BIOTEKNOLOGI FARMASI

PCR DAN
ELEKTROFORESIS
Dr. Oeke Yunita, S.Si., M.Si., Apt.

THE TRIPLET CODE

POLYMERASE CHAIN REACTION

PCR

Polymerase Chain Reaction

DNA/gene amplification

teknik in vitro untuk melipatgandakan asam

nukleat (DNA atau RNA) secara eksponensial
dan cepat dengan bantuan enzim di dalam
suatu mesin / Thermocycler

KEUNGGULAN PCR
Sensitif dan cepat copy fragmen DNA
200.000 kali, 20 siklus selama 220 menit
Jumlah DNA yang akan dicopy sangat sedikit
(~ 5g)
Total volume reaksi untuk PCR, sangat kecil
25-100 l

KETERBATASAN PCR
Kualitas (kemurnian) DNA sangat berpengaruh
terhadap hasil PCR
memerlukan urutan (sequence) DNA utk
mendesain primer

PRIMER

A primer is a strand of nucleic acid that serves as a

starting point for DNA synthesis.
These primers are usually short, chemically
synthesized oligonucleotides, with a length of about
twenty bases. They are hybridized to a target DNA,
which is then copied by the polymerase.
minimum primer length used in most applications is
18 nucleotides.
Replication starts at the 3'-end of the primer, and
copies the opposite strand.

PRINSIP DASAR
a.

b.

c.

ikatan antar untaian DNA dengan

pasangan komplementernya adalah
sangat kuat dan spesifik
Pemisahan untaian menjadi rantai
tunggal dan masing-masing rantai dapat
dipakai sebagai cetakan (template) untuk
mensintesis rantai pasangannya.
Penempelan oligonukleotida penuntun
(primer) pada lokasi DNA yang akan
diamplifikasi

PCR CYCLE
Denaturation: The target DNA (template)
is separated into two stands by heating to
95
Primer annealing: The temperature is
reduced to around 55 to allow the
primers to anneal.
Polymerization (elongation, extension):
The temperature is increased to 72 for
optimal polymerization step which uses up
dNTPs and required Mg++.
deoxyribonucleotide triphosphates (dNTPs)

PCR CYCLES
Temperature

100

Melting

Melting

94 oC
30 s
50

72 oC
60 s

5
5

T i m e

5
3

3
5

94 oC

Extension
Annealing
Primers
45 oC
60 s

40x

5
3
5
5
5

5
3

3
5

ALAT DAN BAHAN

Hardware: mesin PCR / thermocycler, yg dapat

diprogram: suhu dan waktu dalam siklus amplifikasi
Bahan kimia : PCR-buffer, MgCl2, dNTP
(dATP,dCTP, dGTP, dTTP atau dUTP)
Oligonukleotida (primer)
Template (DNA target yang dilipatgandakan)
Air suling ultrapure yang telah disterilkan (nuclease
free water)
Enzym Taq (from: Thermo aquaticus) Polymerase
Ix usage: fragmen Klenow DNA polymerase I
(Escherichia coli) : tdk tahan panas, laju polimerasi
sedang, prosesivitas (kemampuan penggabungan
nukleotida-primer tanpa terdisosiasi) rendah

PCR TECHNOLOGY
A

B C
A
+

+
+

Taq polymerase
Genomic DNA

primers

Nucleotides

PCR
(under relaxed conditions)

Buffer

PCR-BASED DNA MARKERS

Generated by using Polymerase Chain Reaction

Preferred markers due to technical simplicity and cost
PCR Buffer +
MgCl2 +
dNTPS +

PCR

Taq +

Primers +
DNA template

THERMAL CYCLING

GEL ELECTROPHORESIS
Agarose or Acrylamide gels

GEL ELECTROPHORESIS

The basic principle is that DNA,

RNA, and proteins can all be
separated by means of an electric
field.
In agarose gel electrophoresis, DNA
and RNA can be separated on the
basis of size by running the DNA
through an agarose gel.
Proteins can be separated on the
basis of size by using an SDS-PAGE
gel, or on the basis of size and their
electric charge by using what is
known as a 2D gel electrophoresis.

An agarose gel is prepared by

combining agarose powder and a
buffer solution.
Buffer

Flask for boiling

Agarose

Electrophoresis Equipment
Power supply

Cover

Gel tank
Electrical leads

Casting tray
Gel combs

Gel casting tray & combs

Preparing the Casting Tray

Seal the edges of the casting tray and put in the combs. Place the casting
tray on a level surface. None of the gel combs should be touching the
surface of the casting tray.

Agarose

Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.

Melting the Agarose

Agarose is insoluble at room temperature (left).

The agarose solution is boiled until clear (right).

Gently swirl the solution periodically when heating to allow all the grains of agarose
to dissolve.
***Be careful when boiling - the agarose solution may become superheated and
may boil violently if it has been heated too long in a microwave oven.

Pouring the gel

Allow the agarose solution to cool slightly (~60C) and then carefully
pour the melted agarose solution into the casting tray. Avoid air
bubbles.

Each of the gel combs should be submerged in the melted agarose solution.

When cooled, the agarose polymerizes, forming a flexible gel. It should

appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.

Place the gel in the electrophoresis chamber.

DNA

buffer

wells

Cathode
(negative)

Anode
(positive)

Add enough electrophoresis buffer to cover the gel to a depth of

at least 1 mm. Make sure each well is filled with buffer.

Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample.
The sample should sink into the well. Be careful not to puncture the
gel with the pipette tip.

DNA pieces are separated by agarose gel electrophoresis

GEL ELECTROPHORESIS

A method of separating DNA

in a gelatin-like material
using an electrical field
DNA

is negatively charged
when its in an electrical field
it moves toward the positive
side
DNA

swimming through Jello

Factors affecting the rate of migration of

charged molecules:
charge
Size
shape

Nature
Pore size

intensity
Molecules

Electric
current

Supporting
media

Buffer
pH

ANALISIS DNA PADA

HASIL ELEKTROFORESIS
100,000

Size, base pairs

10,000

1,000

100
0

10

15

Distance, mm

20

25

30

USES: EVOLUTIONARY RELATIONSHIPS

Comparing DNA samples from different

organisms to measure evolutionary
relationships
turtle snake rat squirrel

DNA

fruitfly
5

USES: MEDICAL DIAGNOSTIC

Comparing normal allele to disease allele

chromosome
with normal
allele 1

chromosome with
disease-causing
allele 2

DNA

Example: test for Huntingtons disease

USES: FORENSICS

Comparing DNA sample from crime scene

with suspects & victim
suspects
S1 S2 S3

crime
scene
V sample

DNA

DNA FINGERPRINTS

Comparing blood
samples on defendants
clothing to determine if
it belongs to victim
DNA

fingerprinting

USES: PATERNITY

Whos the father?

DNA

Mom

F1

F2

child