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Polymerase Chain Reaction (PCR)

PCR is used in molecular biology to make many copies of (amplify) small sections of DNA or a gene

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Rista Anggriani
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66 views18 pages

Polymerase Chain Reaction (PCR)

PCR is used in molecular biology to make many copies of (amplify) small sections of DNA or a gene

Uploaded by

Rista Anggriani
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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POLYMERASE CHAIN REACTION

(PCR)

Copyright © 2009 Pearson Education, Inc.


Still remember about DNA REPLICATION?

Copyright © 2009 Pearson Education, Inc.


What is PCR?

– Merupakan teknik untuk membuat copian (amplifikasi) dari


sebuah potongan DNA spesifik dalam waktu yang singkat

– Dirintis pada tahun 1980an oleh Kary Mullis

Copyright © 2009 Pearson Education, Inc.


PCR vs DNA Replication

DNA Replication PCR


In vivo (di dalam sel), terjadi secara In vitro (di luar sel), menggunakan
alami bantuan enzim
Butuh makhluk hidup Butuh mesin PCR (thermocycler)

PCR = replikasi DNA


akan tetapi hanya dapat menyalin fragmen pendek DNA,
biasanya sampai dengan 10 kb

Copyright © 2009 Pearson Education, Inc.


Still remember about DNA REPLICATION?

Copyright © 2009 Pearson Education, Inc.


What are components in PCR?

DNA cetakan

Primer (forward & reverse) : panjang 20-30 bp

Nucleotida (dATP, dCTP, dGTP, dTTP)

Taq DNA polimerase (diambil dari bakteri Thermus aquaticus)

Buffer

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How PCR works?

• Dalam proses PCR dikenal dengan adanya


rangkaian reaksi : PCR cycle
• Dalam 1 cycle (siklus) ada 3 tahapan:
- Denaturation : 92 – 95oC, 1-5 menit
- Annealing (hybridization) : 45-60oC, 30 detik
- Extension (elongation) : 72oC, 1-5 menit
• Setiap 1 cycle, jumlah DNA akan mengalami
penggandaan
• Siklus akan diulang setiap sekitar10 menit
• 1 running PCR terdiri dari hampir 35 siklus

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PCR MACHINE

PCR Machine or Thermocycler


A) The thermocycler or PCR machine can be programmed to change temperature rapidly. The heat
block typically changes from a high temperature such as 90°C (for denaturation) to 50°C (for primer
annealing), then back to 70°C (for DNA elongation) in a matter of seconds. This may be repeated for
many cycles.
B) Rows of GeneAmp PCR machines copying human DNA at the Joint Genome Institute, in Walnut
Creek, California, which is a collaboration between three of the U.S. Department of Energy’s National
Laboratories. (Credit: David Parker, Science Photo Library.)

Copyright © 2009 Pearson Education, Inc.


1st CYCLE of PCR

Denaturation of DNA

In the steps of PCR, a very small


amount of template DNA is heated to
90ºC, which separates the two strands
of the double helix.

Copyright © 2009 Pearson Education, Inc.


1st CYCLE of PCR

ANNEALING of PRIMER

When the temperature is lowered to


50–60ºC, the primers can anneal to
the ends of the target sequence.

Copyright © 2009 Pearson Education, Inc.


1st CYCLE of PCR

ELONGATION
Once the primers have
annealed to the template, the
temperature is increased to
70°C.
This is the optimum
temperature for the
thermostable Taq polymerase
to elongate DNA.

The polymerase synthesizes new


strands of DNA using the 39 end
of the primer as a starting point.
A pool of nucleotide precursors is
also necessary for this step of the
reaction.
Copyright © 2009 Pearson Education, Inc.
2nd CYCLE

The entire cycle is repeated starting with


the two DNA pieces produced in the first
cycle.

The two double-stranded pieces of DNA


are denatured into four single-stranded
pieces at 90°C.
The temperature is dropped to 50°C in
order for the primers to anneal.
Finally, the temperature is heated to
70°C, the optimal temperature for
Thermostable polymerase to copy the
templates.

Notice how one DNA molecule has given


rise to four molecules

Copyright © 2009 Pearson Education, Inc.


3rd CYCLE

The products from the second cycle go


through the same process as before.
The four double-stranded pieces are
denatured into eight single-stranded
pieces.
The primers anneal and DNA
polymerase makes the complementary
strands.

After this cycle, the number of


sequences containing only the target
DNA grows exponentially, far
exceeding any other product shown in
this figure.

Copyright © 2009 Pearson Education, Inc.


HOW ABOUT cycle 4th, 5th, 6th,…. etc??

Copyright © 2009 Pearson Education, Inc.


PCR APPLICATION

• Isolasi Gen
• DNA Sequencing
• Forensik
• Diagnosa Penyakit

Copyright © 2009 Pearson Education, Inc.


PCR STRENGTHNESS & WEAKNESS

Kelebihan Kekurangan
Memiliki spesifitas tinggi Sangat mudah terkontaminasi
Sangat cepat prosesnya Biaya dan peralatan mahal
m.o yang didteksi tidak harus hidup Teknik yg kompleks & bertahap shg
butuh keahlian khusus
Mudah di set up

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HOW TO CHECK PCR RESULT?

Applied in ELECTROPHORESIS!!

Copyright © 2009 Pearson Education, Inc.


THANK YOU

Copyright © 2009 Pearson Education, Inc.

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