12/10/2014

Polymerase Chain Reaction (PCR)

PCR
• PCR is a technique widely used in molecular biology.
• Amplify a single or few copies of a piece of
Polimerase Chain Reaction DNA across several orders of magnitude, generating millions or
DNA Amplification Dr. Kary Mullis more copies of the DNA piece.

A DNA polymerase used to amplify a

piece of DNA by in vitro
enzymatic replication

Nastiti Wijayanti Uses 2 oligonucleotide primers

Laboratorium Fisiologi Hewan complementary to opposide strands of a
Fakultas Biologi-UGM duplex DNA
www.themegallery.com

History PCR animation

• PCR invented by Dr. Kary Mullis in 1983
• Mullis and Faloona, 1987.
Specific synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction.
• In vitro amplification of specific DNA fragment
•Nobel Prize 1993

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Thermus Aquaticus (Taq)

PCR
Heat Stable DNA Polymerase

Reaction components:
Template preparation
Primers
MgCl2 concentration – Buffer PCR
dNTP concentration
Taq Polimerase
Setting up PCR reaction
Post-PCR analysis

Reaction components: 2. Primer:

urutan nukleotida dgn panjang tertentu yang berfungsi
1. Template preparation sbg inisiator reaksi PCR.

• The amount of template in a reaction strongly General:

influences the performance of the PCR • Length: 18 to 24 nucleotides
• Avoid to dissolve the template in TE bufffer-
• GC content: 40 – 60%
EDTA chelates Mg2+
• Contain no internal secondary structure
• Use 5-10 mM Tris (pH 7-8) or water to dissolve
• Are not complementary to each other at the 3’
the template.
ends to avoid primer-dimer forming

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Primers lists…………………1 Primers lists…………………2

Primers Primers
 Standard: use of a standard primer leads to simple
DNA amplification.
• Non-complementary sequence: used to
introduce restriction sites for sub-cloning.
 Mismatch: can be used to introduce site-directed
mutations/mutagenesis. GAATTC

• Degenerate sequence: are used to amplify

 Fluorescent or biotin label/Labelled primers: can
be used to amplify labelled DNA, for sequencing, DNA whose sequences is not fully known
mapping or spectroscopic probes.
G
A T

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Primer…….sambungan
Melting temperature:

• Estimation of melting temperature Tm:

Tm = [ 2°C x (number of A and T bases)]+ [
4°C x (number of C and G bases)].
• Design primers with similar Tm values.

Primer…….sambungan Primer…….sambungan
Annealing temperature: suhu dimana primer Concentrations of primers:
akan berikatan pada komplementernya. • Optimal concentration between 0.1 and 0.6
μM.
• Optimal annealing temperatures are ~5 to • How to convert μM values into pmol values of
10°C lower than the Tm values of the primers primers.
and have to be determined empirically. • Higher concentration- promote mispriming and
• Design primers as such that an annealing accumulation of non-specific products.
temperature of 55 - 65°C is allowed, for • Lower concentration-exhausted before the
maximum specificity, use temperatures of 62- reaction is completed, resulting in lower yields
72 °C. of desired product.

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Primer…….sambungan 3. MgCl2 concentration

Conversion of μM to pmol: • Function of Mg2+ ions: form soluble complexes
• 1 μM of a X μM primer solution = X pmol primers.
with dNTPs and template DNA to produce the
actual substrate that the polymerase
• Example: recognizes.
Question: • Concentration of Mg2+ ions: The most optimal
- given primer solution: 2.5 μM concentration should be determined
empirically and may vary from 1 mM to 5 mM.
- amount of primers needed for experiment: 2 • Common: 1.5 mM, with a dNTP 200 μM each.
pmol
• Excess Mg2+: increase non-specific primer
Answer: binding and increase the non-specific
- 1 μl of a 2.5 μM primer solution = 2.5 pmol background of the reaction.
- 2 pmol = 0.8 μl • Too little Mg2+: a lower yield of the desired
product.

4. dNTP concentration

• Use balanced solutions of all four dNTPS to

minimize the error rate.
• The final concentration of each dNTP between 50
and 500 μM, the most commonly used conc. is
200 μM.
• Increase conc.of Mg2+ when increasing the conc.
of dNTPs – increases in dNTP concs reduces the
conc of free Mg2+, interfering with the activity of
the polymerase enzyme.

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5. Taq DNA Polymerase Problem with Taq

• Does not have proof reading ability
• Active in high temperature. • Error rate 1 in 2 X 104 bases
• The optimal amount of thermostable DNA • Seems rare but can be recovered in cloning a single molecule
Polymerase-between 0.5 and 2.5 units per 50 μl • Newer polymerases have high fidelity
reaction volume.

Thermus aquaticus from hot springs in Yellowstone National

Park, USA.

Setting up PCR reaction-Cycling Setting up PCR reaction-Cycling….. cont

• Initial denaturation • Initial denaturation:
• Denaturation during cycling • denature the template DNA, completely by initial heating of the
PCR mixture. 2 minutes at 94-95°C enough to denature complex
• Primer annealing genomic DNA.
• Primer extension/Elongation • Partially denatured, it will tend to “snap back” very quickly,
preventing efficient primer annealing and extension, or leading to
• Cycle number “self priming” which can lead to false positive results.
• Final extension

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Setting up PCR reaction-Cycling….. sambungan Setting up PCR reaction-Cycling….. sambungan

• Denaturation during cycling: • Primer annealing:

• Denaturation at 94-95°C for 20 to 30 seconds is usually sufficient
but must be adapted for the thermal cycler and tubes being used. • Is a very critical factor in designing a high specificity
• Use a longer denaturation time or higher denaturation temperature PCR and – for most purposes-has to be optimized
for GC rich template DNA. empirically.
• Unnecessary long denaturation times decreases the activity of the
• If the temperature is too high, no annealing occurs.
polymerase.
• If the temperature is too low, non-specific annealing will
increase dramatically.
• If the primers have complementary bases, primer-dimer
effect will occur.

Besar produk : 121 – 300 = 180 bp

Primer design
Program PCR:
5‘ --------3‘ Pre-denaturasi : 95C 4 ‘
121 atcagcgaga taatgaacgt tctcttccaa gacccagaca actgcaaatt acattatcat
181 taacggtatc agcagaagca atgaaataaa gaaaaacgca gctaatattt ttctcacacc
Siklus 35 x
241 cactgctgtg tgtttgtcaa aggcaataac agtttatcct gttgagtggg gagaagagga Denaturasi : 95C 30”
3’caactcaccc ctcttctcct 5’ Annealing : 49-54C 30”
Extension : 72C 15” (60 basa/sec- 45’’ 1 kb)
!!! Extra extension : 72C 7’
 Sequence as.nukleat selalu ditulis dengan urutan 5’ – 3’ Fast Start PCR - Roche

 Penulisan primer dengan urutan 5’ - 3’

 Perhatikan persyaratan primer
 Forward primer (= seq. as.nukleat yg tertera): 5’ ATC AGC GAG ATA ATG AAC GT 3’
= 40% GC
 Reverse primer (=seq. komplementernya): 5’ TCC TCT TCT CCC CAC TCA AC 3’ =
55% GC

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Program PCR
Setting up PCR reaction-Cycling….. sambungan
• Besar produk : 121 – 300 = 180 bp
• Primer:
5’ ATC AGC GAG ATA ATG AAC GT 3’ = 40% GC • Primer extension:
5’ TCC TCT TCT CCC CAC TCA AC 3’ = 55% GC
• Tag DNA Polymerase adds approximately 60 bases per
[(12 AT x 2) + (8 CG x 4)] + [(9 AT x 2) + (11 CG x 4)] second at 72°C.
2
• A 45 second extension is sufficient for fragments up to 1
= [24 + 32] + [18 + 44] = 56 + 62 = 59 -------- 5 – 10 < TM
2 2 kb.
• To improve yield, use the cycle extension feature of the
• Program PCR:
• Pre-denaturasi : 94-95 C cycler:
• Siklus 35 x • First 10 cycles, e.g., 45 sec for a 1 kb product
• Denaturasi :
• Annealing : (59C -------- 5 – 10C < TM) • Next 20 cycles: increase the extension time by 2-5 sec per cycle,
• Extension : 15” (60 basa/sec- 45’’ 1 kb) e.g., 50 sec for cycle 11, 55 sec for cycle 12,…
• Extra extension : 72 C

Setting up PCR reaction-Cycling….. sambungan

Post-PCR analysis
• Cycle number: • Electroforesis gel agarose
• Most PCR’s should only include 25-35 cycles.
run an aliquot on an agarose gel – check the size
• As cycle numer increases, non-specific products can
accumulate. and integrity of the DNA
• Sequencing DNA
• Final extension:
• After the last cycle, the reaction tubes are held at
72°C for 5-15 minutes to promote completion of
partial extension products and annealing of single-
stranded complementary products.
• After the final extension, immediately put the
samples on ice or store at 4°C.

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Other PCR methodes

• Quantitative Real-Time PCR (qPCR)
• Reverse Transcriptase PCR (RT-PCR)
• Nested PCR
• Multiplex PCR

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