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PENGGUNAAN PCR
UNTUK KLINIKDebbie S. Retnoningrum
Sekolah Farmasi
Institut Teknologi Bandung
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Penggunaan klinik
Mempelajari atau mendeteksi polimorfisme / mutasi
Menegakkan diagnosis penyakit
Memantau terapi obat
Menentukan kesembuhan
Mengetahui terjadinya breakthrough dan relapse
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Polymorphism
A variation in the DNA that is too common to
be due merely to new mutation. A
polymorphism must have a frequency of at
least 1% in the population.
Examples of polymorphisms include the
genes for sickle cell disease, thalassemia and
G6PD deficiency.
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PCR - RFLP
Fragmen DNA yang mengandung mutasi /
polimorfisme diamplifikasi dengan PCR
Produk PCR dipotong dengan enzim restriksiyang dapat membedakan antara alele mutan
dan non-mutan
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Enzim restriksi?
Enzim endonuklease yang mengenali dan
memotong urutan spesifik dan reproducible
pada framen DNA
Urutan yang dikenali merupakan palindrom Palindrom?
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1000 bp
Varian 1
1000 bp
Varian 2
400 bp 600 bp
Varian 1
dan 2
1000 bp
400 bp 600 bp
600 bp
400 bp
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PCR DAN RT-PCR:
KONVENSIONAL DAN REAL TIME
KONVENSIONAL:
AMPLIFIKASI DAN DETEKSI DILAKUKAN SECARA
KONSEKUTIF
REAL-TIME:
AMPLIFIKASI DAN DETEKSI DILAKUKAN SECARA
SIMULTAN
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PCR KONVENSIONAL
COBAS AMPLICOR
HOME BREW PCR
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PEMANTAUAN PCR
Agarose Gel Blotting LightCycler
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Struktur VIRUS HEPATITIS B
YANG MENJADI TARGET ADALAH DNA HBV preCore/Core
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Perbandingan cobas amplicor vs
taQman hBv
Kinerja / metode Cobas Amplicor Taqman
Batas deteksi 60 IU/ml 6 IU / ml
Rentang linearitas 60 3,8 x 104 IU/ml 29 1,1 x 108 IU/ml
Pembacaan End point Real time
Sampel Serum Plasma (serum kurang
sensitif)
Deteksi Kolorimetri Fluorometri
Genotipe A, B, C, D, E, F A, B, C, D, E, F, G
MUTANT PRECORE
Kontrol kontaminasi Ada AdaKontrol mencegah
negatif palsu
Ada Ada
Spesifisitas 100 % 100 %
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StruKtur VIRUS hepatitis C
(RNA)
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PATTERNS OF VIROLOGIC RESPONSE,
BREAKTHROUGH AND RELAPSE
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EVOLVING DEFINITIONS OF TREATMENT RESPONSE
TERMS DESCRIPTIONS
RAPID VIROLOGIC RESPONSE (RVR) UNDETECTABLE HCV RNA AT WEEK 4
EARLY VIROLOGIC RESPONS (EVR) 2 LOG 10 DROP IN HCV RNA FROM BASELINE OR
UNDETECTABLE HCV RNA AT WEEK 12
COMPLETE EVR NO RVR, UNDETECTABLE HCV RNA AT WEEK 12
PARTIAL EVR NO RVR, 2 LOG 10 DROP IN HCV RNA FROM BASELINE AT
WEEK 12 BUT STILL DETECTABLE RNA HCV
NULL RESPONSE < 2 LOG 10 DROP IN HCV RNA FROM BASELINE OR NO
UNDETECTABLE HCV RNA AT WEEK 12
END OF TREATMENT UNDETECTABLE HCV RNA AT END OF TREATMENT
SUSTAINED VIROLOGIC RESPONSE
(SVR)
UNDETECTABLE HCV RNA AT 24 WEEKS AFTER TREATMENT
BREAKTHROUGH DETECTABLE HCV RNA DURING TREATMENT AFTERINITIALLY ACHIEVING UNDETECTABLE HCV RNA
RELAPSE RECURRENCE OF HCV RNA IN PATIENTS WHO ACCHIEVED
AND MAINTAINED UNDETECTABLE RNA THROUGHOUT THE
DURATION OF TREATMENT
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KRAS and the EGFR pathway
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The EGFR signaling pathway is activated inresponse to ligand binding to the cell-surfacereceptors: these ligands include TGF and EGF
The signaling cascade that is activated isinvolved in regulating genes that control cellcycle progression
Cell cycle progression gives rise to tumorsurvival, growth and proliferation as well asmetastasis and angiogenesis
In the early part of the signaling cascade, theprotein KRAS regulates downstream proteinsinvolved in these effects
The KRAS protein plays a central role in tumor development,
regulating downstream proteins that are involved in
proliferation, survival, metastasis and angiogenesis
Th EGFR h d h i
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The EGFR pathway and the importance
of
KRAS status The KRAS gene may benormal (wild-type) ormutated
Wild-type KRAS protein isactive for a short period
when the EGFR is stimulated The effects of the protein are closely controlled
When KRAS is mutated theprotein is permanentlyturned on, even without
being triggered by EGFR-mediated signaling The effects of KRAS that lead to tumor growth and
spread continue unregulated
The KRAS status of a tumor 25
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The KRAS test
A tumor sample is takenand sent to the laboratory the test can use fresh,frozen or paraffin-
embedded tissue
A pathologist confirmsthat the tissue iscancerous, and a sample
of DNA is prepared for theKRAS test
The polymerase chain 26*Direct DNA sequencing can also be used
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TIB MolBiol Assay
DNA Extracted
from FFPET
Each sample added to 2 or 3
different reaction mixtures;
4 controls/standards
required per carousel run
concon
00LowLow
concon concon concon
HigHig
hh
The PCR reaction and
melting curve take around
60 minutes
The analysis is completed
by analyzing the Melting
curve on the instrument
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PCR untuk kuantitasi EBV