PCR- Real Time

MUH. AZWAR AR (N012181022)

AZIZAH NUR AR (N012181023)

PROGRAM STUDI MAGISTER FARMASI

PASCASARJANA UNIVERSITAS HASANUDDIN
 DNA
Samples 3’ 5’
5’ 3’

d.NTPs
Primers
Thermal Stable
DNA Polymerase
DNA

Add to Reaction Tube

Denaturation

Annealing

The Polymerase Chain Reaction

 3’ 5’

5’ 3’

Extension

3’ Taq 5’

5’ 3’
Taq

Extension Continued

3’ Taq
5’

Taq 3’

Repeat

The Polymerase Chain Reaction

 3’ 5’
3’

3’ 5’
3’
Cycle 2
3’ 5’
3’ 4 Copies
3’
3’

3’ 5’
3’
3’ 5’
3’

3’ 5’

Cycle 3
3’

3’ 5’
3’

3’ 5’
3’
8 Copies
3’ 5’
3’

3’ 5’
3’

3’
3’

The Polymerase Chain Reaction

  PCR methods is use Gel Electrophoresis to
detect PCR amplification in the final phase
or at end point of the PCR reaction

 Real Time PCR allows to detect PCR

product (DNA) during the early phases of
the reaction

Different Real-Time PCR & PCR

Traditional
 Real-Time PCR 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 hr 3-5 hr

Post-PCR steps No Agarose gel electrophoresis

 Reverse Transcription adalah proses yang
mentranskripsikan untai
tunggal RNA menjadi DNA komplemennya
(cDNA) dengan katalisator enzim reverse
transcriptase, primer dNTPs dan enzim
RNAase Inhibitor

RT- PCR definition

  Enzim transcriptase balik yang dapat
digunakan antara lain mesophilic viral
reverse transcriptase (RTase) yang dikode
oleh virus avian myoblastosis (AMV)
maupun oleh virus moloney murine
leukemia (MMuLV) [mensintesis cDNA
sampai sepanjang 10 kb], dan Tth DNA
polymerase [mensintesis cDNA sampai
sepanjang 1 – 2 kb]

Enzyme Reverse Transcriptase

 Tiga jenis primer yang umum digunakan
dalam proses reverse transkripsi;

(1) Primer Oligo(dT) atau dNTPs,

Primer dNTPs menggandakan ekor poly A mRNA dan terfosforilasi pada ujung 5’ untuk
menfasilitasi cloning cDNA

(2) Primer random hexamer

Dengan random primer, ujung 5′ gen-gen yang panjang dapat ditranskripsi balik, tetapi
cDNA yang diperoleh mungkin tidak full dari seluruh gen

(3) Gen spesifik primer

primer spesifik gen yang dapat meningkatkan sensitivitas dengan mengarahkan
seluruh aktifitas enzim RT untuk mentranskripsi balik hanya RNA tertentu saja

Primer
RNase inhibitor should be applied in a working
concentration of 40units (1 μl) per 50 μl reaction
volume. The application of RNase inhibitor is
recommended for:

•cDNA synthesis / RT-PCR

•in vitro transcription
•in vitro translation
•isolation of mammalian cell fractions that contain
mRNA-protein complex
•Separation and identification of specific
ribonuclease activities
•Studies of tumor suppression

RNA-ase Inhibitor
Alur RT-PCR
 Penggunaan titik akhir PCR lebih disukai untuk
mengukur perubahan ekspresi gen dalam
jumlah kecil sampel [deteksi level ekspresi gen
dengan menggunakan pewarna fluoresens
seperti etidium bromida],

RT-PCR real-time telah menjadi metode untuk

memvalidasi hasil yang diperoleh dari analisis
array atau perubahan ekspresi gen. Selain itu
juga untuk mendapatkan hasil dari analisis
susunan dan ekspresi gen pada skala global

PCR (RealTime) : qPCR and RT-qPCR

Protokol RT-PCR
  Non Specific Fluorescent Dye
◦ Sybr Green
 Sequence-Specific DNA probes
◦ Hydrolysis
◦ Hybridization

Detection Chemistry
 1) Denaturation
F 1) Intercalating dye fluoresces
F F
F more brightly when bound to
dsDNA.

2) Annealing
F 2) DNA binding dyes are
F F F
F inexpensive compared to the
other probes.

3) Extension
3) SYBR Green I, EtBr
F F F

Detection Chemistry: SYBR Green dye

 1) Fluorescent reporter dye at the 5’
end is quenched by fluorescent
quencher dye at the 3’ end.

2) When amplification occurs the

TaqMan probe is degraded due
to the 5'-->3' exonuclease activity
of Taq DNA polymerase, thereby
separating the quencher from the
reporter during extension.

3) The TaqMan assay accumulates

a fluorescence signal.

Detection Chemistry: TaqMan Probe

(Hydrolysis)
 1) Denaturation 1) A molecular beacon begins as a
stem-and-loop structure. The
R Q sequences at the ends of the
probe match and bind, creating
the stem
2) Annealing
Taq R Q

2) When the probe binds to a single-

stranded DNA template, the
3) Extension structure unfolds, separating the
quencher from the dye and
R Q allowing fluorescence.

Detection Chemistry: Molecular

Beacon Probe (Hydrolysis)
Detection Chemistry: Scorpion Probe
(Hydrolysis)
 1) Denaturation
1) FRET method designed two
D R specifically probe. It labeled with
different dyes, such as at the 5’
end of donor probe and at the 3’
2) Annealing end of acceptor probe.
Energy
Taq transfer
D R

2) At close proximity, the donor dye

(D)is excited by the light source
3) Extension and the energy is transferred the
D R acceptor dye(R). Subsequently,
fluorescent light is emitted at a
different wavelength.

Detection Chemistry: Hybridization

Probe
 Reverse transcriptase-PCR (RT-PCR)
merupakan metode yang digunakan untuk
mengamplifikasi cDNA dari mRNA. RT-PCR
digunakan untuk mendapatkan kembali dan
menyalin utas 5’ dan 3’ dari mRNA,
menghasilkan kumpulan cDNA yang banyak
dari jumlah mRNA yang sangat sedikit

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