DASAR-DASAR

PCR
K JANUARTHA P PINATIH
DEPT. MIKROBIOLOGI KLINIK
FK UNUD
DEVELOPMENT OF MOLECULAR BIOLOGY
TECHNIQUES

DNA
ISOLATION

PURE DNA

IN-VITRO
MANIPULATION

MOLECULAR BIOLOGY
TECHNIQUES
STRUKTUR DNA
MECHANISM OF DNA REPLICATION
POLIMERASE CHAIN REACTION
(PCR)

Ø KaryMullis (1984)
Ø PENGGANDAAN FRAGMEN
SPESIFIK DNA SECARA INVITROà
JUTAAN KOPI FRAGMEN DNA
SECARA CEPAT DAN AKURAT
REPLIKASI

PCR
 REPLIKASI

PCR
 REPLIKASI

PCR
 Basic Components

Ø TEMPLATE ATAU TARGET (FRAGMEN GEN YANG DIINGINKAN)

• DNA ATAU RNA

Ø dNTPs

Ø PRIMER
l OLIGONUKLETIDA PENDEK (15-30 BP)
l TERIKAT DENGAN SEKUENS SPESIFIK PADA TEMPLATE

Ø ENZIM
l HEAT-STABILE DNA POLIMERASE –Taq DNA POLIMERASE
l REVERSE TRANSCRIPTASE (UNTUK RT-PCR / PCR DENGAN RNA
SEBAGAI TARGET)
 Step 1 : Denaturation
(separation of double-stranded DNA) by Heat Step 2 : Primer Anneals (50 - 65° C)
(95° C)

step 3 End of the 1st PCR Cycle

Primer Extension by Taq Polymerase (72° C) Results in 2 Copies of Target Sequence
 A. Double
50º strand DNA

96º B. Denature

50º C. Anneal
primers

Taq
72º D. Polymerase
binds
Taq
 Taq Taq
72º E. Copy
strands
Taq Taq

96º
2 F.
Denature
3

4
 1

G. Anneal
50º primers

4
 Taq 1

72º

Taq 2

H.
Polymerase
Taq 3 binds

Taq
4
 1
Taq

72º

Taq 2

I. Copy
strands

3
Taq

Taq
4
1

J.
Denature at 96º
Anneal primers
at 50º

4
 1

72º
K. Bind polymerase
(not shown) and
copy strands

4
 1

L.
Denature at 96º
Anneal primers
at 50º

2
3

4
 1

M.
Copy strands at
72º 72º

2
3

4
1

2
3

4
 1

After 5 rounds
there are 32
double strands of
which 24 (75%)
are are same
size
2
3

4
1

2
3

4
CYCLE NUMBER AND AMPLICONS
REPLIKASI

PCR
 1 11 21

5’-ATGAGCGACCTTGCGAGAGAAATTACACC-3’

3’-TACTCGCTGGAACGCTCTCTTTAATGTGG-5’
Elektroforesis Produk PCR
 PCR-RFLP
(Polymerase Chain Reaction – Restriction Fragment
Length Polymorphisms)

PCR à pemotongan dengan enzim

restriksi (restriction endonucleases)
RESTRICTION ENDONUCLEASES
Restriction Endonuclease action
(EcoRI)
 PCR-RFLP Untuk Deteksi Thalassemia

Ø Exon 1 (ukuran 293 bp)

l Normal :
202CTGTGGGGC

l Mutan (Thalassemia) : 202CTGTAGGGC

Ø CTGTAG : recognition site untuk SfcI

…..CTGTAG……. à ….C
TGTAG….
 SfcI restriction site :
…..CTGTAG……. à ….C TGTAG….

Ø Normal :
l Exon 1 : 202CTGTGGGGC

293 bp

+ SfcI

293 bp
 SfcI restriction site :
…..CTGTAG……. à ….C TGTAG….

Ø Mutan :
l Exon 1 : 202CTGTAGGGC

293 bp
+SfcI

202 bp 91 bp