PCR Flashcards
Define PCR and summarise the processes.
PCR is a method by which nucleic acid may be amplified. It is performed by:
- Denaturation of the target DNA
- Annealing of specific primers
- Extension of primers using a thermostable DNA polymerase enzyme from the bacterial species Thermus aquaticus (known as Taq DNA polymerase)
What are the most common uses of PCR?
- When there is insufficient nucleic acid within a sample for reliable detection via direct hybridisation, e.g. dot-blot
- If other methods of aetiological diagnosis are unsuitable (eg direct isolation of organisms, serology and diagnostic identification of antibodies in the serum)
- When a rapid result is desired
- If a large amount of the nucleic acid is required for analytical purposes
- Currently there is no way to amplify proteins. This techniques allows you to start from very low levels and make as many as you like.
What are the three main stages in PCR?
- Strand Separation:
Heat dsDNA to 95°C for 15s to melt and separate the strands, denaturing the hydrogen bonds which hold them together. This denaturation leads to two target single stranded DNA. - Hybridisation of Primer:
Can be specific or non-specific but is usually specific. Involves cooling to 50 - 65°C to allow primers to anneal to the DNA strands. The temperature required at this stage is primer dependant,based on things such as primer stringency. Binding and reading of primer occurs 5’ to 3’. - DNA Synthesis
Heat to 72°C to allow elongation. This extension is usually performed by free nucleotides with taq polymerase.
Typically these steps are performed in a thermocycler and repeated in a cycle about 20 to 30 times generating a large amount of identical DNA copies. Only at stage 2 you get new bits of product, of determined size and this isn’t until the 3rd or 4th cycle.
What reaction components are required for PCR?
- Target DNA/RNA (RNA must first be made ds by reverse transcription (reverse transcriptase PCR - rtPCR), where a cDNA copy is synthesized via incubation of the target sequence with reverse transcriptase, an appropriate primer and dNTPs)
- Specific/ non-specific primers (100nM, up to 95% may be unused after 30 cycles)
- Deoxynucleotides (dNTPs) - terminate reaction at specific terminal lengths (200μM - can be modified, 7-deaza-dGTP, dUTP, And a label (e.g. biotin, digoxygenin) - modify for easy detection).
- Taq DNA polymerase (can have 5’ exonuclease activity but removes incorrect incorporation) - originally, DNA polymerase was added to the PCR reaction but it was denatured by the high temperatures, so had to be added at every cycle. Pfu (Pyrococcus furiosus) DNA Polymerase can also be used as it has better thermostability than Taq polymerase and it possesses 3’ to 5’ proof reading activity. Pfu is from an organism of the archaea that lives in submarine vents, the enzyme survives 100°C
Buffer - makes everything physiologically active (eg; Tris-HCl - dipolar ionic buffer, pH varies during PCR, higher fidelity at lower pH; MgCl2 - Taq requires 1.2-1.3mM Mg2+; KCl - assists primer annealing; Gelatin - stabilises enzyme; Denaturing agents (alters Tm) - DMSO, formamide, glycerol)
How are primers designed?
OBTAIN NUCLEIC ACID SEQUENCE ↓ SELECT GENE ↓ SELECT PRIMER ANNEALING SITES ↓ SELECT PRIMER SEQUENCES ↓ ASSESS HOMOLOGY ↓ TEST (best test is to repeat and see if you get the same single product).”
How are primers selected?
There are online tools available for design.
- Primers are 15 - 30 bases to increase the chances of being a unique sequence.
- They have a balance of bases (A,C,G,T) involving no base repetition of more than 3-4 (AAAA, CCCC, etc), was this could lead to looping out, mismatching and low annealing.
- thee complementarity is regulated as you don’t want primers binding to themselves, I.e must not be not self-complementary, complementary to each other, not complementarity at 3’ end, no runs of ≥ 3 G or C at 3’ end and must avoid T at 3’ end.
- it is possible to incorporate secondary structures (e.g. hairpin loop formation).
- Under special circumstances it is advantages to incorporate degeneracy - sometimes don’t want primers to match specifically as not every genome has been sequenced.
In what ways can you optimise the process of primer annealing?
Design primers with similar Tm (melting temp) as annealing normally occurs 5 deg C below Tm.
Melting temp can be calculated for primer designs, eg a simplified formula is:
Tm = 2°C x (A+T) + 4°C x (G+C), as there are 3 H binds for CG but only 2 for AT.
Why is PCR at high risk of contamination and how do you minimise this?
PCR is capable of detecting a single copy of the target sequence so there is a very high risk of false positive results from contaminant DNA.
To avoid this involves essential scrupulous cleaning of both equipment and laboratories.
Controlled by isolation of pre- and post-PCR procedures
- dedicated equipment
- dedicated rooms or work areas
- particularly for diagnostic purposes
A minimum of 3 rooms or work areas should be used:
1. DNA-free
2. Sample preparation
3. Thermal cycling and detection
Rooms 1 and 2 are normally UV-irradiated between uses and surfaces depurinated.
Negative controls are important to detect contamination if it occurs
- during extraction
- during PCR
Positive controls also required for quality control of processes.
What is reverse-transcriptase PCR?
- for single-stranded targets (RNA/DNA) to produce cDNA from the target sequence using reverse transcriptase.
What is hot-start PCR?
- warm all reagents to 94oC before addition of Taq polymerase
- add Taq polymerase in wax pellet
- use heat-activated Taq polymerase
Hot Start PCR significantly reduces nonspecific priming, the formation of primer dimers, and often, increases product yields.
Wax pellet is used so that it melts. Used when PCR isn’t working as well, this will increase specificity of product and reduce contamination.
What is nested PCR?
2 rounds of amplification:
- 1st round produces large amplicon (means product)
- 2nd round amplifies fragment within 1st round amplicon
Intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. Also increases the selectivity of the PCR product.
What is multiplex PCR?
- amplification of several sequences in one reaction using multiple primer pairs
- useful for single-step differential diagnosis
What is touchdown PCR?
- change the annealing temperature by 1oC every cycle for the first 10 cycles, then apply a constant annealing temperature.
- enhances specificity
- avoids amplifying nonspecific sequences
- increases specificity of the reaction at higher temperatures and increases the efficiency towards the end by lowering the annealing temperature.
What are the methods of post-PCR detection?
- Electrophoresis
- agarose
- polyacrylamide
Though this defines the size of any amplified products but does not confirm the identity.
Identity confirmation performed via hybridisation:
- Southern blot hybridisation
- Dot blot
- HPLC
- ELISA
What is real-time PCR (qPCR)?
THE Q STANDS FOR QUANTITATION
Rapid cycling in small volume format
- 1 cycle in 30-60 seconds
- ∴ 30 cycles in 15-30 minutes
Rapid detection of fluorescent dye during cycling
- no need for electrophoresis
- confirmation of product with specific probe
Relative/absolute quantitation
- standard curve from known concentration of target DNA
- compare with unknown sample - housekeeping gene.
Non-specific/specific platforms
- SYBR Green - non-specific
- Hybridisation
