PolymeraseChainReaction(PCR)Polymerase Chain Reaction (PCR) · 2010-10-17 · PolymeraseChainReaction(PCR)Polymerase Chain Reaction (PCR) • A technique for the in vitroamplification

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)

• A technique for the in vitro amplification of specific DNA sequences by the simultaneous primer extension ofsequences 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).

DNA polymerization: DNA polymerase extends a primer by using p y p y p y ga complementary strand as a template.

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

5) Mg++


1) TEMPLETE3’ 5’

…

4) dNTP’s poolC

A dATP

dCTP G

CA A

AC

C

T

G

T G

G

T

dGTP

dTTPT

A

A

A

C

C

C

G

T

G

G

T

Newly synthesised strand

GT T T TC CA A G G G


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

Schematic diagram of PCR5’… …3’

5’…3’

GenomicDNA

…5’

3’

… …3’ 5’

… …

3’ 5’

…5’ 3’Denature

94 ℃

…

5’ 3’

3 5

5’… …3’

… …Denature

94 ℃

3’ 5’… … 5’3’

5’

…5’ 3’

Anneal primers

ca. 50 ℃

5’

…3’

5’3’

5’… …Anneal primers

ca. 50 ℃5’3’

3’

3’ 5’… …

5’3’5’ 3’

3’ 5’… …

ca. 50 ℃

…5’ 3’

3’ 5’

……

…5’ 3’

5’ 3’

…3’ 5’

…Synthesize

Newstrand72 ℃

…

5’

SynthesizeNew

strand72 ℃

3’ 5’

3 55’

…3’ …

…3’ 5’

… 72 ℃

5’3’

…5’ 3’

… …3’ 5’

………

…Denature

94 ℃

… ……

… …

…

…

94 ℃ …

… …

…

…

SynthesizeNew

strand72 ℃

…

……

…Anneal primers

ca. 50 ℃ … ……

… …

… …

Hydrothermal ventsHydrothermal ventsHydrothermal ventsHydrothermal vents

Taq DNA Polymerase:Taq. DNA Polymerase:

• Most commonly used DNA polymerase for PCRIsolated from Thermus aquaticusHeat stable polymeraseHeat 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.

ThermophilicThermophilic DNA polymerasesDNA polymerasesThermophilicThermophilic DNA polymerasesDNA polymerases

( )Thermus aquaticus (Taq)Thermus thermophilus (Tth)Thermus thermophilus (Tth)Bacillus stearothermophilus (Bst)P f i i (Pf )Pyrococcus furiosis (Pfu)

Reaction componentsReaction components

1) Thermophilic DNA polymerases

• Thermus aquaticus (Taq)Thermus thermophilus (Tth)B ill t th hil (B t)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)

• Genetically modified• Genetically modified for improving proof reading function

(3’ to 5’ exonuclease function)and for hot start PCRand 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/ul0.2-0.5ul / 100ul reaction


1) Thermophilic DNA polymerases

• Thermus aquaticus (Taq)Thermus thermophilus (Tth)B ill t th hil (B t)Bacillus stearothermophilus (Bst)Pyrococcus furiosis (Pfu)

• Genetically modified• Genetically modified for improving proof reading function

(3’ to 5’ exonuclease function)and for hot start PCRand 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/ul0.2-0.5ul / 100ul reaction

An example of a PCR methodAn example of a PCR method

100

Pre-denature95 ℃

Denature95 ℃

Final-extend72 ℃80

100

Extend72 ℃

40

60

Temp.℃ Anneal

55 ℃

20Cycle 1 Cycle 2 • • • Cycle 30 Soak

4 ℃

0

Holdprogram

Cycleprogram

Holdprogram


2) Deoxynucleotide Triphosphates

• General concentration for PCR reaction: 200uM

Working solution: 10X (2mM of each dNTP)• Working solution: 10X (2mM of each dNTP)use 10ul of working solution for 100ul reaction

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

• Low dNTP concentrations minimize mispriming at nontarget sitesand reduce the likelihood of extending misincorporatedg pnucleotides (Innis et al. 1988)


3) Primers

• Generally, use over 18 nucleotides

Genome size of higher plants:

Probability of presence of same nucleotide sequences

6 mer 1/46= 1 / 4X103

9 mer 1/49= 1 / 2 6X105• Genome size of higher plants:5X108-6X109

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

• Check list for primer design:1) Similar Tm values are recommended for two primers2) Avoid self-complement sequences2) Avoid self complement sequences3) Avoid primer dimer formation

• General concentration for PCR reaction: 0.1-0.5 uMGeneral concentration for PCR reaction 0.1 0.5 uM

• Working solution: 10X (10-20 uM)e.g. use 2.5 ul of working solution for 100ul reaction (0.5uM)g g ( )

P i D i d O dPrimer Design and Order

- Degenarate letters- Inosine

프라이머디자인사이트 1. (Bioneer) http://www.bioneer.co.kr/

프라이머디자인사이트 2. (COSMO) http://www.cosmo4.com

Reaction components

4) R i b ff4) Reaction buffer

• Mainly modified from Saiki et al. (1988)Components of PCR buffer

Saiki et al. (1988): Suh lab:

• Low conc. of Mg++: reaction failedHigh conc. of Mg++: mis-pairing

pseudo bands

Tris pH 8.4 10mM 20mMKCl 50mM 50mMMgCl2 1.5mM 1.5mMgelatin 0.01% -NP40 0 01% -pseudo bands

• General Mg++ concentration for PCR reaction: 1 5 mM

NP40 0.01%Tween-20 0.01% 0.001%

General Mg concentration for PCR reaction: 1.5 mM

Reaction components

5) Template DNA

• General amount of template DNA: 105-106 target molecules

• 1 ug of human DNA1 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 A260 of 1.0 = 50 mg/ml concentration of DNA.

• DNA의 순도: OD260/280280nm는 protein이 흡수하는 파장

UV N dUV-spec vs. Nanodrop

Gel electrophoresis

침강제+Dye+DNA침강제+Dye+DNA

An example of spot test

• Marker: PCR Marker (Promega G3161)

- Marker concentration: 5 ul contains 30-40 ng of each DNA fragmentsg

- Sample DNA concentration:6-8 ng/ul

∴ dilute to 1/10 use 2 ul of DNA for 100ul reaction (1.2-1.6 ng)

An example of a PCR methodAn example of a PCR method

100

Pre-denature95 ℃

Denature95 ℃

Final-extend72 ℃80

100

Extend72 ℃

40

60

Temp.℃ Anneal

55 ℃

20Cycle 1 Cycle 2 • • • Cycle 30 Soak

4 ℃

0

Holdprogram

Cycleprogram

Holdprogram

Depending on reaction conditions and thermal cycling, one or more of the following may influence plateau:one or more of the following may influence plateau:

1) Utilization of substrates (dNTPs or primers)2) Stability of reactants (dNTPs or enzymes)2) Stability of reactants (dNTPs or enzymes)3) End-product inhibition (pyrophosphate, duplex DNA)

…

• Cloning

SequencingSequencing1) DNA sequencing: Maxam-Bilbert methodhttp://www.nd.edu/~aseriann/maxam.htmlhttp://en.wikipedia.org/wiki/DNA_sequencing

2) DNA sequencing: Sanger methodq g ghttp://www.bio.davidson.edu/Courses/Bio111/seq.htmlDNA Secret of Life #3 21:20 25:30 25:30

3) Highthroughput sequencing (Pyro-sequencing)http://en.wikipedia.org/wiki/Pyrosequencinghttp://kr.youtube.com/watch?v=kYAGFrbGl6Ehttp://www.google.co.kr/imgres?imgurl=http://www.biotagebio.com/graphics/8214.gif&imgrefurl=http://www.biotagebio.com/CpG&h=297&w=600&sz=19&tbnid=Ic39gKJGHkNhSM::&tbnh=67&tbnw=135&prev=/images%3Fq%3Dpyrosequencing&hl=ko&usg=__vZK0WE86eeEDNM7cLlJD2MKM3Qc=&sa=X&oi=image_result&resnum=5&ct=image&cd=1=5&ct=image&cd=1

Maxam-GilbertGilbert method

Sanger method

Manual method: 1 di i t S351. radio-isotope S352. Silver staining

Automated 1. Plate type2. Capillary type

i. one capillaryii. Multiple capillariesp p

Sanger methodSanger and Gilbert shared a Nobel prize in 1980

One-dye (or isotope) four-lane system

Acrylamide Gel Electrophoresis with S35 for Sequencing

Gel dryer

IntensifyingCacetteCacette

Four-dye one-lane system

ABI 377: Gel-type Automatic sequencer

ABI 3730: Capillary-type Automatic sequencerABI 3730 Capillary type Automatic sequencer

ABI 3100

P S iPyro-Sequencing

1) Emulsion PCR2) Sequencing by synthesis (SBS)3) Microtiter plate

Three Big Companies

Roche GS-FLX 100MIll i S l 80GIllumina Solexa 80GABI SOLid 200G

Solexa Sequencingq g

50 Million Clusters Per Flow Cell

20 Microns20 Microns

100 Microns100 Microns

Krause et al. 2006. Multiplification of the mammoth mitochondial genome and the evolution of Elephantidae. Nature 439: 724 727439: 724-727.

Poinar et al. 2006 Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. q gScience 311: 392-394.

개인 유전체 시대의 시작:각자의 전체 유전체를 밝각자의 전체 유전체를 밝혀 개인식별, 개인적 유전병 치료, 궁극적으로는 클로닝에 이용될 수 있음

미래 사회를 장악하고 있는 DNA 염기 배열이다. 우주선을 발사하는회사 <가타카>를 출입하기 위해본인 확인을 하려면 매일 약간양의혈액을 엄지 손가락으로부터 뽑아내야 한다내야 한다.

5000불개인유전체 시대개인유전체 시대

RFLP Restriction Fragment Length Polymorphism

PCR-mediated RFLP

Documents

PolymeraseChainReaction(PCR)Polymerase Chain Reaction (PCR) · 2010-10-17 · PolymeraseChainReaction(PCR)Polymerase Chain Reaction (PCR) • A technique for the in vitroamplification