Upload
nungkii-nilasarii
View
37
Download
4
Embed Size (px)
DESCRIPTION
biotek
Citation preview
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