POLYMERASE CHAIN REACTION
AMPLIFIKASI FRAGMEN DNA
Agustina Setiawati, MSc., Apt
Background on the Polymerase Chain Reaction (PCR)

Ability to generate identical high copy number DNAs made possible
in the 1970s by recombinant DNA technology (i.e., cloning).

Cloning DNA is time consuming and expensive (>>$15/sample).

Probing libraries can be like hunting for a needle in a haystack.

PCR, “discovered” in 1983 by Kary Mullis, enables the amplification
(or duplication) of millions of copies of any DNA sequence with
known flanking sequences.

Requires only simple, inexpensive ingredients and a couple hours.
DNA template
Primers (anneal to flanking sequences)
DNA polymerase
dNTPs
Mg2+
Buffer

Can be performed by hand or in a machine called a thermal cycler.

1993: Nobel Prize for Chemistry
The polymerase chain reaction (PCR) can
selectively and rapidly amplify a given DNA
sequence to large amounts
Used in cloning, sequencing, forensics, diagnosis
Specific primers hybridize on each side of the DNA
sequence to be copied
Enzyme – Taq DNA polymerase – from Thermus
aquaticus – resistant to high temperatures
Very sensitive – can amplify a sequence present in
very low copy number
How PCR works:
1.
Begins with DNA containing a sequence to be amplified and a pair
of synthetic oligonucleotide primers that flank the sequence.
2.
Next, denature the DNA at 94˚C.
3.
Rapidly cool the DNA (37-65˚C) and anneal primers to
complementary s.s. sequences flanking the target DNA.
4.
Extend primers at 72˚C using a heat-resistant DNA polymerase
(e.g., Taq polymerase derived from Thermus aquaticus).
5.
Repeat the cycle of denaturing, annealing, and extension 20-45
times to produce 1 million (220)to 35 trillion copies (245) of the
target DNA.
6.
Extend the primers at 72˚C once more to allow incomplete
extension products in the reaction mixture to extend completely.
7.
Cool to 4˚C and store or use amplified PCR product for analysis.
Example thermal cycler protocol used in lab:
Step 1 7 min at 94˚C
Initial Denature
Step 2 45 cycles of:
20 sec at 94˚C
20 sec at 64˚C
1 min at 72˚C
Denature
Anneal
Extension
Step 3 7 min at 72˚C
Final Extension
Step 4 Infinite hold at 4˚C
Storage
BIOL 362 samples processed in:
MJ Research DNA Engine Dyad
Hot water bacteria:
Thermus aquaticus
Taq DNA polymerase
Life at High Temperatures
by Thomas D. Brock
Biotechnology in Yellowstone
© 1994 Yellowstone Association for Natural Science
http://www.bact.wisc.edu/Bact303/b27
Fig. 7.23
Denature
Anneal PCR Primers
Extend PCR Primers
w/Taq
Repeat…
10_27_1_PCR_amplify.jpg
The polymerase chain reaction – used to amplify a specific
DNA sequence with cyclical changes in temperature
10_27_2_PCR_amplify.jpg
PCR – applications:
1) The method of choice for cloning relatively short
DNA sequences (under 10,000 nts) – can use to get
genomic clone or cDNA clone
10_28_PCR_clones.jpg
PCR – applications:
1) The method of choice for cloning relatively short
DNA sequences (under 10,000 nts) – can use to get
genomic clone or cDNA clone
2) Can detect infectious pathogens at very early
stages
3) PCR is used in forensic medicine to generate a
DNA fingerprint – based on amplifying areas of the
genome that contain VNTRs (variable number
tandem repeats)
10_29_PCR_viral.jpg
Using PCR to detect a viral genome in a drop of blood
10_30_1_PCR_forensic.jpg
Primers are used to amplify areas with VNTRs, which differ
in different chromosomes, different individuals
10_30_2_PCR_forensic.jpg
Three areas
amplified to
generate a DNA
fingerprint
Basic idea…lets say we want to amplify 1 gene of 500 bp from
some bacterial DNA.
DNA
* must know the sequence at the limits/ends of the
* design complementary primers  anneal to template
5
3
5
3
3
5
3
5
Melt template, then rapidly cool
* some primers will anneal to complementary sequence
5
3
3
5
5
3
3
5
Melt template, then rapidly cool
* some primers will anneal to complementary sequence
5
3
3
5
Add DNA polymerase
* provide substrate + time to extend
5
3
3
5
Melt template, then rapidly cool
* some primers will anneal to complementary sequence
5
3
3
5
Add DNA polymerase
* provide sunstrate + time to extend
These 3 steps constitute 1PCR ‘cycle’, which will be repeated
many times (usually 25-30)
1) melt template
2) anneal oligonucleotide primers
3) extend with DNA polymerase
If ever confused about how PCR works…
* draw out first three cycles
25-30x
Limitations – finite amounts of
* dNTPs
* primers
* DNA pols
Exhaustion after 30
PCR
AKUMULASI EKSPONENSIAL FRAGMEN
TERAMPLIFIKASI

Setelah 30 siklus

2 pangkat 28 = 268 345 456 fragmen
Good Primer’s Characteristic





A melting temperature (Tm) in the range of 52 C to
65 C
Absence of dimerization capability
Absence of significant hairpin formation (>3 bp)
Lack of secondary priming sites
Low specific binding at the 3' end (ie. lower GC
content to avoid mispriming)
Uniqueness
There shall be one and only one target site in the template DNA
where the primer binds, which means the primer sequence shall be
unique in the template DNA.
There shall be no annealing site in possible contaminant sources,
such as human, rat, mouse, etc. (BLAST search against
corresponding genome)
Template DNA
5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’
CAGTCAACTGCTAC
TGCTAAGTTG
5’-TGCTAAGTTG-3’
Primer candidate 2
5’-CAGTCAACTGCTAC-3’
A
TGCT AGTTG
Primer candidate 1
NOT UNIQUE!
UNIQUE!
Length
Primer length has effects on uniqueness and
melting/annealing temperature. Roughly speaking, the
longer the primer, the more chance that it’s unique; the
longer the primer, the higher melting/annealing
temperature.
Generally speaking, the length of primer has to be at
least 15 bases to ensure uniqueness. Usually, we pick
primers of 17-28 bases long. This range varies based
on if you can find unique primers with appropriate
annealing temperature within this range.
PANJANG PRIMER

Panjang primer 8
4
pangkat 8 = 65.536 pb
 Ukuran kromosom 3.000.000 kb ada 46.000
kemungkinan situs
Panjang primer 17
= 17.179.869.184 pb diharapkan hanya menempel
pada 1 situs
Base Composition
Base composition affects hybridization specificity and
melting/annealing temperature.
• Random base composition is preferred. We shall avoid long (A+T)
and (G+C) rich region if possible.
Template DNA
5’...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3’
TGCCCGATCATGCT
• Usually, average (G+C) content around 50-60% will give us the
right melting/annealing temperature for ordinary PCR reactions, and
will give appropriate hybridization stability. However,
melting/annealing temperature and hybridization stability are
affected by other factors. Therefore, (G+C) content is allowed to
change.
Melting Temperature
Melting Temperature, Tm – the temperature at
which half the DNA strands are single stranded and
half are double-stranded.. Tm is characteristics of
the DNA composition; Higher G+C content DNA has
a higher Tm due to more H bonds.
Calculation
Shorter than 13: Tm= (wA+xT) * 2 + (yG+zC) * 4
Longer than 13: Tm= 64.9 +41*(yG+zC-16.4)
/(wA+xT+yG+zC)
(Formulae are from
http://www.basic.northwestern.edu/biotools/oligocalc.html)
Annealing Temperature
Annealing Temperature, Tanneal – the
temperature at which primers anneal to the
template DNA. It can be calculated from Tm .
Tanneal = Tm_primer – 4C
Primer Pair Matching
Primers work in pairs – forward primer and reverse
primer. Since they are used in the same PCR reaction, it
shall be ensured that the PCR condition is suitable for
both of them.
One critical feature is their annealing temperatures,
which shall be compatible with each other. The
maximum difference allowed is 3 C. The closer their
Tanneal are, the better.
Summary ~ when is a “primer” a primer?
5’
3’
5’
3’
5’
3’
3’
5’
Summary ~
Primer Design Criteria
1. Uniqueness: ensure correct priming site;
2. Length: 17-28 bases.This range varies;
3. Base composition: average (G+C) content around 50-60%;
avoid long (A+T) and (G+C) rich region if possible;
4. Optimize base pairing: it’s critical that the stability at 5’ end be
high and the stability at 3’ end be relatively low to minimize
false priming.
5. Melting Tm between 55-80 C are preferred;
6. Assure that primers at a set have annealing Tm within 2 – 3 C
of each other.
7. Minimize internal secondary structure: hairpins and dimmers
shall be avoided.
Macam PCR
PCR spesial
 RFLP-PCR
 Reverse
Transcriptase-PCR (RT-PCR)
 Nested-PCR
 Real Time-PCR (RT-PCR)
RT-PCR Procedure





mRNA was isolated from a tissue cell culture
Reverse transcriptase used to synthesize cDNA from
mRNA
PCR performed on cDNA using Taq polymerase
and primers specific for Syk gene
Beta-actin used as an external control for the RTPCR
Southern blot used a biotynlated internal Syk
probe used to confirm amplification of Syk mRNA
RT-PCR Results




Syk is expressed in normal
mammary gland tissue and
breast epithelial cells
Several carcinoma cell lines
expressed Syk strongly
Some carcinoma cell lines
poorly expressed Syk
Some carcinoma cell lines
completely lack Syk
expression
Advantages

Disadvantages
RT-PCR is much faster
and more sensitive that
RNase protection
assays



RT-PCR is very sensitive
and can detect low
levels of mRNA in cells
RT-PCR requires a lot of
preparation and must be
strictly controlled
Non-competitive RT-PCR may
result in false conclusions
because experimental results
are caused by differences in
PCR conditions
REAL TIME PCR
RT PCR
Apakah Real-Time PCR ?



‘Real Time’ dapat dikatakan sebagai mengoleksi dan menganalisa data
yang terjadi selama proses reaksi
‘Real Time PCR’ berarti amplifikasi dan analisa terjadi bersamaan
Dikenal sebagai ‘Rapid Cycle PCR’ dengan siklus temperatur antara 2060 detik

Produk PCR dapat dianalisa selama proses amplifikasi

Menggunakan ‘pewarna DNA’ dan ‘probe fluoresensi’

Data dikoleksi dari tabung yang sama didalam instrumen yang sama

Tidak ada transfer sampel, penambahan reagensia atau gel separasi
Apakah Real-Time PCR ?

Format deteksi :
SYBR Green I
 Hybridization Probes (HybProbe Probes)
 Hydrolysis probes / Taqman Probes
 SimpleProbe Probes



Dapat melakukan ‘real-time quantitative PCR’
‘Real Time PCR’ adalah metode yang ‘powerful’,
sederhana dan cepat
Applications For Detecting and
Quantifying Transcripts
Quantifying viruses
Pathogen detection
Gene expression
Drug therapy
DNA damage
Immune response
Genotyping
Monitoring PCR Reaction
Agarose Gel Blotting
LightCycler
What’s Wrong With Agarose Gels?
Poor precision.
 Low sensitivity.
 Short dynamic range < 2 logs.
 Low resolution.
 Non-automated.
 Size-based discrimination only.
 Results are not expressed as numbers.
 Ethidium bromide staining is not very
quantitative.

Approaches in quantifying by PCR
.
3. Real-time RT-PCR (QPCR). Measurement
.occurs during exponential phase.
Available Chemistries for detecting
PCR product (amplicon).
1. Intercalating dyes that fluoresce, e.g.
SYBR Green I.
2. Hybridization probes, Scorpions. Molecular
Beacons. Donor probe excites acceptor by FRET.
3. Hydrolysis probes, Quench is by nonradiative transfer: Taqman system.
4. Simple probes
1. SYBR Green I


Ketika SYBR Green I berikatan dengan dsDNA,
akan terjadi peningkatan fluoresensi
Selama tahapan PCR yang berbeda, intensitas dari
sinyal fluoresensi akan berbeda, tergantung dari
jumlah dsDNA yang ada
SYBR Green I
Fluoresces when bound
to dsDNA
2. HybProbe Probes

Hybridization probe merubah fluoresensi pada saat hibridisasi
dengan ‘fluorescene resonance energy transfer (FRET)’
Fluorescence Resonance
Energy Transfer (FRET)
E
E
E
E
FRET is Nonradiative energy transfer
Energy Transfer
R
>>
R
o
A
No Transfer
A
D
R
D
3. Taqman probes are “hydrolysis”
probes
Polymerization
5’
Forward
Primer
TaqMan®
R Probe Q
3’
5’
5’
3’
5’
Reverse
Primer
Displacement
5’
Forward
Primer
R
Q
3’
5’
5’
3’
5’
Reverse
Primer
Hydrolysis
R
Q
5’
3’
5’
5’
3’
5’
Polymerization Completed
R
Q
5’
3’
5’
5’
3’
5’
4. Simple Probes




Simple Probe adalah bentuk sederhana dari
hybridization probe dan hanya menggunakan 1 probe
saja
Ketika terjadi hibridisasi akan memancarkan sinyal
fluoresensi yang lebih besar
Perubahan sinyal fluoresensi tergantung dari status
hibridasi dari probe, semakin stabil hibridisasinya
semakin tinggi temperatur melting
Untuk aplikasi SNP genotyping dan deteksi mutasi
Molecular Beacons are
“hybridization” probes
Masalah PCR



Hasil PCR tidak ada atau sedikit
Terlalu banyak pita
Pita-pita tidak jelas
Suhu hibridisasi primer

Hasil PCR sedikit atau tidak ada
 Turunkan

suhu hibridisas
Muncul terlalu banyak pita
 Naikan
suhu hibridisasi
Masalah preparasi DNA



Larutan DNA berwarna (kontaminasi)
Tidak ada DNA
Rasio A260/A280 rendah (gula, fenol, protein)
TERIMA KASIH