Pengenalan proses

Polymerase Chain
Reaction (PCR)
 Definisi
Polymerase Chain Reaction (PCR)
adalah proses memperbanyak
(copy/amplifikasi) rangkaian spesifik
doble helix DNA secara identik (urutan
dan panjang yang sama) dengan
metoda enzimatik dan prosesnya
berulang.
 Prinsip kerja reaksi PCR
1. Denaturasi : proses pembukaan unti double helix DNA
dengan pemanasan
2. Annealing : proses penempelan primer ke untai DNA
3. Elongasi : proses sintesis untai DNA baru

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 Denaturasi step
94-98C for 20-30 seconds.
Denaturasi DNA template atau pembukaan
untai double helix DNA template oleh panas
adalah dengan merusak ikatan hidrogen yang
menghubungkan kedua untai DNA
 Annealing step
50-65C for 20-40 seconds
Penempelan untaian primer ke untaian DNA
template terjadi dengan pembentukan ikatan
hidrogen yang stabil antara kedua untai tersebut.
Elongation / Extension step
Final elongation
70-74C for 5-15 minutes
Untuk memastikan semua untai DNA
single-stranded telah penuh terisi.

Final hold
4-15C for an indefinite time
Untuk menyimpan hasil reaksi sementara
 i
r as
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De
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DN
DNA Elongasi
g
in
al
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An
 PCR amplification rate

One DNA molecule can be

amplified into 68 billion copies
fragment DNA

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Deteksi, Kuantifikasi dan Visualisasi
DNA hasil PCR
Spektrophometri
-nanodrop-

elektrophoresis
Components PCR
Peralatan

Cooling block
Instruments

Centrifuge

Ice maker
 DNA Template
NA template diperoleh dari hasil isolasi DNA dari berbagai sumber
DNA Isolation
Protocols from
cells
 Primers - oligonucleotida
Apa itu Primer ?

Primer adalah untaian pendek sintetik nukleotida yang urutannya merupakan

komplemen dari daerah spesifik DNA target.

3 5
TGACCTGAAAAGAC Primer

GATGGACTGATTACCGATGACTGGACTTTTCTG Template
5 3

3 5
TGACCTGAAAAGAC
:: ::: : : : : : : : :
GATGGACTGATTACCGATGACTGGACTTTTCTG Annealing
5 3
 Urutan nucleotida primer bisa
diperoleh dari literatur dan juga di
desain sendiri.
Primer terdiri dari satu pasang
- primer forward 5 3
- primer reverse 5 3
 dNTPs
De oxy nucleotide triphosphate yang terdiri
dari :
(dATP, dGTP, dTTP, dCTP)
Mereka adalah kompenen dasar pembentuk
DNA yang disusun/disintesis oleh DNA
polymerase membentuk untaian DNA baru.
Range - 0.5l (for 10l reaction mixture)
Struktur dNTPs
DNA Polymerase
 Taq DNA Polymerase

Thermus aqaticus
Range 0.2ul of (in 10l of reaction mix)
It assebles a new DNA strand from dNTPs
 Buffer solution
Contains Divalent cations like Mg+2
Provides suitable chemical environment for
optimum activity and stability of the DNA
polymerase
Range - 1l ( for a total reaction mixture of
10l)
Sterile deionized water
Its quantity is variable
PCR MIX Solutions
PCR Amplification Settings
ELECTROPHORESIS
 ELECTROPHORESIS
Principle of Separation
the movement of molecules by electric current in
a matrix
proteins and nucleic acids have naturally occurring
negative and positive charge
the sum of these charges determines the overall
charge
Two formats of gel
electrophoresis Molecules with a
negative charge
(anions) will be
attracted to the
positively charged
Horizontal vertical node (anode)
+
Molecules with a
positive charge
+
(cations) will be
attracted to the
negatively charged
 ELECTROPHORESIS UNITS
Horizontal gel electrophoresis
units

Vertical gel electrophoresis units

 GEL SYSTEMS / MATRICES
Functions:
provide resistance to the movement of molecules
under the force of the electric current.
prevent diffusion and reduce convection currents
so that the separated molecules form a defined
group, or band.
serve as a support medium for analysis of the
separated components.
These matrices must be simple to prepare and amenable to
modification.

Agarose and
polyacrylamide
are polymers thatmake
they both meet these
porous gels criteria.
 AGAROSE GELS
Agarose is a highly purified uncharged
polysaccharide derived from agar (red seaweed)
Agarose dissolves when added to boiling liquid. It
remains in a liquid state until the temperature is
lowered to about 40C at which point it gels
The pore size may be predetermined by adjusting
the concentration of agarose in the gel
Agarose gels are fragile, however. They are
actually hydrocolloids, and they are held together
by the formation of weak hydrogen and
hydrophobic bonds
 Agar vs Agarose
Agarose is a linear polymer made up of the repeating unit of
agarobiose, which is a disaccharide made up of D-galactose and 3,6-
anhydro-L-galactopyranose.
Agarose is one of the two principal components of agar, and is
purified from agar by removing agar's other component,
agaropectin

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 Agarose
A linear polymer made up of the repeating unit of
agarobiose, which is a disaccharide made up of
D-galactose and 3,6-anhydro-L-galactose.
Agarose gel polymerization
 POLYACRYLAMIDE GEL
Polyacrylamide gels are tougher than
agarose gels
Acrylamide monomers polymerize
into long chains that are covalently
linked by a crosslinker
Polyacrylamide is chemically
complex, as is the production and
use of the gel
 CROSSLINKING ACRYLAMIDE CHAINS

Ammonium persulfate

and

Tetramethylethylenediamine
(CH3)2NCH2CH2N(CH3)2

catalyse of
acrylamide
polimerisation
Agarose vs acrylamide gel structure
 Agarose gel electrophoresis

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Apparatus
Gel preparation, with/out EtBr

UV detection
Running the electric
current

gel electropherogram

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 Staining and Visualization DNA on agarose gel

The gel with UV

illumination: DNA
stained with ethidium
bromide appears as
glowing orange bands.

Fluorescent dye molecules such as EtBr,

methylene blue, SYBR green intercalates into the
major grooves of the DNA and fluoresces under
UV light
Electrophoresis DNA Visualisation