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Polymerase Chain Reaction (PCR)
PCR protocols
Polymerase Chain Reaction (PCR)
• A technique for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA (>105).
• PCR method was devised and named by Mullis and colleagues at the Cetus Corporation (Mullis and Faloona, 1987)
• The principle had been described in tetail by Khorana et al. (Kleppe et al., 1971) over a decade earlier.
• Kary Mullis received the Novel prize (1993).
3) DNA POLYMERASE
GT T T TC CA A G G G
2) PRIMER3’5’
AC TA C G TA CG TA C GT G T C CT AC G G TT CT T CT T A G T TG T A C CA CA G TA C TCG TA C T CG TA C T AAA
1) TEMPLETE3’ 5’
…
4) dNTP’s pool
G
C
A
T
dATP
dCTP
dGTP
dTTP
5) Mg++
G
CA
T
A
A
A
A
C
C
C
C
G
T
G
T
G
T G
T
GT T T TC CA A G G G
AC TA C G TA CG TA C GT G T C CT AC G G TT CT T CT T A G T TG T A C CA CA G TA C TCG TA C T CG TA C T AAA
3’
5’
5’
…
Newly synthesised strand
C A A G G A CG T C CA A A T G C A A G A A A A A A AG G G G GT T T T T TC C C CA AG G
DNA polymerization: DNA polymerase extends a primer by using a complementary strand as a template.
Taq. DNA Polymerase:
• Most commonly used DNA polymerase for PCRIsolated from Thermus aquaticusHeat stable polymerase
• PCR technique put to practical use by finding of Taq.
• The use of a heat stable enzyme has two major advantages:
1) replenishment after each heating step is not required, thus simplifying the process
2) the enzyme is active at high temperatures, where annealing of the oligonucleotide primers is more specific and DNA synthesis more rapid.
Schematic diagram of PCR
… …Denature
94 ℃
3’
5’
5’… …
3’
3’
5’
5’
…
…
…
…
Anneal primers
ca. 50 ℃5’3’
3’
5’…3’
GenomicDNA
…5’
3’
… …
…5’ 3’
…3’ 5’
…
5’ 3’
…3’ 5’
…Synthesize
Newstrand72 ℃
…
3’
5’
5’
…
…
…
…
5’3’
3’
5’
…5’ 3’
Anneal primers
ca. 50 ℃
5’
…3’
5’
5’3’5’ 3’
3’
5’
5’
SynthesizeNew
strand72 ℃
3’ 5’
…
3’
…5’ 3’
… …
5’ 3’
3’ 5’
……
5’
…3’ …
3’ 5’
3’ 5’… …
5’… …3’
3’ 5’
…5’ 3’Denature
94 ℃
…
…
… …
……
…
…
…
Denature94 ℃
…
… …
…
…
…
… …
Anneal primers
ca. 50 ℃
…
… …
…
…
…
… …
…
…
SynthesizeNew
strand72 ℃
…
Reaction components
1) Thermophilic DNA polymerases
• Thermus aquaticus (Taq)Thermus thermophilus (Tth)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)
• Genetically modified for improving proof reading function
(3’ to 5’ exonuclease function)and for hot start PCR.
• General concentration for PCR reaction: 1-2.5 U / 100ul reaction
• One unit: the amount of enzyme that will incorporate 10 nmolesof dNTPs into acid insoluble material in 30 min at 74℃.
• General concentration provided: 5 unit/ul 0.2-0.5ul / 100ul reaction
Reaction components
1) Thermophilic DNA polymerases
• Thermus aquaticus (Taq)Thermus thermophilus (Tth)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)
• Genetically modified for improving proof reading function
(3’ to 5’ exonuclease function)and for hot start PCR.
• General concentration for PCR reaction: 1-2.5 U / 100ul reaction
• One unit: the amount of enzyme that will incorporate 10 nmolesof dNTPs into acid insoluble material in 30 min at 74℃.
• General concentration provided: 5 unit/ul 0.2-0.5ul / 100ul reaction
Reaction components
1) Thermophilic DNA polymerases
• Thermus aquaticus (Taq)Thermus thermophilus (Tth)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)
• Genetically modified for improving proof reading function
(3’ to 5’ exonuclease function)and for hot start PCR.
• General concentration for PCR reaction: 1-2.5 U / 100ul reaction
• One unit: the amount of enzyme that will incorporate 10 nmolesof dNTPs into acid insoluble material in 30 min at 74℃.
• General concentration provided: 5 unit/ul 0.2-0.5ul / 100ul reaction
Final-extend72 ℃
An example of a PCR method
0
20
40
60
80
Temp.℃
100
Holdprogram
Pre-denature95 ℃
Anneal55 ℃
Extend72 ℃
Cycleprogram
Holdprogram
Cycle 1 Cycle 2 • • • Cycle 30
Denature95 ℃
Soak4 ℃
Reaction components
1) Thermophilic DNA polymerases
• Thermus aquaticus (Taq)Thermus thermophilus (Tth)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)
• Genetically modified for improving proof reading function
(3’ to 5’ exonuclease function)and for hot start PCR.
• General concentration for PCR reaction: 1-2.5 U / 100ul reaction
• One unit: the amount of enzyme that will incorporate 10 nmolesof dNTPs into acid insoluble material in 30 min at 74℃.
• General concentration provided: 5 unit/ul 0.2-0.5ul / 100ul reaction
Reaction components
2) Deoxynucleotide Triphosphates
• General concentration for PCR reaction: 200uM
• Working solution: 10X (2mM of each dNTP) use 10ul of working solution for 100ul reaction
• Low dNTP concentrations minimize mispriming at nontarget sitesand reduce the likelihood of extending misincorporatednucleotides (Innis et al. 1988)
e.g.100mM dATP 20 ul100mM dCTP 20 ul + D.W. 920 ul 10X working dNTP solution100mM dGTP 20 ul 1000ul (each 2mM)100mM dTTP 20 ul
Reaction components
3) Primers
• Generally, use over 18 nucleotides
• Genome size of higher plants:5X108-6X109
• Check list for primer design:1) Similar Tm values are recommended for two primers2) Avoid self-complement sequences3) Avoid primer dimer formation
• General concentration for PCR reaction: 0.1-1 uM
• Working solution: 10X (10-20 uM) e.g. use 2.5 ul of working solution for 100ul reaction (0.5uM)
Probability of presence of same nucleotide sequences
6 mer 1/46= 1 / 4X103
9 mer 1/49= 1 / 2.6X105
12 mer 1/412= 1 / 1.7X107
15 mer 1/415= 1 / 1.1X109
18 mer 1/418= 1 / 6.9X1010
21 mer 1/421= 1 / 4.4X1012
An example of primer design: OLIGO program
Reaction components
4) Reaction buffer
• Mainly modified from Saiki et al. (1988)
• Low conc. of Mg++: reaction failedHigh conc. of Mg++: mis-pairing
pseudo bands
• General Mg++ concentration for PCR reaction: 1.5 mM
Components of PCR buffer
Saiki et al. (1988): Kim lab:Tris pH 8.4 10mM 20mMKCl 50mM 50mMMgCl2 1.5mM 1.5mMgelatin 0.01% -NP40 0.01% -Tween-20 0.01% 0.001%
Reaction components
5) Template DNA
• General amount of template DNA: 105-106 target molecules
• 1 ug of human DNA10 ng of yeast DNA ≒ 3X105 molecules of single copy gene1 ng of E.coli DNA
c.f. rbcL gene (chroloplast genome) amplification in angiosperm:1-50 ng of DNA
• DNA elution (final step of the extraction): use D.W. or TLE (Tris-low EDTA; TE0.1)
• Quantification of extracted DNA:1) spectrophotometer: OD260
2) spot test in agarose gel
An example of spot (band) test
• Marker: PCR Marker (Promega G3161)
- Marker concentration: 5 ul contains 30-40 ng of each DNA fragments
- Sample DNA concentration:6-8 ng/ul
∴ dilute to 1/10 use 2 ul of DNA
for 100ul reaction (1.2-1.6 ng)
Thermal condition and cycle number
e.g. PCR amplification of ndhF gene in Magnoliaceae (Kim et al., 2001)
Pre-denaturation: 95 ℃ 3 min
Denaturation: 95 ℃ 1 minPrimer annealing: 55 ℃ 1 min 30 cyclesPrimer extension: 72 ℃ 1 min
Final extension: 72℃ 7 min
Final-extend72 ℃
An example of a PCR method
0
20
40
60
80
Temp.℃
100
Holdprogram
Pre-denature95 ℃
Anneal55 ℃
Extend72 ℃
Cycleprogram
Holdprogram
Cycle 1 Cycle 2 • • • Cycle 30
Denature95 ℃
Soak4 ℃
Depending on reaction conditions and thermal cycling, one or more of the following may influence plateau:
1) Utilization of substrates (dNTPs or primers)2) Stability of reactants (dNTPs or enzymes)3) End-product inhibition (pyrophosphate, duplex DNA)
…
Typical result of PCR
PCR of rbcL in Scutellaria sp.
S. st
rigill
osa
1
S. st
rigill
osa
2
S. peki
nensi
s va
r. a
lpin
a
S. in
dic
a
Isodon inflexs
us
500bp
1,000bp
1,500bp
PCR Product Purification
• For removing primers, dNTPs, and pyrophosphates
Method 1.Purification by glass milk
Method 2.Column membrane method
Method 3.Treatment of exonuclease and pyrophosphatase
