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Taq DNA Polymerase

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Taq DNA Polymerase Polymerase Chain Reaction (PCR) • PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested. • As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in different samples. Polymerase Chain Reaction (PCR) • PCR carry out for – DNA cloning – sequencing, – DNA-based phylogeny, or – functional analysis of genes; – the diagnosis of diseases; – genetic fingerprints and – the detection and diagnosis of infectious diseases. PROCEDURE ….. PCR Reagents • Template DNA (e.g., plasmid DNA, genomic DNA). • Forward and reverse PCR primers. • MgCl2 (25 mM). • dNTPs (a mixture of 2.5 mM dATP, dCTP, dGTP, and dTTP). • PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 25°C. PCR Reagents • Thermal stable DNA polymerase – DNA polymerases without 3′ → 5′ proofreading activity • Taq DNA polymerase • Tth DNA polymerase • Tfl DNA polymerase – DNA polymerases with 3′ → 5′ proofreading activity: • Pwo DNA polymerase • Pfu DNA polymerase • Tli DNA polymerase • Vent DNA polymerase PCR Reagents • Optional: PCR additives /cosolvents (optional; e.g., • betaine, glycerol, DMSO, formamide, bovine serum albumin, ammonium sulfate, polyethylene glycol, gelatin, Tween-20, Triton X-100, β-mercaptoethanol, or tetramethylammonium chloride). Polymerase Chain Reaction (PCR) • Initialization step: 94–96 °C (or 98 °C if extremely thermostable polymerases are used), for 1–9 minutes. – the complete separation of the DNA strands and unfolding secondary structures – This stage also required for DNA polymerases that require heat activation by hot-start PCR. Polymerase Chain Reaction (PCR) • Denaturation step: 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. – it is appropriate to use a higher denaturation temperature and a longer incubation time for some templates, such as those templates with high GC content, to achieve complete denaturation for more complete denaturation of the DNA template • Although a higher temperature and a longer incubation time reduce lifetime of enzyme. Polymerase Chain Reaction (PCR) • Annealing step: 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3–5 °C below the Tm of the primers used. • Extension/elongation step (72 °C): The temperature at this step depends on the DNA polymerase used; – Taq polymerase has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme. Polymerase Chain Reaction (PCR) • In elongation step, – the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, – banding the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand (Phosphodiester band). – Primer extension time depends on the length and concentration of the target sequence, as well as the extension temperature. Extending the Chain DNA elongation DNA elongation DNA polymerases synthesize DNA using 5’dNTP substrates, a DNA template, and elongate the chain at the 3’ end: Base1 Base 2 Base1 Base2 + OH PPP 3’ OH PPP OH PPP OPO 5’ B1 B2 B3 B4 B5 B6 B7 … etc… PPP OH Polymerase Chain Reaction (PCR) • In elongation step, – Taq DNA polymerase extends at a rate of 0.25 nucleotides per second at 22°C, 1.5 nucleotides per second at 37°C, 24 nucleotides per second at 55°C, greater than 60 nucleotides per second at 70°C, and 150 nucleotides per second at 75 to 80°C. – at 72°C, Taq DNA polymerase is expected to extend at the rate of greater than 3500 nucleotides per minute. – Thus, in common, an extension time of 1 min per kilobase is more than sufficient to generate the expected PCR product. Polymerase Chain Reaction Polymerase Chain Reaction (PCR) • Final elongation: – This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended • (A tail for cloning). Polymerase Chain Reaction (PCR) • Two-step cycling programs are generally applied when a high annealing temperature is used, such as 65 to 70°C. • Because a higher annealing temperature improves amplification specificity, it is argued by some investigators that better PCR results may be obtained using a two-step cycling program . Polymerase Chain Reaction (PCR) • In long PCR, – Two-step cycling programs are more frequently used than three-step cycling programs. – For example, denaturation at 92 to 95°C for 10 to 30 s, followed by annealing and extension at 65 to 68°C for 1 min per kilobase will increase the probability of obtaining the desired product. Melting PCR 100 94 oC 50 Temperature 0 T i m e 3’ 5’ 5’ 3’ Melting PCR 100 94 oC 50 Temperature 0 T i m e 3’ 5’ Heat 5’ 3’ Melting PCR 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C 50 50 oC Temperature 0 T i m e 3’ 5’ 5’ 5’ 5’ 3’ Temperature control in a PCR thermocycler Temperature 0C 94 0C - denaturation 50 – 70 0C - primer annealing 72 0C - primer extension 94 0C - denaturation Melting PCR 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C 50 50 oC Temperature 0 T i m e 3’ 5’ Heat 5’ 5’ Heat 5’ 5’ 3’ Melting PCR 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C 50 50 oC Temperature 0 T i m e 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ Melting PCR 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C 50 50 oC Temperature 0 3’ 5’ 5’ T i m e 5’ 5’ 5’ 3’ Heat 5’ 5’ Heat 5’ Melting PCR 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C 50 50 oC Temperature 0 3’ 5’ 5’ T i m e 5’ 5’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ PCR Melting 30x 100 Melting 94 oC 94 oC Annealing Extension o Primers 72 C 50 50 oC Temperature 0 3’ 5’ 5’ T i m e 5’ 5’ 5’ 5’ 3’ 5’ Fragments of 5’ defined length 5’ 5’ 5’ 5’ PCR Round 1 target DNA 5' 3' Double-stranded DNA 3' 5' 5' 3' Denaturation 3' 5' 5' 3' Primer annealing 3' 5' 5' 5' 3' 3' 5' 5' 3' 3' Extension 5' 5' 3' 3' 3' 3' 5' 5' repeat PCR cycles DNA polymerase always adds nucleotides to the 3’ end of the primer 5' 3' 5' 3' PCR Round 2 5' 3' 3' 5' After the second round of 5' 3' 3' 5' PCR, the number of long denaturation strands increases 5' 3' arithmetically and the 3' 5' number of short strands increases exponentially 5' 3' (the number of chromosomal strands is 3' 5' primer annealing 3' always the same). 5' 3' 5' 5' 3' 5' 3' 3' 5' 5' 3' extension Short strand 3' 5' 5' 3' 3' 5' Long strand Chromosomal strand • The confirmation of PCR products 1. Correct product size (expected-size product) 2. Sequencing the products. 3. Use a gene probe to confirm the product.s 4. Use nested PCR PCR • PCR amplifications can be grouped into three different categories: – standard PCR, – long PCR, and – multiplex PCR. • Standard PCR – amplification of a single DNA sequence that is less than 5 kb (my experience, 2kb) in length and – applications, such as sequencing, cloning, mutation detection, etc. PCR Optimisation • Long PCR is used for – the amplification of a single sequence that is longer than 5 kb (2kb) and up to 40 kb in length. – Its applications include • long-range sequencing; • amplification of complete genes; • molecular cloning; • assembly and production of larger recombinant constructions for PCR-based mutagenesis. Long PCR • Programming an increase in extension time automatically in later cycles may also improve the yields of the amplification. – Forexample: 3M*10s for extension time • increasing the extension time in each of the later PCR cycles could increase the likelihood of synthesizing long PCR products. PCR Optimisation • Multiplex PCR, – is used for the amplification of multiple sequences that are less than 5 kb in length. – Its applications include • pathogen identification (detecting in quarantine); • template quantitation; • genetic disease diagnosis; • population genetics. Multiplex-PCR • targeting multiple genes at once with multiple primer sets within a single PCR mixture which produce amplicons of varying sizes that are specific to different DNA sequences. • Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction (very close Tm) • amplicon length should be different enough to form distinct bands when visualized by gel electrophoresis. Multiplex PCR • Use of multiple sets of primers • to detect more than one organism or • to detect multiple genes in one organism. E. Coli Salmonella sp. genome genome or Multiplex PCR • Therefore, Multiplex PCR enabling simultaneous amplification of some genes in one reaction by using more than one pair of primers. PCR Optimization • Optimization of PCR depend on: – (1) Quality and concentration of DNA template; – (2) Design and concentration of primers; – (3) Concentration of magnesium ions; – (4) Concentration of the four deoxynucleotides (dNTPs); – (5) PCR buffer systems; – (6) Selection and concentration of DNA polymerase; – (7) PCR thermal cycling conditions; – (8) Addition and concentrations of PCR additives (enhancements) – (9) Use of the “hot start” technique. PCR Optimization- DNA template • The quality and concentration of DNA templates can directly affect the outcome of PCR amplifications.
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