PCR and Applications Flashcards
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)
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
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
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
5
Q
4 deoxy Nucleotide Tri-Phosphates (dNTPs)
A
- dATP
- dCTP
- dGTP
- dTTP
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+
7
Q
Conditions in In vitro DNA Replication
A
- Reaction buffer (correct pH, salt concentration etc.)
- Thermocycler (PCR machine)
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
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
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+
11
Q
Conditions for PCR
A
- Reaction buffer (correct pH, salt concentration etc.)
- Thermocycler (PCR machine)
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
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
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
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
16
Q
Typical additives
A
- Dimethyl sulfoxide (DMSO): disadvantage: increases error rate of the PCR reaction
- Glycerol
- Proprietary mixes
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
Examples of PCR in Biosciences
- 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
Additional Developments in PCR
- 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
Diagnostic PCR
- 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
Preparatory PCR
- 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
Multiplex PCR (example PCR)
- 2-20,000 simultaneous reactions
- Similar primer annealing temperatures
- Products known
- Different sizes: electrophoresis
- Different sequences: multiparallel sequencing
30
Reverse Transcription PCR (RT-PCR) (example PCR)
- 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
Priming for RT-PCR
- 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
Quantitative PCR (qPCR) (examples PCR)
- 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
Real-time qPCR (example PCR)
- 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
Quantitative Real-time PCR
- 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
Absolute Quantification
- 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
Relative Quantification
- 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
Site-directed Mutagenesis using PCR
- 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
Normal cloning PCR for creating protein overexpression construct
(self-directed mutagenesis by PCR)
- 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
Site-directed mutagenesis by PCR, step 1
- 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
Site-directed mutagenesis by PCR, step 2
- 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
Site-directed mutagenesis: Primers
- 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
