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AP0511 Molecular Biology and Genetics > PCR and Applications > Flashcards

PCR and Applications Flashcards

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1
Q

History of PCR

A
  • 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)
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2
Q

In vivo DNA Replication

A
  • 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
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3
Q

Following synthesis of Okazaki fragments in in vivo DNA replication

A
  • Ribonuclease removes the primer
  • Gap filled by a DNA Pol
  • DNA Ligase binds
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4
Q

Biochemistry of DNA Synthesis (In vivo and In vitro)

A
  • 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
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5
Q

4 deoxy Nucleotide Tri-Phosphates (dNTPs)

A
  • dATP
  • dCTP
  • dGTP
  • dTTP
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6
Q

Reagents in In vitro DNA Replication

A
  • 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+
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7
Q

Conditions in In vitro DNA Replication

A
  • Reaction buffer (correct pH, salt concentration etc.)

- Thermocycler (PCR machine)

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8
Q

DNA synthesis

A
  • 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
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9
Q

Equation for PCR DNA synthesis

A
  • 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
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10
Q

Reagents in PCR

A
  • 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+
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11
Q

Conditions for PCR

A
  • Reaction buffer (correct pH, salt concentration etc.)

- Thermocycler (PCR machine)

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12
Q

A typical protocol for setting up a PCR reaction

A
  • 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
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13
Q

PCR: Primers

A
  • 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
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14
Q

Thermostable DNA polymerases

A
  • 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
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15
Q

𝑦 mL additive for high GC-content targets

A
  • 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
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16
Q

Typical additives

A
  • Dimethyl sulfoxide (DMSO): disadvantage: increases error rate of the PCR reaction
  • Glycerol
  • Proprietary mixes
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17
Q

Thermocycler

A
  • 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
18
Q

95oC for 5-15mins (PCR Temperature program)

A
  • When using whole cells, extra long denaturation is needed to fully open up the cells
19
Q

~55oC for 30-60secs (PCR temperature program)

A
  • 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
20
Q

72oC for >30secs (PCR temperature program)

A
  • 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
21
Q

72oC for 10mins (PCR temperature program)

A
  • Finishes any ‘loose ends’, improving yield of full-length amplicon
22
Q

Advantages of PCR

A
  • 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
23
Q

Disadvantages of PCR

A
  • 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
24
Q

PCR applications

A
  • Hundreds of PCR applications, derivations
  • Perfect for small or valuable samples
  • Quicker than cloning
  • Many specialised techniques are PCR-dependent
  • Diagnostic or preparatory
25
Q

Examples of PCR in Biosciences

A
  • Multiplex PCR: 2-20K reactions in 1 tube
  • Combined Reverse transcription-PCR (RT-PCR) – convert RNA to cDNA product
  • Quantitative real-time PCR: quantify DNA or RNA
  • in situ PCR: detect low copy number RNA in tissue sections
  • Introduction of mutations at specific positions in recombinant proteins
26
Q

Additional Developments in PCR

A
  • dUTP used vs dTTP (also used for labelling probes):
  • use in reaction 1
  • treat reaction 1 with uracil-N-glycosylase (UNG): removal of the uracil base stops amplification of DNA in reaction 2
  • Reuse sample for reaction 2 (different product)
27
Q

Diagnostic PCR

A
  • All contamination must be avoided: from operator, environment and previous PCR reactions
  • Sterile tubes, barrier pipette tips and new reagents used
  • Gloves worn (hairnets, masks)
  • Pre- and Post-PCR: separate areas or rooms
  • Reaction controls critically important
28
Q

Preparatory PCR

A
  • Sterility and isolation: less critical, but may be appropriate
  • Aim is usually high yield of the desired, specific product.
  • Uses: subcloning, blotting, sequencing, labelled and used as probes, expressed in vivo, make products
29
Q

Multiplex PCR (example PCR)

A
  • 2-20,000 simultaneous reactions
  • Similar primer annealing temperatures
  • Products known
  • Different sizes: electrophoresis
  • Different sequences: multiparallel sequencing
30
Q

Reverse Transcription PCR (RT-PCR) (example PCR)

A
  • Allows study of mRNA i.e. expressed gene sequences
  • Uses retroviral reverse transcriptase enzyme - makes cDNA copy of mRNA
  • many different RNAs in cells and tissues
  • rRNA, tRNA and mRNA
  • first make complementary DNA (cDNA) copy of mRNAs using either: gene specific primer, oligo-dT, random hexamer [p(dNTP)6]
31
Q

Priming for RT-PCR

A
  • Reverse transcriptase synthesises a cDNA copy of the mRNA
  • RNAse H used to digest RNA if necessary
  • First PCR cycle synthesises DNA using cDNA strand as template
  • Double-stranded cDNA with sequence of mRNA produced
32
Q

Quantitative PCR (qPCR) (examples PCR)

A
  • dye binds double stranded DNA non-specifically
  • most commonly used is SYBR GREEN.
  • Melt curve analysis is performed at end of reaction involving a dye
  • allowing assessment of the quality and characteristics of the resulting amplicon
  • Optimally, only the targeted region of a template is amplified during qPCR, resulting in a specific product
  • Quantitative assuming exponential amplification, single endpoint measurement
33
Q

Real-time qPCR (example PCR)

A
  • During strand synthesis, the fluorescent reporter is removed using polymerase 5’-3’ exonuclease activity
  • Removal from probe/quencher facilitates the measurement of fluorescence
  • Essentially synthesis of every dsDNA fragment equates to 1 fluorescent probe to be ‘unquenched’ - the fluorescence intensity is directly proportional to the number of dsDNA molecules formed during the PCR making it ‘Real-time’
34
Q

Quantitative Real-time PCR

A
  • Fluorescence is proportional to PCR product
  • Requires dedicated instrumentation that can measure fluorescence and ‘thermocycle’ to drive PCR reaction - excitation of fluorescence needed
  • 96-1536 well microtitre plates allow high sample throughput
  • > 1 colours allow >1 reactions (m-plex)
35
Q

Absolute Quantification

A
  • measured by comparison to samples of known DNA concentrations
  • Production of a standard curve
  • Used for determination of actual transcript number (the gene expression level)
36
Q

Relative Quantification

A
  • Use a reference gene to identify the difference between multiple treatments
  • Typically compared to a housekeeping gene
  • Used to identify the relative change in gene expression
37
Q

Site-directed Mutagenesis using PCR

A
  • Site-directed mutagenesis: mutate specific residues in a protein by changing coding sequences of a recombinant protein
  • Why?: study the role of individual amino acids in protein function
  • How can this be done?: PCR with mutagenic primers
38
Q

Normal cloning PCR for creating protein overexpression construct
(self-directed mutagenesis by PCR)

A
  • Forward (F) and reverse (R) primers used to result in DNA fragment that is the exact copy of a certain DNA region with flanking restriction sites for cloning into an expression vector
39
Q

Site-directed mutagenesis by PCR, step 1

A
  • Perform two separate PCR reactions (A and B) to introduce a mutation into a fixed position in the gene using a sense (mS) and anti-sense (mAS) mutagenic primer
  • Combination of mS and mAS primers with respective reverse (R) and forward (F) cloning primers you can produce PCR products A and B
40
Q

Site-directed mutagenesis by PCR, step 2

A
  • Combine fragments A and B (both containing mutation) with forward and reverse primers
  • Purify the resultant PCR product AB with mutation in exactly the correct position and ligate into the correct expression vector (as with ‘normal’ cloning)
41
Q

Site-directed mutagenesis: Primers

A
  • mS sequence is created by making a copy of coding strand and ‘mutating’ the sequence to make sure the desired mutation is created
  • mAS sequence is simply the reverse complement of mS

AP0511 Molecular Biology and Genetics (11 decks)

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