
-1 Acid Glycoprotein
-1 T Glycoprotein
-1 Antitrypsin
Transcortin
-1 Antichymotrypsin
-1 B glycoprotein
9,5-s -1 Glycoprotein
Vitamin-D binding protein
-1 Lipoproteins

-2
Retinol binding protein
-2 HS Glycoprotein
Histidine-rich 3,8 S 2 Glycoprotein
Haptoglobin
Pregnancy zone protein
2 Macrogobulin
Prothrombin
Antihemophilic factor
C1 inactivator
C1s
STRUKTUR PROTEIN
SIFAT PROTEIN
FUNGSI PROTEIN
PEMBENTUKAN PROTEIN
DISTRIBUSI PROTEIN
PEMERIKSAAN PROTEIN

STRUKTUR PROTEIN :
Struktur Primer : Sequensi asam amino
Struktur Sekunder : -helix, lipatan
Struktur Tertier : sub-unit protein (tiga dimensi)
Struktur Kwaterner : gabungan bbrp struktur tertier
SIFAT PROTEIN :
Ditentukan oleh sifat asam amino
FUNGSI PROTEIN :
Sangat bervariasi
PEMBENTUKAN PROTEIN :
Berdasarkan gen / DNA di inti sel
Berlangsung di Organella (Ribosome)
Proteins are composed of subunits called amino acids
Biokimia : DNA adalah Polymer dari
Desoxyribonucleotide (Basa, zat Gula dan 1
atau lebih gugus Phosphat)
Zat Gula : -D-2 Desoxyribose (Ribose)
Ikatan N-Glykosida antara Desoxyribose
(C1) dengan Pyrimidin (N1) atau Purin (N9)
Sanger dan Gilbert (1975) : methode
sequensi Basa Nukleotida (A, T, C, G)
Nukleotida : 2,9 milyar (990 mm) di
Chromosome (inti sel)
Telah selesai disequensi pada Juli 2000
Gen : Sepotong DNA (Intron atau Exon)
A - T G - C
Satuan DNA : bp (base pair)
A G C G A T C T G G
T C G C T A G A C C
DNA Base Pairing
Double helix consists of 2
complimentary strands of DNA.
Chromosomes
Long strands of DNA packaged and
compressed very tightly
Everyone has 2 sets (1 pair) of
chromosomes
1 pair of each of the 22 autosomes
plus XX for a female (46XX)
or XY for a male (46XY)
1 is inherited from mum, 1 from dad
You pass 1 of each pair onto each child
The Human Genome
The haploid human genome is made up
of 3 x 10
9
base pairs of DNA

This contains 50,000- 100,000 genes
arranged on 46 chromosomes

Packaged within the nucleus of the cell

DNA Replication
Each of the 2 DNA strands is copied by
machinery in the cell
Each new daughter strand has a sequence
complimentary to the original template
strand
Replication essential to allow cell division
(Mitosis) where 1 cell becomes 2
A
T
C
T
A
G
A
T
C
T
A
G
T
A
G
A
T
C
DNA Replication
semi-conservative
2 daughter cells
DNA Replication
DNA Replication
Replication fork : leading strand and lagging strand
DNA synthesized in the 5 3
The 5-3 synthesis of the leading strand is
continuous.
The lagging strand is also synthesized in the 5-3
direction but in small segments
This segments referred to as Okazaki fragments
Okazaki fragments has 100 200 nucleotides
DNA ligase joined the Okazaki fragments.
5 DNA Polymerase : , , , and
The DNA Replication Fork
DNA Replication in Meiosis
During the replication of chromosomes, there is a
cross-over of portions of one DNA strand to
another (of the same chromosome).
This cross-over, along with randomization assures
that offspring differ from the parents.
+
meiosis
Genes
Segments of DNA code for proteins (or
parts of proteins)
Each coding segment is called a gene
One gene codes one protein (or part of)
Genes contain the information which
makes us what we are
Gene Structure
Every three bases of DNA is called a codon
Each codon specifies an amino acid which
join together to form the protein
eg ATG = methionine = START
TAA = STOP
TAG = STOP
TGA = STOP
Exons
Introns
Gene Structure
Promoter
TAA
TAG stop
TGA
ATG
start
Exon = coding sequence
Intron= intervening sequence
(non-coding)
DNA
RNA
Protein
transcription
translation
Protein Synthesis
Transcription
3 Nuclear RNA Polymerase : mRNA transcribed by
RNA Polymerase II
The initiation of transcription involves binding RNA
Polymerase to a specific DNA sequence called a
Promoter
Many promoters for RNA Polymerase II contain
consensus sequences, referred to as the TATA box
( T A T A A/T A A/T A/G) which occur about 25-35
bp upstream from the transcription initiation site.
The activity of many promoters is affected by
Enhancers (regulatory sequences that may occur
thousands of base pairs upstream or downstream of
the gene they affect.
Protein Synthesis - Transcription
Each gene codes for a protein
DNA sense strand acts as template
and is transcribed into messenger
RNA (mirror image of the DNA but
Uracil instead of Thymine)

DNA
mRNA
A T C G G
U A G C C
Protein Synthesis- Translation
Introns are spliced out of the mRNA
mRNA leaves the nucleus
In the cytoplasm, ribosomes attach to the
mRNA ensuring the correct amino acid, for
each codon, is added to a growing chain of
amino acids which forms the resulting
protein.
rRNA : 40s particle (sebuah 18S RNA dan 55 %
protein) ; 60S particle (28S; 5,8S; 5S rRNA dan protein)
Translation: 1. Initiation
2. Elongation
3 Termination
Translational initiation signal : AUG
mRNA become translated through 5 3 direction
Elongation : Peptidyl transferase.
Termination : Stop Codon (UAG, UAA, UGA)
Amino acid will be activated and linked to the tRNA by
Aminoacyl-tRNA synthetase.

Amino acid assembly during translation occurs on ribosomes;
tRNA serves as the crucial adaptor molecule
Nukleotida 1. Nukleotida 2. Nukleotida 3.
(5) (3)
U C A G

U Phe Ser Tyr Cys U

U Phe Ser Tyr Cys C

U Leu Ser STOP STOP A

U Leu Ser STOP Trp G

C Leu Pro His Arg U

C Leu Pro His Arg C

C Leu Pro Gln Arg A

C Leu Pro Gln Arg G

U C A G

A Ile Thr Asn Ser U

A Ile Thr Asn Ser C

A Ile Thr Lys Arg A

A Met Thr Lys Arg G

G Val Ala Asp Gly U

G Val Ala Asp Gly C

G Val Ala Glu Gly A

G Val Ala Glu Gly G

Perbedaan Sandi Nukleotida
Nukleotida : Chr. : Mit. :

UGA Stop Trp
AUA Ile Met
AGA Arg Stop
AGG Arg Stop
MITOCHONDRIAL ENERGY TRANSDUCTION
I
II
NADH
III
Succinate
coQ Cytc IV
ATP synthase
Proton Motive
Force
ATP ADP + Pi
O
2
H
2
O

Human body synthesizes body weight of
ATP per day
motoric functions
biosynthetic activities
heat maintenance
Matrix side
Cytosolic side
H
+
I III II IV
V
ATP
ADP
ANT UCP
ADP+Pi ATP
Succinate
H
2
O
H
+
+O
2
c
NADH
NAD + H
2
Q Q
MITOCHONDRIAL RESPIRATORY
ENZYME COMPLEXES
Gen Mitochondria

Gen yang berbentuk sirkuler, terdiri dari 16569 bp
Diturunkan secara maternal, mudah bermutasi
Menyandi : 7 sub unit kompleks I (NADH Q-
Reduktase), 3 sub unit kompleks IV (Sitokrom
Oksidase), 2 sub unit ATP Synthase dan 1 sub unit
kompleks III (Apositokrom B)
Mutasi noktah (point mutation) pada gen mitochondria
:
A3243G G3316A
A3260G T3394C
A3256G A3252G
luas dijumpai : T16189C
MITOCHONDRIAL BIOLOGY AND
GENETICS
Semi-autonomous
organelles, contain multiple
copies of mtDNA
Double membrane structure,
cristae containing respiratory
chain enzymes
Most mitochondrial proteins
encoded by nuclear genome
and imported into
mitochondria
Functions in cellular
metabolism and the
regulation of cell death
Outer
Compartment
Inner
Membrane
Inner
Compartment
Outer
Membrane
Cristae Matrix
MITOCHONDRIAL PROTEINS mtDNA
Circular DNA - 16,569 bp
Encodes 13 polypeptides - for OXPHOS 22 tRNA 2 rRNA
D-loop - initiation of replication and transciption
Evolves at higher rate than nDNA
Maternally inherited
Pathogenese NIDDM
Patophysiologi secara genetik yang berkorelasi dengan
metabolisme energi
Timbul oleh karena perobahan cara hidup dengan cepat
(terutama dalam hal nutrisi)
Sel Pankreas berfungsi untuk mensekresikan Insulin
bergantung pada energi yang dibentuk di Mt.
Phosphorilasi oksidatif pada rantai respirasi Mt
ATP ATP dependent Potassium Channel
tertutup Calcium Channel terbuka sekresi
Insulin
Mutasi MtDNA penurunan ATP


MITOCHONDRIAL ENERGY METABOLISM
AND INSULIN SECRETION
MODY2
Transmembrane Protein Synthesis

Mutations
A change in the DNA sequence of the
gene
All cells acquire mutations as they
divide
rate of approx 10
-6
per gene per cell

Mutations can alter protein product of
DNA, stop gene working or activate
gene
Types of Mutation
Deletion - DNA missing
Insertion - extra DNA inserted
Expansion (Amplification) - DNA
repeat size has increased
Point Mutation - change in one base
Types of Mutation
(in coding sequence)
AGC TTC GAC CCG Wild type
AGC TCG ACC CG Deletion
AGC TTC CGA CCC G Insertion
AGC TTC TTC GAC CCG Expansion
ATC TTC GAC CGG Point mutation

POINT MUTATION

U A A
(Termination Codon)

U C A
(Codon for Serine)
U C U
(Codon for Serine)
C C A
(Codon for Proline)
Polimerase Chain Reaction (PCR)

Tahun 1985, Kary Mullis, California
Metode untuk meng-amplifikasi (melipat
gandakan) fragment DNA (Gen)
Dibutuhkan :
DNA atau RNA
Oligonucleotidprimer (PRIMER)
Enzym Taq-Polimerase
Campuran dari 4 Basa Nukleotida
(dNTPs)
10 x Reactions Buffer
Larutan MgCl
2

Alat : Thermal Cycler
Prinsip : perobahan temperatur secara
otomatis dengan waktu yang telah ditentukan
Dapat diatur (Program)
Contoh : 95 C------ Denaturasi
55 C------ Hybridisasi
(Annealing)
72 C------ Synthese DNA
(Extension)
Lama reaksi, bervariasi tergantung panjang
fragment DNA (2 min. : < 1000 Nukleotida)

DNA
DNA di-isolasi dari sel (darah atau jaringan)
DNA menjadi template atau matrix untuk
proses amplifikasi
Sense : 5- ATG(Start) -GGT-TCT-GTT-GCT-
GCT-TGG-TAA(Stop)- 3
Antisense : 3 - TAC-CCA-AGA-CAA-CGA-
CGA-ACC-ATT- 5
Exon dan/atau Intron dapat berfungsi
sebagai Matrix untuk amplifikasi
RNA
Single strand (Uracil pengganti Thymin)
Transkripsi dari DNA mRNA
Mengandung informasi genetik dari Exon
Dengan Enzym Reverse Transkriptase
diperoleh DNA dari RNA cDNA
Reaksi PCR nya disebut RT-PCR
Taq-Polimerase
Klenow - DNA Polymerase dari E.Coli
1988 : Taq-Polymerase dari Bakteri Thermus
aquaticus
Hybridisasi dan Polimerisasi berlangsung pada
temp. 50-70 C
Perhatikan : Buffer yang digunakan
(10 x RB) dan diperlukan MgCl
2

Primer
Sequence dari Nukleotida tertentu (Intron
atau Exon) : 20 30 bp
Prinsip : merupakan complementare dari
kedua strand DNA (Forward Primer dan
Reverse Primer).
Dari kedua Primer ini disinthese DNA yang
baru dan seterusnya berfungsi sebagai
matrix untuk siklus berikutnya.
Penentu bagi fragment DNA yang akan
diamplifikasi
PCR-REACTION
PCR-Reaction
Polymerase Chain Reaction
PCR Product (Amplifikat)
Gel-elektrophorese (Agarose)
Southern Blot (Hybridisasi dengan Sonde
DNA spesifik)
Dot - Blot (deteksi : Enhanced Chemie
Luminescense = ECL)
Denaturating Gradient Gel Electrophorese
(DGGE) atau Pulse Field Gel
Electrophorese (PFGE)
Enzym Restriksi : Restriction
Endonuclease
Sequence analysis (DNA Sequencing)
RER
free
ribosomes
cytoplasmic
proteins
Protein
Traffic

MOLECULARE MICROBIOLOGY
Aplikasi teknologi DNA
INFEKSI SALURAN CERNA:
Membedakan jenis : pathogen non pathogen
(Eschericia coli)
Untuk bakteri yang sulit dikultur oleh karena
memerlukan syarat tertentu (Campylobacter)
Membedakan jenis bakteri dari toxin yang
diproduksinya (E. coli dan Shigela sp.)
Subklas bakteri : Campylobacter, Helicobacter
Mengidentifikasi jenis Rotavirus (A, B, C)

Aplikasi teknologi DNA
INFEKSI SALURAN NAFAS:
Mycobacterium tuberculosis :
Membedakan jenis atypic, dengan mikroskop hal
ini tidak mungkin
Kultur : waktu yang lama dan bakteri harus
banyak (terutama untuk sensitivity test)
Diagnose cepat dibutuhkan, mis. pada penderita
AIDS.
Ditemui jenis yang multi drug resistant (MDR)
Diagnosa dengan PCR dan Hybridisasi (contoh :
dot-blot)

RESULT
MOLECULARE ONKOLOGY
PROTOONKOGEN : gen yang normal pada
Genom yang berperan penting dalam proliferasi
dan differensiasi sel
ONKOGEN : protoonkogen yang oleh karena
mutasi atau gangguan pada ekspresinya
menyebabkan proliferasi sel yang neoplastis
TUMORSUPPRESSOR GEN : gen yang berperan
pada proliferasi dan differensiasi sel, dimana bila
gen ini di-inaktivasi atau tidak terdapat, akan
terbentuk sel neoplastis
MOLECULARE ONKOLOGY
Contoh Neoplastic Transformation :
1. Gentranslocation : bcr-abl (chr. 9 dan 22)
2. Genamplification : N-myc gen 300 x pada
Neuroblastoma pada anak-anak
3. Point mutation : ras mengontrol
GTP(aktif) GDP (inaktif)
4. Insertion gen virus : virus Hepatitis B
5. Tumorsuppressorgen : p53 dan gen
retinoblastoma : regulasi siklus sel (stop
pada G
1
untuk DNA - repair)

Second-Messenger Mechanism
Adenosine 3,5-cyclic monophosphate (cAMP)
cAMP
Adenylate cyclase
Hormone
Receptor
Transducer G Protein
Protein Kinase A
Structural Proteins
Enzymes
Membrane
Channels
ATP
(+,-)
(+,-)
Produk Protoonkogen
Inti Sel
FOS
MYC
JUN
Orga
nella
ERB-B1
FMS
SIS
MOS
ABL
FMS
SRC
RAS
Carcinogenesis (Colorectal Cancer)
Produk dari gen untuk therapy dan
prophylaxis :
Erythropoietin
Insulin
Hormon pertumbuhan
Faktor pembekuan darah VIII
Plasminogen aktivator
Vaksin Hepatitis B
Penerapan Teknologi Gen/DNA dalam Therapy
Aplikasi gen dalam Forensik
Sebelum teknologi DNA diterapkan (1978)
biasanya digunakan protein, misalnya
antigen gol.darah, HLA, dll.
1985 : DNA Polymorphismus.
Nov.1987 : DNA sebagai barang bukti di
pengadilan di Inggris.
Sampai akhir 80-an : lebih dari 1000 perkara
dibantu oleh bukti-bukti DNA
Juga dapat menentukan Paternity
Profil DNA tiap individu berbeda