PCR

Polymerase Chain Reaction

Metode enzimatis untuk melipatgandakan

secara eksponensial suatu sekuen nukleotida
tertentu
It was invented in 1983
by Dr. Kary Mullis, for
which he received the
Nobel Prize in Chemistry
in 1993.

PCR
Metode yg mengamplifikasi atau
mengkopi sejumlah kecil rangkaian
DNA atau satu molekul DNA.
Memisahkan gen-gen berkopi tunggal
dri sekelompok sekuen genom.

Why Polymerase?
It is called polymerase because the only enzyme
used in this reaction is DNA polymerase.

Why Chain?

It is called chain because the products of the

first reaction become substrates of the following
one, and so on.

The Reaction

PCR tube

THERMOCYCLER

PCR Machine / Thermocycler

 Components of PCR
Template DNA
dNTPs (dATP, dTTP, dCTP & dGTP)
primers
Taq DNA polymerase
MgCl2
PCR buffer

DNA template
dNTP(deoxynucleotide
triphosphate)

MgCl2

KCl

The DNA which will be amplified by the PCR

provide both the energy and nucleosides for the
synthesis of DNA.
It is important to add equal amounts of each
nucleotide (dATP, dTTP, dCTP, dGTP) to the
mixture to prevent mismatches of bases.
The role of Mg++ in PCR forms complexes with
dNTPs that are the actual substrates for Taq
Polymerase.
When Mg++ is too low, primers fail to anneal to
the target DNA.
When Mg++ is too high, the base pairing becomes
too strong and the amplicon fails to denature
completely when you heat to 94C.
promotes specific annealing of primers to the PCR
template. K+ binds to phosphate groups on doublestranded DNA, stabilizing primer annealing.

Primers
Short pieces of DNA (20-30 bases) that bind to the DNA
template allowing Taq DNA polymerase enzyme to initiate
incorporation of the deoxynucleotides.
Both specific and universal primers can be used.

Taq DNA polymerase

A heat stable enzyme that adds the deoxynucleotides to the
DNA template.

Buffer
keeps the mixture at the proper pH so the PCR reaction will
take place.

 Three major phases in PCR:

Denaturing (94C)
Annealing (55C)
Extension (72C)
The total time to perform a standard
PCR is approximately 4 hours.

 Three major phases in PCR:

Denaturing (94C)
Annealing (55C)
Extension (72C)
The total time to perform a standard
PCR is approximately 4 hours.

PCR cycling program

Denaturation

At temperatures above 90C, double-stranded DNA denatures or "melts".

That means the weak hydrogen bonds that usually hold the two
complementary strands together at normal temperatures are disrupted
resulting in two single stranded DNA strands

DNA melting
The three parts of the polymerase chain reaction are carried out in
the same vial, but at different temperatures.
The first part of the process separates the two DNA chains in the
double helix.
This is done simply by heating the vial to 95 oC for 30 seconds.

double-stranded DNA

single-stranded DNA

Annealing
After separating the DNA strands, the temperature is lowered so the
primers can attach themselves to the single DNA strands. The
temperature of this stage depends on the primers and is usually 5C
below their melting temperature (45-60C).

Primer annealing
The primers cannot bind to the DNA strands at such a
high temperature, so the vial is cooled to 55 oC. At
this temperature, the primers bind or "anneal" to the
appropriate location in the DNA strands.
This takes about 20 seconds.

single-stranded DNA + primer

annealed DNA

The final step of the reaction is to make a complete copy of the

templates.

Primers
Primers are short, artificial DNA strands often not more than 50 and
usually only 18 to 25 base pairs long that are complementary to the
beginning or the end of the DNA fragment to be amplified
Examples of bacteria universal primer sequences are:
Forward 5' GAT CCT GGC TCA GGA TGA AC 3' (20 mer)
Reverse 5' GGA CTA CCA GGG TAT CTA ATC 3' (21 mer)
Estimation of the melting and annealing temperatures of primer:
If the primer is shorter than 25 nucleotides, the approx. melting
temperature (Tm)
Tm=4(G+C) + 2 (A+T)
GAGGTAACCACACCAGA 4Gs, 5Cs, 7As, 1T
Tm = {4(4+5)}+{2(7+1)}
= (49)+(28)
= 36+16
= 52
Tm = 52

Annealing temperature should be approx. 5 lower than the Tm

Extension
Finally, the DNA polymerase has to copy the DNA strands.
It starts at the annealed primer and works its way along the DNA
strand. The extension temperature depends on the DNA
polymerase.
Taq polymerase extends optimally at a temperature of 72C. The
time for this step depends both on the DNA polymerase itself and
on the length of the DNA fragment to be amplified

Primer extension
The Taq polymerase begins adding nucleotides to the primer and
eventually makes a complementary copy of the section of the
template
that lies between the primers. This completes one PCR cycle.

primer-annealed DNA
with Taq polymerase and
dATP, dTTP, dGTP, dCTP

primer-extended DNA

(1) 90C, open the DNA doublestranded helix.

(2) 50C, hybridization with
primer.
(3)75C, DNA synthesize.

Repeating for 20~30 times.

The amount doubles in every cycle.

PCR Animation
Please click here.

Process
Denature

Anneal Primer

Replicate
DNA
1st cycle

2nd cycle

3rd cycle

Extraction of DNA

Add protease
Add lysis buffer

Vortex each tube for 15 sec. to ensure proper mixing.

Incubate each tube for 10 min. at 56C.

Centrifuge each to remove any

mixture that may be on the lid.

Add 210l of ethanol,vortex

and then centrifuge again.

Collection DNA

The plate is then placed in the automated reader,where each

well is read spectrophotometrically.

Preparation the master mix

Master Mix
10x Buffer - 10 l
MgCl2 - 6 l
dNTP mix - 0.8 l of each
nucleotide
F5F primer - 2 l
F5R primer - 2 l
Taq polymerase - 0.5 l
Sterile H2O - 73.7 l

Place 5l of sample and 95l of master mix in vials and place

these vials in a PCR panel, whichwill then be placed in the
thermocycler for theDNA amplification cycles.

Designing PCR programs

APLIKASI PCR
- Deteksi dini penyakit infeksi dan herediter
- Test Paternity & Maternity
- Uji GMO (genetically modified organism)
- Uji keseragaman/kemurnian genetis
- Test forensik
- Peningkatan kualitas & kuantitas produk biologis
- Pembuatan produk rekombinan (vaksin, hormon,
dll.)