POLYMERASE CHAIN REACTION

(PCR)

TINA DEWI ROSAHDI

POLYMERASE CHAIN REACTION
(PCR) ADALAH TEKNIK UNTUK
MEMPERBANYAK
(MENGAMPLIFIKASI) DNA
SECARA CEPAT DI LUAR SEL.
WHAT DOES PCR DO?
PCR membuat ribuan salinan DNA
Menggunakan mesin PCR
Sebuah fotokopi DNA
 PEMBELAHAN SEL

DNA polymerase memperbanyak DNA selama pembelahan sel

 SIAPA YANG MENEMUKAN PCR?

EUREKA!!! I stopped the car. Somehow, I

thought, it had to be an illusion.
Otherwise it would change DNA chemistry
forever. Otherwise it would make me famous. It
was too easy. Someone else would have done it
and surely I would have heard of it.
We would be doing it all the time.

Kary Mullis - inventor of PCR, Nobel Prize 1993

PCR Thermocycler
TIGA TAHAPAN DALAM SIKLUS PCR

Denaturasi DNA pada 95C.

Primer annealing at 50-60C.
Polimerisasi DNA oleh DNA Polimerase
termostabil pada 72C.
DNA POLYMERASE IN ACTION!

Tanda bintang menunjukkan DNA polymerase terikat pada DNA

 BAGAIMANA REAKSI PADA
PCR?

Use 5l from tube labelled Use 20l from tube labelled The PCR bead
Template DNA primers

Template DNA Primers are two short

The starting material in a
pieces of DNA (0-15 bases
PCR reaction.
long) that determine the
region of DNA to be
copied.
Nucleotides
As, Ts, Gs and Cs to
make up the new DNA
strands
Taq DNA polymerase
The enzyme that makes
new DNA strands

MgCl2
Required for Taq DNA
polymerase to function
 BAGAIMANA PCR BEKERJA?

Mix the following:

Template DNA
Nucleotides
Primers
Taq DNA polymerase

MgCl2 ++ ++ ++
 JUMLAH DNA YANG DIPRODUKSI

Successive cycles double

amount of DNA
 TAQ DNA POLYMERASE

Bakteri Thermus aquaticus

yang hidup di suhu tinggi

DNA polymerase sangat

penting untuk PCR
 DIMULAI DENGAN MOLEKUL
TUNGGAL DNA, 25 SIKLUS PCR
AKAN MENGHASILKAN
SEKITAR 10 JUTA MOLEKUL
DNA YANG IDENTIK!!
 APLIKASI PCR

Forensic medicine.
Preimplantation Genetic Diagnosis (PGD).
Arkeologi.
Paternity testing.
FORENSICS!
 PENGGUNAAN PCR UNTUK
FORENSIK
PCR bisa digunakan untuk mengamplifikasi DNA dari
sejumlah kecil sel (sekitar 1000 sel).
DNA yang telah diamplifikasi bisa digunakan untuk
menganalisis DNA fingerprinting untuk menentukan
tersangka pelaku kejahatan.
DNA FINGERPRINTING MENGGUNAKAN
PCR DALAM INVESTIGASI FORENSIK

DNA diisolasi dari darah tersangka dan diamplifikasi melalui

PCR.
DNA yang telah diamplifikasi dipotong dengan enzim
restriksi dan diidentifikasi melalui gel agarosa.
Analisis Southern Bloth digunakan untuk mengidentifikasi
lebih lanjut DNA fingerprint.
PATERNITY TESTING
GENETIC TESTING
THE
EXPERIMENT
QUICK PCR SET UP
Carefully transfer PCR bead to 0.2ml tube & label
Add 5ul template DNA
Add 20ul primer mix
Place in PCR machine
 QUICK PCR CYCLES

Initial denaturation
94C for 180 seconds
Then 20 cycles of:
94C for 30 seconds
71C for 15 seconds (annealing)
71C for 15 seconds (extension)
Annealing & extension are same temperature
EDVOCYCLER
PCR machine
Easy to use
Select cat no
Programmable
CHOOSE PROGRAMME
PRESS TO SELECT
LID WILL HEAT UP
THEN CYCLES START
THEN CYCLES START

Programme selected
= Cat no of kit
THEN CYCLES START

Number of cycles
to go
THEN CYCLES START

Number of cycles
completed
THEN CYCLES START

Temperature for each step

THEN CYCLES START

Time in seconds for each step

 Temperature
PCR 100
Melting
94oC

50

0
Time

3 5
5 3
Temperature
100
Melting
94oC

50

0
Time
3 5

Heat

5 3
Temperature
100
Melting Melting
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC

0
Time
3 5
5

5
5 3
Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC

0
Time
3 5

Heat
5

Heat
5
5 3
Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC

0
Time
3 5
5

5
5

5
5

5
5 3
 Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC

0
3 5 5 Time
5
5
5 3

Heat
5

Heat
5
 Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC

0
3 5 5 Time
5
5 5
5 3

5
5

5
5

5
5
 Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC

0
3 5 5 Time
5
5 5
5 3

5
Fragmentsof 5

definedlength
5
5

5
5
DNA
ELECTROPHORESIS
GEL CASTING
RUNNING BUFFER
TAE buffer
20ml 50x buffer to 1 litre with water

Distilled water ideal but tap ok

Can be reused a few times
Store unused for future use ok
AGAROSE
0.8% Agarose
3g in 375 ml dilute buffer
Melt in microwave/autoclave
Pour when hand hot
AGAROSE
Store gels for 1-2 weeks in fridge wrapped in cling film or
plastic bag
Keep any left over gels and remelt next time
REMOVE COMB & ENDS
FIXED VOLUME
MINIPIPETTE
ADJUSTABLE
MICROPIPETTE
DRY LOADING

You can load gel dry instead of through buffer!

Otherwise load wet through buffer

Either way, remember

Do not puncture bottom of well
Change tips between each sample
HEXAGEL AND EVT
300 POWER SUPPLY
QUICK PCR KIT GEL
LOADING
After PCR reaction add 5l loading dye
Load 40 l of each sample into the wells

A B C D E F

M LG1 LG2
RUN GELS
Put on lid and attach to power supply
Run for 30 minutes at 150 volts
Or 75 volts for 40-50 minutes
Check for bubbles at electrode
Run until tracking dye halfway across gel
AFTER GEL RUN
STAIN THE GEL
STAIN PCR GEL
MetBlue card blue side down 5 mins
Weigh with gel tray
Destain in warm water 10-15 mins
Leave overnight in fridge for best result
Keep long term in bag in fridge
STAIN GEL
GEL STORAGE
Can store gels in fridge before staining over weeknight or
weekend

Cannot store dye kits overnight before viewing as diffuse!

QUICK PCR RESULT