POLITEKNIK KESEHATAN SEMARANG

Bernet Agung Saputra

Semarang, Januari 2019
 Materi
• Dasar Biologi Molekuler
• Preparasi sample
• Dasar Polymerase Chain Reaction (PCR)
• Bioneer Molecular Diagnostic
• MDx: Real-Time PCR Kit
– AccuPower®- Bioneer, Korea Selatan
– AbTes – AITbiotech, Singapore
– Amoy – AmoyDx®, China
• Good practice laboratory
 Molecular Biology

Biologi molekuler merupakan cabang ilmu biologi yang

mempelajari struktur dan fungsi gen pada tingkatan molek
uler.

Biologi molekuler berkaitan erat dengan cabang keilmuan

lain, khususnya ilmu genetikadan biokimia.

Pendekatan biologi molekuler memungkinkan sebuah pen

elitian yang dapat memprediksi peristiwa-peristiwa alami
yang akan terjadi pada makhluk hidup dimasa mendatang
 Genome

• Keseluruhan informasi genentik yang beradapada suatu organisme

• Pada umunya dapat berupa DNA, namun untuk beberapa virus ber
upa RNA

Perbandingan ukuran genome makhluk hidup

 Asam Nukleat

 DNA dan RNA merupakan rant

ai polimer yang panjang dari ik
atan kimia yang disebut nukleot
ida.
 Nukleotida tersusun oleh 3 kom
ponen utama, yaitu 5-carbon su
gar (pentose), nitrogen base dan
phosphate group (P)
 Phosphate group membentuk ra
ngkaian yang menguhubungkan
satu dengan lainnya
 Bentuk Asam Nukleat

1. Ribonucleic acids (RNA)

Ikatan gula pentose dalam bentuk Ribose (hydroxyl group
terletak pada rantai karbon ke 3---OH)

2. Deoxyribonucleic acids (DNA)

Ikatan gula pentose dalam bentuk Deoxyribose (hanya me
miliki hydrogen pada semua ikatannya--- H) Deoxy = “ta
npa ikatan oksigen”
 DNA Double Helix

• Merupakan model yang diajukan oleh

Watson dan Crick pada tahun 1953
• Terdiri dari nukleotida double strand
• Nukleotida berikatan dalam bentuk ikat
an kovalen.
– Adenine (A) berikatan dengan Thym
ine (T).
– Cytosine (C) berikatan dengan Guan
ine (G)
 Gen

 Gen merupakan segment tertentu dari double stranded DNA yang

sangat panjang
 Gen merupakan informasi dasar yang dapat diturunkan
 Pada organisme eukaryote, gen teletak di lokus pada setiap kromo
som.
 Gen memiliki struktur , exon (coding sequence) dan intron (non-c
oding sequence)
Central Dogma Biologi Molekuler
 Preparasi Sample
(Purifikasi Asam Nukleat)
Peralatan Penunjang

Centrifuge berpendingin Centrifuge meja Mini spin centrifuge

Vortex Heating block Micropipette

11
Investor Relations 2010 11
Laminar Air Flow Biosafety Cabinet

12
Investor Relations 2010 12
Laminar Air Flow Biosafety Cabinet

13
Investor Relations 2010 13
 Biosafety Cabinet Laminar Air Flow

Level perlindungan Melindungi user dan sample dari a Melindung sample dari cem
gen biologis yang ada di dalam rua aran agen biologis yang ada
ng kerja di dalam ruang kerja namun
tidak memberikan perlindun
gan kepada user
Sirkulasi udara Udara dimurnikan dan disirikulasik Udara dimurnikan dan dibua
an kembali ke dalam ruang kerja ng keluar ruang kerja

Fungsi penggunaan Digunakan untuk pengerjaan samp Digunakan untuk pengerjaan

el yang infeksius dan non infeksiu sampel non infeksius : Prep
s: Mikroorganisme pathogen, samp arasi media
el manusia

14
Investor Relations 2010 14
Perlengkapan Keamanan

Jas laboratorium Sarung tangan latex Masker

Protective slipper Protective gloves Protective glass

15
Investor Relations 2010 15
How to Obtain Highly Pure and
High Yield Nucleic Acid

 Lysis
 Precipitation
 Purification
 Elution
 Tujuan Ekstraksi Asan Nukleat

Untuk memperoleh DNA/RNA dalam bentuk

yg relatif murni sehingga dapat digunakan
untuk uji lanjutan, contoh : PCR, sekuensing, dll.
 Pemanfaatan Asan Nukleat

NGS (Next Generation Sequencing)

• Multi-region DNA/RNA sequencing.

Molecular Diagnostics of Viral Infections

• Highly pure Nucleic acid purification for reproducible results.

Cancer Diagnostics

• Circulating gDNA purification from very limited amount.

 Nucleic acid purification
• Pure nucleic acid is very fundamental matter
– Appropriate removal of proteins, polymerase inhibitors or nucleases from various samples

• Quality of purified DNA and RNA can provide successful experiment for almost all of modern
molecular biology applications such as molecular diagnostics, NGS, molecular inspection etc.

Wide range of starting materials

Clinical : Whole Blood, Buccal swab, Urine, Saliva
Academic : Animal tissue and cell, bacteria, Fungi, Insect
Agriculture : Plant, Soil, GMO
Forensic : Hair, Sperm, Nail, cigarette butts
Protokol dasar ekstraksi asam nukleat
4 tahap utama untuk memisahkan dan memurnikan NA dari
komponen sel lainnya :
1. Lisis
2. Pengendapan / precipitation
3. Pencucian / washing
4. Pelarutan / resuspension
PURIFIKASI

Perusakan / Lisis Sel

Perusakan dan homogenisasi sampel
– Mekanis (Gerus, sonikasi, vortex)
– Fisik (Pembekuan)
– Enzimatik (Proteinase K, Lysozyme, Collagenase)
– Kimiawi
(Guanidinium isothiocyanate (GITC), Alkali treatment, CTAB)
Traditional Nucleic acid Isolation methods

Phenol-
chloroform
method

Lithium Traditional
Ethanol
chloride Nucleic acid
precipitation
precipitation Isolation
method
method methods

CsCl density
gradient
method
Most Popular Ways of Nucleic Acid Purification

Magnetic Beads Silica Membrane Spin Colum

Mechanisms

Magnetic Beads Silica Membrane

Fishing hook Fishing net

Working Flow of Magnetic Beads System
 Silica Membrane System

Lysate & Buffers

Nucleic Acid

Silica Membrane
Pros and Cons

Magnetic Beads Silica Membrane

Wash Wash Wash Elute

Bind
I II III

Pros: Fast operation time and more

economical Wash away
Cons: Highly contamination of residuals.

Pros: Highly Pure nucleic acid purification

Cons: Resistively slow operation
and less economical
 Selection of Optimal Silica Types

gDNA
RAW cell ( <3 x 10^6) Silica Beads
260/280 Yield
28s rRNA (ng/ul)
Silica bead type A 2.10 181.4
18s rRNA 2.11 161.0
Silica bead type B 2.16 131.3
2.16 109.3

Matrix
type A
Matrix
type B
Matrix
Type C
Matrix
Type D Silica Membranes
Sample ID Nucleic Acid Conc. Total yield(ug/ul) 260/280 260/230
Silica matrix 1444.60 72.23 2.15 2.23
type A 1482.00 74.10 2.15 2.22
gDNA
Silica matrix 1477.50 73.88 2.16 2.25
28s rRNA type B 1513.20 75.66 2.14 2.25
Silica matrix 1381.50 69.08 2.13 2.24
18s rRNA type C 1263.50 63.18 2.14 2.23
Silica matrix 1405.20 70.26 2.14 2.26
type D 1354.70 67.74 2.14 2.23
 Finding Optimal Composition in buffers

First washing buffer

gDNA
Jurkat cell ( < 4 x 10^6)

28s rRNA 260/230 260/280 Yield

(ng/ul)
18s rRNA Buffer A 1.33 2.07 77.5
Buffer B 2.19 2.06 83.4
Buffer C 1.18 2.05 80.5
Buffer D 2.29 1.96 174.5

Optimal Combination of silica matrix with salt, ethanol, pH etc.

 Dasar PCR

Overview 1. Tujuan PCR

2. Pengantar
3. Komponen reaksi PCR
4. Variabel yg mempengaruhi PCR
 Polymerase Chain Reaction

• PCR, polymerase chain reaction, merupakan tekhnik in-

vitro untuk memperbanyak fragmen DNA tertentu yang
dikehendaki

• Sebelum ditemukannya metode PCR, DNA hanya dapat

diperbanyak melalui metode over ekspresi pada sel sehi
ngga jumlahnya sangat terbatas
 1985
• Enzymatic amplification of beta-globin genomic se
quences and restriction site analysis for diagnosis
of sickle cell anemia.
Science. 1985 Dec 20;230(4732):1350-4.

• Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlic

h HA, Arnheim N.
•
Cetus Corporation, Department of Human Genetics, Emeryville
, CA 94608.
 Tujuan PCR

• Mengamplifikasi fragment DNA dari jumlah yang

sedikit

• Menganalisis fragmen DNA tertentu dalam

kompleks campuran DNA.

• Mengubah sekuens DNA – mutagenesis langsung

Kenapa PCR??
• 1. Simple
• 2. Powerful
– A. sensitive – sensitivity
– B. specific – specificity
– C. reliable – reliability; fidelity
• 3. Fast
 Komponen Reaksi PCR

• DNA Template
• Primer DNA (Forward, Reverse)
• Polimerase thermo-stabil
• dNTP
– (dATP, dTTP, dCTP, dGTP) Thermus aquaticus
• Buffer PCR (Mg++)
1. Template DNA

• DNA containing region to be sequenced

• Size of target DNA to be amplified: up to 3 Kb
2. Primer DNA

• Pasangan primer yg sesuai (Forward + Reverse)

• Panjang (15-30bp)
• %GC= 50-60%
• Tm antara 55-80°C
• Hindari urutan basa yang mudah terdenaturasi – e.g. untaian basa
G
• Hindari primer yang mungkinkan untuk melakukan self compleme
ntary
• e.g. hairpins, homodimers, heterodimers
3. Polymerase thermo-stabil

• Taq (Thermus aquaticus), Pfu (Phyrococcus furi

osis), KOD (Thermococcus kodakaraensis)
• Native atau Cloned
• Half-life
– e.g. Taq 40 min half-life, Vent 7 hour half-life
• 3’5’ Exo nuclease – proofreading
• Fidelity (Error Rate)
– Taq 1/10,000nt, Pfu 1/1,000,000
• Processivity
• Extra bases at end (A-overhang)
4. Buffer PCR

• Komponen dasar
– 20mM Tris-HCL pH 8.4
– 50mM KCl
– 1.5 mM MgCl2
• Magnesium – Ion Mg2+ dapat membentuk kompleks dg dNTP, primer
dan templat DNA, sehingga konsentrasi ion Mg2+ perlu dioptimasi
dlm setiap eksperimen.
Mg2+ terlalu sedikit : hasil produk PCR tidak banyak;
Mg2+ terlalu banyak : kesalahan inkorporasi basa, produk non spesifik.

• Bahan Additives yg potensial :

– Helix Destabilisers - baik digunakan jika DNA target memiliki %GC yg tin
ggi.
• dimethyl sulphoxide (DMSO),
• dimethyl formamide (DMF),
• urea
• formamide
– Panjang target>1kb. Formamide and glycerol
– Konsentrasi DNA templat rendah: Polyethylene glycol (PEG)
 Kondisi Reaksi PCR

1. Denaturasi, suhu dimana dsDNA akan mengalami penuruna

n interaksi hidropobik antar sehingga terpisah menjadi ssDNA

2. Annealing, kondisi hibridisasi dimana primer akan menempel

pada pilinan DNA yang sudah terpisah pada proses denatura
si

3. Extension, proses sintesis DNA polymerase dimana penamb

ahan complement deoxyribonucloside pada template DNA da
ri arah ujung-5’ ke ujung-3’
Siklus Reaksi PCR
Protokol PCR umumnya :
Step Time/Temp
1 3 min at 95°
2 30 sec at 95°
3 1 min at 55°
4 2 min at 72°
5 Go to step 2 - 35 times
6 8 min 72°
7 ~ min 4°
8 End
End-point PCR
End-point PCR
Prinsip kerja end-point PCR

Cockerill FR III. Arch Pathol Lab Med. 2003;127:1112

 Kekurangan end-point PCR

What is Wrong with Agarose Gels?

• Poor precision
• Low specificity
• Size-based discrimination only
• Low sensitivity/Resolution
• Short dynamic range (< 2 logs)
• Possibility of human errors
• Cross-contamination
• Results are not expressed as numbers
• Ethidium bromide staining is not very
quantitative
 Real-Time PCR
Next generation diagnosis
 In vitro diagnostics
1. Definition: Detection, Quantification, & Identification of target pathogens in
patient’s specimens

2. Methods: (1) Pathology, (2) Clinical chemistry, (3) Microbiology,

(4) Immunology (5) Molecular Diagnostics
Principle of ELISA Test
Rapid Test
Method
ELISA Rapid Conventional PCR Real-Time PCR

Micro-plate Elisa
Intstrument ※ Thermal cycler Real-Time PCR
Reader

Target Antigen & Antibody Antigen & Antibody Viral RNA Viral RNA

Principle of Immunoassay + Immunoassay + RT-PCR +

Real-Time PCR
measurement Color reaction Color reaction Electrophoresis

Sensitivity 102 pg/mL 102 pg/mL 102 copy/rxn 101 copy/rxn

Test 96 T 1T 96 T 96 T

Time 3~4hrs 40 min 4 hrs 3 hrs

Contamination Risk ↓
Hands on times↑
Sensitivity ↑
Etc. Sensitivity ↓ Contamination Risk ↑
Specifity↑
Specifity↓
Real-time PCR : the best diagnostic tool?
 Real-Time PCR
Real-time PCR monitors the fluorescence emitted during the
reaction as an indicator of amplicon production at each PCR
cycle (in real time) as opposed to the endpoint detection
 What is Real-Time PCR has become a
cornerstone of molecular biology:
Real-Time
• Gene expression analysis
PCR used – Medical research
for? – Drug research
• Disease diagnosis
– Viral quantification
• Food testing
– Percent GMO food
• Transgenic research
– Gene copy number
 Prinsip kerja Real-time PCR

Key components of Real-Time qPCR

1. Fluorescence
Dyes
Real-
Time 2. Optical
Components
qPCR
3. Thermal
Cycler

Fast, Accurate and Quantitative Results

 Real-time PCR
DNA Extraction PCR PreMix ExicyclerTM 96

: The best choice of diagnostic methods

- High sensitivity : even one copy of virus can be detected
- Early detection : detected immediately after virus infected into
serum No blind period until antibodies are produced
- Rapid results / High-throughput assay : 96 samples in one run
(1-2 hr)
- High specificity : sequence-specific assay
Basics of PCR ?
Ideal graph

1 Cycle
End-Point PCR analysis is not quantitative

2 Cycle

Real graph

3 Cycle

N Cycle
 Keuntungan Real-time PCR

Real-time PCR vs. Conventional PCR

Real-Time PCR End-point PCR
Sensitivity High Low
Specificity High Low
-use specific probes -only size discrimination
Quantitative Yes No
results -Specific fluorescence -EtBr staining
Detection method Probe-specific Fluorescence Agarose gel
Electrophoresis
Detection range Wide range Short range (<2 log)

Reaction time 1-2 hr 3-5 hr

Post-PCR steps No Agarose gel electrophoresis

Cross- No Yes
contamination -Closed system -Open system
- Single step - Multiple steps
Bioneer Molecular Diagnostic
Produk Molecular Diagnostics Bioneer
• Development & commercialization of MDx instruments and kits for gene extra
Competitive ction, and amplification and diagnostics
Edge • Secured cost advantage through in-house production from raw materials
• Streamlined R&D to mass-production facility (17,734 m2)
Real Time Quantitative Thermal Block

Exicycler ™96
• Light Tunnel Technology (LTT) untuk eksitasi
dan iluminasi plate yang lebih homogen
• Modul pencitraan 2D-CCD yang ultra-sensitif
• 5 filter eksitasi dan emisi
• Dg mercury short arc lamp utk iluminasi yg
lebih baik dan merata
• Modul analisis :
Absolute Quantification,
Relative Quantification,
Existence/Nonexistence,
SNP genotyping
• Fitur diagnosis-diri untuk mencegah error
sebelum eksperimen dimulai
Exicycler ™96 Technolog
y
Exicycler™ 96 Company A’s system
Sistem Ekstraksi Asam Nukleat secara Otomatis

ExiPrep™16Dx
 2 Mode pengaturan: layar sentuh / komputer
 Dapat digunakan untuk mengekstraksi
DNA/RNA dari berbagai jenis sampel klinis:
darah, serum/plasma, cairan tubuh, sputum,
swab, dll
 Kapasitas 1-16 sampel
 Protokol ekstraksi telah dioptimasi dan
tersimpan dalam instrumen
 Dilengkapi dengan Lampu UV, blok pemanas
& blok pendingin
 Waktu pengerjaan 1h15m – 1h40m
Exiprep™ 16Dx Technology
Bioneer Molecular Diagnostic
AccuPower ® Diagnostics Kits
AccuPower ® Genotyping Kits
Bioneer Viral load Quantification
Kit contents Experimental flow charts
Bioneer Chronic Myelogenous Leukemia Detection
Results Interpretation
AccuPower HBV Quantitative

Bioneer Existation HBV LoQ = 15 IU/ml

AccuPower HCV Quantitative

Bioneer Existation HCV LoQ = 15 IU/ml

AccuPower HIV Quantitative

Bioneer Existation HIV LoQ = 50 IU/ml

AccuPower MTB&NTM Real-Time PCR Kit

Benefit:

1. Mampu membedakan Mycobacterium tuberculosis dan non-tuberculosis

2. Sample uji variatif (Sputum, BAL, Urine)
3. Mendeteksi 57 species Mycobacterium non-tuberculosis sehingga meminimalkan pelua
ng false negatif
AccuPower TB&MDR Real-Time PCR Kit
TB/MDR Bioneer vx GeneXpert:

1. Mampu membedakan Mycobacterium tuberculosis, Rifampicin dan Isoniazid resisten

2. Sample uji variatif (Sputum, BAL, Urine)
• Test Multiplex  satu tabung PCR utk mendeteksi bbrp patogen
• Reagen MasterMix
• Hasil PCR dalam 3.5 Jam
• Telah dioptimasi untuk beberapa instrument Real-Time PCR
 Bio-Rad, CFX96TM Real-Time PCR System
 Agilent, Stratagene Mx3005P QPCR System
 Thermo Fisher, ABI Prism® 7500 SDS/7500 Fast
 QIAGEN, Rotor-GeneTM Q5/6Plex
 Bioneer, Exicycler TM 96
Tropical Viruses & Parasites
Kit
abTESTM Den5 qPCR Kit
abTESTM Den4 qPCR Kit
abTESTM CHIKU qPCR Kit
abTESTM Malaria qPCR Kit
abTESTM Malaria 5 qPCR Kit
abTESTM Leptospira qPCR Kit
abTESTM Leptospira / B.pseudomallei Leptospira sp. / B. pseudomallei / IC
qPCR Kit
Deteksi
DEN1/DEN2/DEN3/DEN4/Chiku
DEN1/DEN2/DEN3/DEN4/IC
Chiku/IC
Plasmodium sp./IC
P. Falciparum / P. vivax / P. ovale /
P. Malariae / P. knowlesi
Leptospira sp. / IC
• Amoy Dx  Pelopor diagnostik molekuler di China,
terutama di bidang personalized medicine
• Berkolaborasi dengan perusahaan farmasi global,
spt Pfizer, AstraZeneca, Merck
• Sensitivitas tinggi, mendeteksi 1% alel mutan
• Open system, dapat digunakan pada berberapa tipe
instrument PCR
– ABI StepOne/StepOnePlus/7300/7500/7900
– Stratagene Mx3000P/3005P
– Roche LightCycler 480
– Bio-Rad CFX96
– Rotor-Gene 6000/Q
– Bioneer Exicycler TM 96
 Kit Detection
EGFR 29 EGFR mutations
EGFR ctDNA 42 EGFR mutations
EML4-ALK 21 eml4-alk fusions
ROS1 14 ros1 gene fusions
KRAS 19 KRAS mutations, exon 2,3&4
NRAS 16 NRAS mutations, exon 2,3&4
BRAF 6 BRAF mutations, codon 600
KRAS/NRAS 19 KRAS mutations, 13 NRAS mutations
KRAS/NRAS/BRAF 17 KRAS mutations, 13 NRAS mutations, 6 BRAF mutations
HPV Genotyping 19 HighRisk & 1 LowRisk HPV
FFPE DNA DNA from FFPE tissue sample
FFPE RNA RNA from FFPE tissue sample
Circulating DNA DNA from pleural effusion, serum & plasma
Cell DNA DNA from urogenital tract secretion and cultured cell
Laboratorium Biomolekuler PCR
Layout ruang kerja molekuler PCR
Layout ruang kerja molekuler PCR

RUANG A (Ruang Preparasi Sample)

• Meja preparasi sample

• Lemari reagent ekstraksi
• Sink
RUANG B RUANG A
RUANG B (Ruang PCR dan analisa data)

Trensfer Box • Biosafety cabinet/Laminar Air Flow

• Refrigerator reagent PCR
• Meja kerja PCR
• Sink

93
Investor Relations 2010 93
TERIMA KASIH