Académique Documents
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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
Gene engineering
recombinant DNA, cloning
Methods .
Finger printing
PCR, RFLP, enzyme digestion, sequensing,
microsatelit analysis, etc
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
Calpain 10
NEUROT1 (2q32), HNF4A DM2 late
onset
GLUT4, GLUT2
GPD2
dll
2. Obesity
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
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
Exon
45
46
53
54
P: Patient DNA
FISH
to see the specific chromosomal
abnormality
Teknik Pemeriksaan
RNA
Control of gene
expression
tRNA
rRN
A
snRNA
RNA Synthesis
(Transcription)
Using DNA as a template
Transcription mRNA
maturation
Simpulan
(from introduction)
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)
mRNA Level
MCP
CPCPCPM
Exon 71 72 73 74
M:Marker
C: Normal Control
P:Patient
M: Marker C
Muscle mRNA
: Normal Control
M C P
P: Patient
Isolasi RNA
Isolasi RNA.
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.
RT-PCR
(Reverse Transcriptase-PCR)
Pengertian
Konversi RNA DNA (cDNA)
(
Alasannya
RNA tidak stabil/mudah rusak
RNA membentuk struktur
sekunder atau tersier
Microarray
Microarray
also known as gene or genome chip,
DNA/RNA chip, or gene array
Expression profiling
i.e., monitoring expression levels of thousands of
genes simultaneously, or for comparative genomic
hybridization
Transgenic Mice
3. Analisis Hubungan
Kekerabatan
4. Therapy
Gene Therapy