PCR

(Polymerase Chain reaction)

Mesin PCR
 Peralatan umum

Mikropipet dan mikrotip

Microcentrifuge/
Refrigerred microcentrifuge
 Peralatan Umum

• Laminar air flow

• Blower
 PCR
◼ Polymerase Chain Reaction
◼ Allows amplification from nucleic acid
templates
◼ enzymatic reaction
– relies on fidelity of enzyme to produce a
complementary sequence to the template
strand in the presence of primers and
deoxyribonucleotides
 Reaction
Once a round is completed, the
new DNA products are
subjected to denaturation and
Double stranded re-enter the amplification
template is Denature procedure.
dissociated by Re-enter cycle
heating to 95oC

Anneal Primers
On the final cycle, finish
with an extension step.

Extend

Temperature is decreased to
Extension from primer sites at optimal temperature of 68-74oC.
optimal hybridisation
Extension time varies according to product size: enzyme activity
temperature for duplex
averages 1Kb per min. 68 oC is preferred for longer templates to
formation in the presence of
conserve enzyme activity over multiple cycles.
extension primers.
 PCR
Komponen PCR

• DNA templat
• Sepasang oligonukleotida primer
• Campuran dNTP (dATP, dGTP, dTTP dan dCTP)
• Enzim DNA polimerase termostabil
• Buffer
• Program

DNA templat
primer
 KOMPONEN PCR
Templat DNA → keutuhan &  DNA
Primer :
➢ Desain primer optimal :
- Panjang primer  20 nukleotida
- % GC = 50 – 60 %
- Tm = 60 – 70 oC
- Minimalkan self complementary
terutama pada ujung 3’ primer
➢ Kons. & Komposisi primer setimbang (@ 0,1-0,4M)
dNTP :
➢ Kons. & Komposisi dNTP optimal (@ 0,1-0,4mM)
......komp. PCR

Enzim DNA polimerase:

➢ 3’ → 5’ eksonuklease
➢ Taq polimerase
➢ jumlah enzim optimal
Ion Mg2+ → Sensitifitas & spesifitas
 Programs
◼ Initial denaturation 95oC (3-5min)
◼ Denaturation (15-30s)
– template
◼ Anneal (20-30s)
– Duplex melting temp
◼ Extend (from 30s)
– Length of template (15 bases/s)
– Temp
◼ Final extension and hold A final extension at 72oC will ensure that all
the molecules in the final product mixture are
full length. The hold is normally at 4oC.
Products are then stored in a refrigerator or
freezer before analysis.
 Prosedur PCR :
Kondisi PCR :
• Denaturasi awal : 94oC, 1 menit
• Denaturasi : 94oC, 1 menit
• Annealing : 50oC, 1 menit
• Polimerasi : 72oC, 1 menit
• Polimerasi akhir : 72oC, 4 menit
• Siklus : 30
• Enzim : 1,25 unit Taq Polimerase
• dNTP : 0,8 mM dNTP total
• MgCl2 : 1,5 mM
• Primer : @. 0,4 M
 Siklus PCR :
◼ Denaturasi DNA template: 94-96oC
◼ Annealing → Tannealing = (Tm – 5)oC
◼ Extension →suhu ~ aktivitas optimal
enzim DNA polimerase
Waktu ~panjang fragmen

Tm = 4 (G + C) + 2 (A + T) C
 Siklus PCR

Cycle 1 Cycle 2 Cycle 3

94 oC 94 oC 94 oC
T
72 oC 72 oC 72 oC

55 oC 55 oC 55 oC

Time (seconds)
Perbanyakan DNA : secara
eksponensial

16

220
 -
P
C Polimerisasi DNA
Template
BASA’ BASA’
BASA BASA
dNTP
3’
Primer 3’  C5’ OH
5’ C P 
P -

M2+ C M2+ 
P
A B -
D882
-
C
DNA polimerase C
D705
 Mekanisme PCR ( animasi )
Mg2+
5‘ 3‘
5‘ 3‘

5‘
DNA
3‘ 3‘ 5‘

94oC (Denaturasi)
5‘ 3‘

3‘ 5‘
5‘ 3‘
Mg2+

Mg2+ Mg2+

5‘ 3‘

3‘ 5‘

3‘ 5‘
3‘
5‘

65oC (Annealing) 3‘ 5‘

Mg2+ Mg2+

3‘
5‘
5‘ 3‘

3‘ 5‘
3‘ 5‘
Primer 5‘
3‘
Primer
5‘
Prod. 3‘ 5‘

5‘ 3‘
3‘ = (2n-2n)x 5‘

3‘ 5‘
3‘
5‘

72oC (extension) 3‘ 5‘
 Optimasi Suhu Annealing
5‘ 3‘

3‘ 5‘
Primer Primer 3‘ 5‘
5‘
Primer
Primer Tidak ada Produk
5‘

3‘ 5‘

Annealing >> Tm
3
5‘

5‘ 3‘ 3‘ 5

3‘
Annealing 3‘ 5‘ 5‘
Primer
Tm-5OC Primer 3‘ 5‘
5‘
3‘
5‘
3‘ 5‘
3‘ 5‘
3‘
5‘

Annealing << Tm 3‘ 5‘

5‘ 3‘

3‘ 5‘
5‘ 3‘
3‘
5‘
3‘ 5‘ 3‘ 5‘
3‘ 5‘ Produk
Primer Primer 5‘ 3‘ non spesifik
Primer Primer 3‘ 5‘
5‘ 5‘ 3‘
5‘

3‘ 5‘ 5‘
3‘
 Optimasi Kondisi Reaksi
Bahan Jumlah (µl) Step Temperatur Duration (Menutes)
KAPA”SYBR 10 e (0C)
FAST
Incubation 50 02:00
PCR water 9
Polymerase 95 10:00
Primer 0,4
(forward) Activation
Primer 0,4 PCR Cycling 95 00:10
(reverse) PCR Cycling 60 00:30
DNA template 0,5 Melt Curve 95 00:15
Total volume 20
Melt Curve 55 00:15
Melt Curve 95 00:15

Total Program
Length: 1 Hour 59
Seconds