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 ℃

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Anneal primers

ca. 50 ℃5’3’

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GenomicDNA

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Newstrand72 ℃

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Anneal primers

ca. 50 ℃

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SynthesizeNew

strand72 ℃

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Anneal primers

ca. 50 ℃

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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 or trnH~psbA (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

DNA quantification by “relative intensity test”

Lambda phage DNA: 500ng/ul

- Prepare diluted lambda DNA: 1/10, 1/20, 1/50, 1/100, 1/200, 1/500, 1/1000

- Dilute extracted DNA (e.g., 1/10 ~ 1/100)

- Load same amount of 1) a series of diluted lambda and 2) diluted sample DNAs (e.g. 5ul each):

- Compare band densities among a series of diluted lambda and diluted DNA

- Sample concentration looks similar to 1/500 diluted lambda.

- Therefore sample concentration = 1ng/ul

1/10 1/20 1/50 1/100 1/200 1/500 1/1000

samples

An example of intensity 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)

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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