Polymerase Chain Reaction (PCR)

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1 Polymerase Chain Reaction (PCR)

2 PCR protocols

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8 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 (>10 5 ). 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).

9 DNA polymerization: DNA polymerase extends a primer by using a complementary strand as a template. 3) DNA POLYMERASE 2) PRIMER 5 3 T T T G C A A G G G C T A A A C G T T C C C G A G T T C C T C G A G T G T T A C G T T C T T C T C T A G T G T T A C A A A C G T T C C A C G T T C A A A 3 5 1) TEMPLETE 4) dntp s pool A C G T datp dctp dgtp dttp A A 5) Mg ++ T C G T G G T C A T C C G AT A G C Newly synthesised strand 3 5 T T T G C A A G G G C T C A A G G A G C T C A C A A T G C A A G A A G A G A T C A C A A T G T T T G C A A G G T G C A A A C G T T C C C G A G T T C C T C G A G T G T T A C G T T C T T C T C T A G T G T T A C A A A C G T T C C A C G T T C A A A 5

10 Taq. DNA Polymerase: Most commonly used DNA polymerase for PCR Isolated from Thermus aquaticus Heat 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.

11 5 Schematic diagram of PCR Genomic DNA Denature Denature Anneal primers ca Anneal primers ca. 50 Synthesize New strand Synthesize New 3 strand 72

12 Denature 94 Synthesize New strand 72 Anneal primers ca. 50

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14 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: U / 100ul reaction One unit: the amount of enzyme that will incorporate 10 nmoles of dntps into acid insoluble material in 30 min at 74. General concentration provided: 5 unit/ul ul / 100ul reaction

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16 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: U / 100ul reaction One unit: the amount of enzyme that will incorporate 10 nmoles of dntps into acid insoluble material in 30 min at 74. General concentration provided: 5 unit/ul ul / 100ul reaction

17 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: U / 100ul reaction One unit: the amount of enzyme that will incorporate 10 nmoles of dntps into acid insoluble material in 30 min at 74. General concentration provided: 5 unit/ul ul / 100ul reaction

18 An example of a PCR method 100 Pre-denature 95 Denature Extend 72 Final-extend Temp. 40 Anneal Cycle 1 Cycle 2 Cycle 30 Soak 4 0 Hold program Cycle program Hold program

19 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: U / 100ul reaction One unit: the amount of enzyme that will incorporate 10 nmoles of dntps into acid insoluble material in 30 min at 74. General concentration provided: 5 unit/ul ul / 100ul reaction

20 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 e.g. 100mM datp 20 ul 100mM dctp 20 ul + D.W. 920 ul 10X working dntp solution 100mM dgtp 20 ul 1000ul (each 2mM) 100mM dttp 20 ul Low dntp concentrations minimize mispriming at nontarget sites and reduce the likelihood of extending misincorporated nucleotides (Innis et al. 1988)

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22 Reaction components 3) Primers Generally, use over 18 nucleotides Genome size of higher plants: 5X10 8-6X10 9 Probability of presence of same nucleotide sequences 6 mer 1/4 6 = 1 / 4X mer 1/4 9 = 1 / 2.6X mer 1/4 12 = 1 / 1.7X mer 1/4 15 = 1 / 1.1X mer 1/4 18 = 1 / 6.9X mer 1/4 21 = 1 / 4.4X10 12 Check list for primer design: 1) Similar Tm values are recommended for two primers 2) Avoid self-complement sequences 3) Avoid primer dimer formation General concentration for PCR reaction: um Working solution: 10X (10-20 um) e.g. use 2.5 ul of working solution for 100ul reaction (0.5uM)

23 An example of primer design: OLIGO program

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28 Reaction components 4) Reaction buffer Mainly modified from Saiki et al. (1988) Low conc. of Mg ++ : reaction failed High conc. of Mg ++ :mis-pairing pseudo bands Components of PCR buffer Saiki et al. (1988): Kim lab: Tris ph mM 20mM KCl 50mM 50mM MgCl2 1.5mM 1.5mM gelatin 0.01% - NP % - Tween % 0.001% General Mg ++ concentration for PCR reaction: 1.5 mm

29 Reaction components 5) Template DNA General amount of template DNA: target molecules 1 ug of human DNA 10 ng of yeast DNA 3X10 5 molecules of single copy gene 1 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; TE 0.1 ) Quantification of extracted DNA: 1) spectrophotometer: OD 260 2) spot test in agarose gel

30 An example of spot (band) test Marker: PCR Marker (Promega G3161) - Marker concentration: 5 ul contains ng of each DNA fragments - Sample DNA concentration: 6-8 ng/ul dilute to 1/10 use 2 ul of DNA for 100ul reaction ( ng)

31 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 min Primer annealing: 55 1 min 30 cycles Primer extension: 72 1 min Final extension: 72 7 min

32 An example of a PCR method 100 Pre-denature 95 Denature Extend 72 Final-extend Temp. 40 Anneal Cycle 1 Cycle 2 Cycle 30 Soak 4 0 Hold program Cycle program Hold program

33 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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53 Typical result of PCR

54 PCR of rbcl in Scutellaria sp. S. strigillosa 1 S. strigillosa 2 S. pekinensis var. alpina S. indica Isodon inflexsus 1,500bp 1,000bp 500bp

55 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

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