PCR DAN

ELEKTROFORESIS

Dr. Oeke Yunita, S.Si., M.Si., Apt.

BIOTEKNOLOGI FARMASI

THE TRIPLET CODE

POLYMERASE CHAIN REACTION

teknik in vitro untuk melipatgandakan asam

nukleat (DNA atau RNA) secara eksponensial

dan cepat dengan bantuan enzim di dalam

suatu mesin / Thermocycler

PCR Polymerase Chain Reaction

DNA/gene amplification

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. ikatan antar untaian DNA dengan pasangan komplementernya adalah sangat kuat dan spesifik

b. Pemisahan untaian menjadi rantai tunggal dan masing-masing rantai dapat dipakai sebagai cetakan (template) untuk mensintesis rantai pasangannya.

c. 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

Melting

94 oC

Melting

94 oC

30 s Annealing

Primers

45 oC

60 s

Extension

72 oC

60 s

Tem

per

atu

re 100

0

50

T i m e

40x

5 3

3 5

3 5

5

5 3 5

3 5

5

5

5

5 3

3 5

3 5

5 3

5 3

5

Tem

per

atu

re 100

0

50

T i m e

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

Genomic DNA

+

Taq polymerase

+

primers

A

+

Nucleotides

+

Buffer

PCR

(under relaxed conditions)

PCR-BASED DNA MARKERS

Generated by using Polymerase Chain Reaction

Preferred markers due to technical simplicity and cost

GEL ELECTROPHORESIS

Agarose or Acrylamide gels

PCR

PCR Buffer +

MgCl2 +

dNTPS +

Taq +

Primers +

DNA template

THERMAL CYCLING

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.

Agarose

Buffer

Flask for boiling

Casting tray

Gel combs

Power supply

Gel tank Cover

Electrical leads

Electrophoresis Equipment

Gel casting tray & combs

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.

Preparing 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.

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.

Melting the Agarose

Allow the agarose solution to cool slightly (~60C) and then carefully

pour the melted agarose solution into the casting tray. Avoid air

bubbles.

Pouring the gel

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.

buffer

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

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

Cathode (negative)

Anode (positive)

wells

DNA

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:

pH Nature

Pore size

intensity

charge

Size

shape

Molecules Electric current

Buffer Supporting

media

ANALISIS DNA PADA

HASIL ELEKTROFORESIS

100

1,000

10,000

100,000

0 5 10 15 20 25 30

Distance, mm

Siz

e,

ba

se

pa

irs

B

A

USES: EVOLUTIONARY RELATIONSHIPS

Comparing DNA samples from different organisms to measure evolutionary relationships

+

DNA

1 3 2 4 5 1 2 3 4 5

turtle snake rat squirrel fruitfly

USES: MEDICAL DIAGNOSTIC

Comparing normal allele to disease allele

chromosome with

disease-causing

allele 2

chromosome

with normal

allele 1

+

DNA

Example: test for Huntingtons disease

USES: FORENSICS

Comparing DNA sample from crime scene

with suspects & victim

+

S1

DNA

S2 S3 V

suspects crime scene

sample

DNA FINGERPRINTS

Comparing blood

samples on defendants

clothing to determine if

it belongs to victim

DNA fingerprinting

USES: PATERNITY

Whos the father?

+

DNA

child Mom F1 F2