PCR and Applications

• Devised by Kary Mullis et al, Cetus Corporation (~1985) • Awarded Nobel prize for chemistry 1993 • Based on DNA polymerase-catalysed DNA synthesis originally using Klenow DNApol • In essence PCR is in vitro DNA cloning • Adapted principles of Khorana HG (first to synthesise artificial gene)

• leading strand template 5’-3’ • lagging strand template 3’-5’ • leading strand synthesis - requires 1 priming event • lagging strand synthesis - each Okazaki fragment requires a separate primer • DNA Topoisomerase: relieves stress of twisting downstream of replication fork • Single-strand binding (SSB) protein: binds to and stabilizes unpaired DNA strands

• Ribonuclease removes the primer • Gap filled by a DNA Pol • DNA Ligase binds

• Biochemical DNA synthesis requires a primer, i.e. a short stretch of RNA/DNA with a free 3’-OH • Biochemical DNA synthesis is always based on a template • Biochemical DNA synthesis occurs at the 3’-end of a growing DNA chain • Incoming deoxy Nucleotide Tri-Phosphates (dNTPs) required as building blocks • Catalysed by DNA polymerase

• dATP • dCTP • dGTP • dTTP

• Template material (genomic or plasmid DNA, synthetic DNA fragments, whole cells) • 2 oligonucleotide primers (forward and reverse) • DNA polymerase (thermostable): thermus aquaticus (Taq): (slightly) error prone PCR, e.g. Pyrococcus furiosus (Pfu) - high fidelity (3’-5’ exonuclease) • 4 deoxyribonucleotides: dATP, dCTP, dGTP and dTTP (dNTPs) • Cofactor: Mg2+

• Reaction buffer (correct pH, salt concentration etc.) - Thermocycler (PCR machine)

• Primer anneals to template strand • DNA polymerase binds to primed template • dNTPs are used to synthesise new complementary strand • After 1 denaturation-annealing-elongation cycle, number of DNA molecules has doubled • total number of DNA molecules doubles every time a full denaturation-annealing-elongation cycle is completed

• 𝑁_𝐷𝑁𝐴𝑚𝑜𝑙 (𝑥)=𝑁_𝑡𝑒𝑚𝑝𝑙𝑚𝑜𝑙×2^𝑥 • 𝑁_𝑡𝑒𝑚𝑝𝑙𝑚𝑜𝑙: number of template DNA molecules • 𝑁_𝐷𝑁𝐴𝑚𝑜𝑙 (𝑥): number of DNA molecules after 𝑥 PCR cycles

• Template material (genomic or plasmid DNA, synthetic DNA fragments, whole cells) • 2 oligonucleotide primers (forward and reverse) • DNA polymerase (thermostable): thermus aquaticus (Taq): (slightly) error prone PCR, e.g. Pyrococcus furiosus (Pfu) - high fidelity (3’-5’ exonuclease) • 4 deoxyribonucleotides: dATP, dCTP, dGTP and dTTP (dNTPs) • Cofactor: Mg2+

• Reaction buffer (correct pH, salt concentration etc.) - Thermocycler (PCR machine)

• 𝑥 mL template material (at least 104-107 copies of the amplicon) • 2mL 10mM forward primer • 2mL 10mM reverse primer • 1mL 10mM dNTPs each • 10mL 5× reaction buffer • 1mL 5UmL-1 Thermostable DNA polymerase • 𝑦 mL additive for high GC-content targets • 34-𝑥-𝑦 mL Ultrapure H2O

• Single-stranded synthetic DNA oligonucleotides • Designed to complement unique sequences of template DNA either side of the target sequence • 18-20 nt and designed to be specifically binding to the target region • Midpoint melting temperature (Tm) determined by length and strength of basepairing (higher GC-content means higher Tm all else being equal) and applies to region that actually matches the target • Annealing temperature: 5°C below Tm • Balance specificity and sufficient annealing required

• needed for efficient PCR to prevent enzyme inactivation during each denaturation (95 oC)-annealing-extension cycle • Use enzymes from thermophilic organisms • Thermus aquaticus (Taq): isolated from hot springs e.g. Yellowstone (USA) • Pyrococcus furiosus (Pfu): name roughly translates as ‘rushing fireball’, discovered in Geothermally heated marine sediments near Vulcano Island just north of the Sicilian coast

• Template material with high GC content (>60%) can be difficult to denature completely, in particular genomic DNA, which is the dominant template material during the initial cycles • Formation of GC-rich hairpins may dislodge DNA polymerase • Additional optimizations: touch-down PCR (start from a high annealing temperature), some DNA polymerases deal with high GC targets better than others

• Dimethyl sulfoxide (DMSO): disadvantage: increases error rate of the PCR reaction • Glycerol • Proprietary mixes

• Reliable temperature control • Heated lid to prevent condensation on the lid due to evaporation • Fast cooling and heating • Use thin-walled small volume reaction vials (0.1-0.2 mL) for effective and fast transfer of heat • Programmable

• When using whole cells, extra long denaturation is needed to fully open up the cells

• Actual temperature depends on primers • Touchdown PCR can be used to improve yield with high GC amplicons: start at 68oC and lower annealing temperature 0.5oC every cycle

• Extension time depends on DNA polymerase speed and amplicon length • Taq DNApol: 30 sec/kb, Pfu DNApol: 2 min/kb. For amplification of a 1.5 kb amplicon extension times of 45 sec and 3 minutes respectively would be used • Minimal of 30 sec is recommended by most suppliers

• Finishes any ‘loose ends’, improving yield of full-length amplicon

• Extremely sensitive: 10^9-fold amplification of target sequence, highly specific with optimal primers and optimal annealing temperature • Pure DNA is not needed (e.g. colony PCR, whole cell suspension) • High throughput compatible (robotics) • Fast

• Target sequence must be known: less and less of an issue in the modern next generation sequencing era • Extremely sensitive: risk of contamination, in particular in medical diagnostics • Possible artifacts (although tools to minimize effects are available): primer mis-annealing (multiple PCR products), primer dimers

• Hundreds of PCR applications, derivations • Perfect for small or valuable samples • Quicker than cloning • Many specialised techniques are PCR-dependent • Diagnostic or preparatory