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Aplikasi Klinis

Biologi Molekuler
S. Yudha Patria
Pediatrics Dept, Fac. Medicine,
Gadjah Mada University

Introduksi

Techniques Approach in
Molecular Genetic Studies

PCR based
classic PCR, RACE, SSCP, RT-PCR, insitu-PCR,
qPCR,etc

Cloning subcloning

Enzym digestion
RFLP, haplotyping

Hybridization
Southern, Northern, Western bloting

Sequensing
enzymatic, radioactive

Methods in Molecular Genetic


Studies

Mutation exploration and analysis of gene


structr.
PCR, Southern blot, enzyme digestion, RFLP,
sequensing, homology analysis

Gene expression analysis (gene function


analysis)
PCR of cDNA, Western blot, recombinant DNA,
Transgenic mice, Regenerative medicine, etc

Gene engineering
recombinant DNA, cloning

Methods .

Finger printing
PCR, RFLP, enzyme digestion, sequensing,
microsatelit analysis, etc

Molecular genetic epidemiology Methods


Variation analysis, Polymorphism analysis

Gene therapy methods


Cell therapy, Vector-mediated gene transfer, Lipid
carrier systems, Antisense oligonucleotides, Cloning
modification

How to enter
molecular-biological
research ?

Pattern of inheritance ?
Simple or Complex disease ?
Gold standard clinical diagnosis ?
Unknown or well-known mechanism ?
What the purposes of research ?

An example case : DM

Diabetes is a chronic metabolic disorder that


is inability to manufacture and use insulin
failed the conversion of food into energy.

DM greatly increases the risk of blindness,


heart disease, kidney failure, neurological
disease, and other conditions

Type 1, or juvenile onset diabetes, is the


more severe form of the illness.

In Type 1 diabetes, the body's immune


system assault on its own insulin and the
pancreatic cells, however, the mechanism of
how this happens is not yet understood.

About 10 loci in the human genome have


been found to confer susceptibility to IDDM

Among these are:


1) a gene at the locus IDDM2 on chromosome
11
2) the gene for glucokinase (GCK), an enzyme
of glucose metabolism which helps modulate
insulin secretion, on chromosome 7.

At DM2 involving many genes


(race, environment, lifestyle, etc)

Calpain 10
NEUROT1 (2q32), HNF4A DM2 late
onset
GLUT4, GLUT2
GPD2
dll

2. Obesity

Obesity is an excess of body fat that known as risk


factor for chronic diseases including heart
disease, diabetes, high blood pressure, stroke and
some forms of cancer

The body weight that more than 25% body fat in a


man and women with more than 30% are obese.

Evidence, suggests that obesity has more than


one cause: genetic, environmental, psychological
and other factors may all play a part.

The hormone
leptin, is act as a
lipostat

Most overweight
people have high
levels of leptin in
their bloodstream.

Clinical Application

Molecular Analysis

Cytogenetics

DNA level

mRNA level

Expressivity
(transcription, translation)

1. Prevensi Polimorfisme
variasi nukleotid tunggal / ulangan pada sekuens
DNA
Konsekuensi Polimorfisme
- Netral (fenotipe normal):
tidak menunjukkan perbedaan fenotip atau
berbeda bentuk/struktur protein (isozym) yang baru
tampak aktif pada kondisi tertentu (misalnya pada
tekanan udara atau suhu atau pH tertentu)
- Penyakit genetik multifaktorial
seseorang mempunyai kecenderungan untuk
mempunyai
penyakit tertentu dipengaruhi oleh lingkungan tertentu

Contoh analisis polimorfisme

Penentuan alel T235 gena Angiotensinogen


Alel T235 sangat berhubungan dg
tingginya
kejadian hipertensi
Alel T235 terletak pada nukleotid 704
ekson 2 gena angitensinogen
transisi C T (Thr Met)

Teknik analisis alel T235 pada pre-eklamsi

PCR ekson 2 + RE (Tth111-I GAC NNGTC)


Prediksi hasil :
246 bp

1 = AA
2 = Aa
3 = aa

Frekuensi Alel
Eklamsia
(n=20)
Genotipe

AA
Aa
aa

Alel

A
a

Non-Eklamsia
(n=100)

4
10
6

14
48
38

0.45
0.55

0.38
0.62

2. Diagnosis
mencari perbedaan
susunan
gena (mutasi)
membandingkan susunan gena
orang normal dan gena pasien

DNA Analysis of Dystrophin Gene


M

Exon

45

46

53

M: Marker C: Normal control DNA

54
P: Patient DNA

Diagnosis Cytogenetic Analysis


to see the chromosomal abnormality

FISH
to see the specific chromosomal
abnormality

Teknik Pemeriksaan
RNA

Suryono Yudha Patria


Pediatrics Dept, Fac. Medicine,
Gadjah Mada University

Control of gene
expression

Struktur RNA - DNA

Struktur RNA & DNA

Comparison with DNA

RNA is a single-stranded molecule

Much shorter chain of nucleotides

Contains ribose (two hydroxyl


groups attached to the pentose
ring) hydroxyl groups make RNA
less stable than DNA because it is
more prone to hydrolysis.

Comparison with DNA

Biologically active RNAs (tRNA, rRNA, snRNAs


and other non-coding RNAs) are extensively
base paired to form double stranded helices
(secondary-, tertiary- structure)

RNA can achieve chemical catalysis, like


enzymes

RNA function as active form of gene


expression

Struktur sekunder RNA

Three major types of RNA


messenger RNA (mRNA)
has an information storage function, like DNA.
Carries information from DNA to the ribosome sites
of protein synthesis in the cell

transfer RNA (tRNA)


a small RNA chain of about 74-95 nucleotides that
transfers a specific amino acid to a growing
polypeptide chain at the ribosomal site

ribosomal RNA (rRNA)


rRNA and ribozymes have catalytic functions, like
proteins, 80% of the RNA molecules found in a
typical eukaryotic cell

tRNA

rRN
A

Other types of RNA


noncoding RNA

A number of other types of RNA


identified until now, present in smaller
quanitites

small nuclear RNA (snRNA)


small nucleolar RNA (snoRNA)
the 4.5S signal recognition particle
(SRP) RNA

snRNA

RNA Synthesis
(Transcription)
Using DNA as a template

Catalyzed by an enzyme - RNA polymerase


Initiation of synthesis begins with the binding
of the enzyme to a promoter sequence in the
DNA (usually found "upstream" of a gene)
The DNA double helix is unwound by the
helicase enzyme
The progresses along the template strand in
the 3 -> 5 direction, synthesizing a
complementary RNA molecule with
elongation occurring in the 5 -> 3 direction

Transcription mRNA
maturation

Simpulan

(from introduction)

RNA is very similar to DNA, but differs in:


nucleotides contain ribose sugars and RNA uses
predominantly uracil instead of thymine present
in DNA. RNA is transcribed (synthesized) from
DNA by enzymes called RNA polymerases and
further processed by other enzymes. RNA serves
as the template for translation of genes into
proteins, transferring amino acids to the
ribosome to form proteins, and also translating
the transcript into proteins.

RNA is the only biological polymer that serves as


both a catalyst (like proteins) and as information
storage (like DNA)

Aplikasi Pemeriksaan
RNA
- Penelitian biologi molekuler secara
umum (PCR, kloning/subkloning, dsb)
- Diagnostic tool
- Genetic expression study
- Microarray application
(to understand processes as diverse as the mechanisms of
toxicity, embryogenesis and disease progression)

Contoh Aplikasi Pemeriksaan


RNA
DNA Level

mRNA Level

MCP

CPCPCPM

Exon 71 72 73 74

M:Marker

Exon 65-74 74-79 70-75

C: Normal Control

P:Patient

Contoh aplikasi pemeriksaan RNA


Ekspresi gen
Lymphoblast mRNA
C

M: Marker C

Muscle mRNA

: Normal Control

M C P

P: Patient

Isolasi RNA

Pengambilan materi RNA untuk


keperluan pemeriksaan

Isolasi RNA isolasi RNA total

Isolasi RNA.

The successful isolation of intact RNA requires four


steps:
1) effective disruption of cells or tissue
2) denaturation of nucleoprotein complexes
3) inactivation of endogenous ribonucleases
(RNases)
4) removal of contaminating DNA and protein

The most important step is the immediate


inactivation of endogenous RNases that are released
from membrane-bound organelles upon cell
disruption

Contoh Langkah Isolasi


RNA

The Maxwell 16 Total RNA Purification Kit combines the


disruptive and protective properties of guanidine thiocyanate
(GTC) to lyse samples, denature nucleoprotein complexes and
inactivate ribonucleases.

Adding -mercaptoethanol (BME) is strongly recommended for


samples containing a high level of nucleases (e.g., spleen or
pancreas tissue).

The genomic DNA is removed from the sample lysate using the
Clearing Agent.

The total RNA is captured from the cleared sample lysate using
MagneSil PMPs and is further purified from contaminating
salts, proteins and cellular impurities by ethanol washes.

Finally, total RNA is eluted in Nuclease-Free Water.

Evaluasi Hasil Isolasi


RNA

RT-PCR

(Reverse Transcriptase-PCR)
Pengertian
Konversi RNA DNA (cDNA)
(
Alasannya
RNA tidak stabil/mudah rusak
RNA membentuk struktur
sekunder atau tersier

The Principle of RT-PCR

An RNA could be converted into DNA,


allowing manipulation and cloning

Use of reverse transcriptases to perform


first-strand cDNA synthesis

Application of PCR to amplify DNA.


It does not take long before first strand
cDNA synthesis

Microarray

Microarray
also known as gene or genome chip,
DNA/RNA chip, or gene array

is a collection of microscopic DNA


spots, commonly representing
single genes, arrayed on a solid
surface by covalent attachment to
chemically suitable matrices

How does DNA microarray


technology work?

Skema cara kerja microarray

DNA microarrays are commonly used for:

Expression profiling
i.e., monitoring expression levels of thousands of
genes simultaneously, or for comparative genomic
hybridization

Comparative genomic hybridization (Array CGH)


Assessing large genomic rearrangements

SNP detection arrays


Looking for Single nucleotide polymorphism in the
genome of populations

Chromatin immunoprecipitation (chIP) studies


Determining protein binding site occupancy
throughout the genome, employing ChIP-on-chip
technology

Aplikasi klinis yg lain

Transgenic Mice

3. Analisis Hubungan
Kekerabatan

4. Therapy

Some recombinant DNA products used in human


therapy
insulin for diabetics
factor VIII for males suffering from hemophilia A
factor IX for hemophilia B
human growth hormone (GH)
erythropoietin (EPO) for treating anemia
Interferons several interleukins
granulocyte-macrophage colony-stimulating factor (GM-CSF) for
stimulating the bone marrow after a bone marrow transplant
tissue plasminogen activator (TPA) for dissolving blood clots
adenosine deaminase (ADA) for treating some forms of severe
combined immunodeficiency (SCID)
angiostatin and endostatin for trials as anti-cancer drugs
parathyroid hormone

Gene Therapy

Modulation of gene expression


Gene Replacement Therapy:
1. Cell-mediated gene transfer (cell
therapy)
2. Vector-mediated gene transfer (gene
therapy)
3. Combine techniques