PCR

Question 1

Define PCR and summarise the processes.

Answer 1

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)

Question 2

What are the most common uses of PCR?

Answer 2

• 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.

Question 3

What are the three main stages in PCR?

Answer 3

1. 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.
2. 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’.
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.

Question 4

What reaction components are required for PCR?

Answer 4

• 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)

Question 5

How are primers designed?

Answer 5

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).”

Question 6

How are primers selected?

Answer 6

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.

Question 7

In what ways can you optimise the process of primer annealing?

Answer 7

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.

Question 8

Why is PCR at high risk of contamination and how do you minimise this?

Answer 8

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.

Question 9

What is reverse-transcriptase PCR?

Answer 9

• for single-stranded targets (RNA/DNA) to produce cDNA from the target sequence using reverse transcriptase.

Question 10

What is hot-start PCR?

Answer 10

• 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.

Question 11

What is nested PCR?

Answer 11

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.

Question 12

What is multiplex PCR?

Answer 12

• amplification of several sequences in one reaction using multiple primer pairs
• useful for single-step differential diagnosis

Question 13

What is touchdown PCR?

Answer 13

• 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.

Question 14

What are the methods of post-PCR detection?

Answer 14

• 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

Question 15

What is real-time PCR (qPCR)?

Answer 15

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

Question 16

What are the advantages of using qPCR?

Answer 16

Traditional PCR is measured at End-Point (plateau), while Real-Time PCR collects data in the exponential growth phase

• An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated
• Increased dynamic range of detection
• No-post PCR processing
• Can detect even a 2-fold change in initial loading
• Is automated so has all the advantages of automation.

Question 17

How is quantitation performed for real-time PCR?

Answer 17

The threshold line, which is drawn on the fluorescence vs cycle number graph, is the level of detection. Meaning it is the point at which the reaction reaches a fluorescent intensity above background.
The Cycle Threshold (CT) is the cycle at which the sample reaches this level.

Question 18

How does SYBR green work for qPCR?

Answer 18

SYBR green is a fluorescent dye that binds to the minor groove of DNA (DOUBLE STRANDED ONLY). This means it will bind very weakly before the after denaturation and before the amplification has occurred, producing only a very weak signal.
After the three stages of PCR have been performed it gives a strong fluorescence to to all the replicated strands of new dsDNA.
In this way we can see that the PCR product is amplified over time.

This method of quantitation is equivalent to electrophoresis.

The advantages to SYBR green are that it is cheap and doesn’t require probe design. However it can bind unspecifically to different regions of the DNA. This leads to false positives with non-specific proteins. Plus it is not attuned for complex protocols.

In melting curve analysis there is a mismatch that reduces Tm.

Question 19

What is the Taqman assay?

Answer 19

This is an alternative to SYBR green.

It exploits the 5’ nuclease activity of Taq DNA polymerase using an oligonucleotide probe with a:

• Fluorescent reporter at 5’ end
• Quencher at 3’ end

The probe anneals downstream from primer site if the target is present and extension begins as usual.
Once the enzyme reaches the probe (at the reporter 5’ end) the 5’ nuclease activity of the enzyme begins to displace the probe.
Cleavage of the probe begins and the Reporter starts to fluoresce as it is separated from Quencher.
When the Reporter dye is cleaved and released, there is an increase in fluorescence intensity proportional to amount of amplicon produced.

Question 20

How does Fluorescence Resonance Energy Transfer (FRET) Hybridisation occur?

Answer 20

FRET uses 2 adjacent probes 1-5 bases apart

• one labelled 3’ fluorescein (green)
• one labelled 5’ red marker (red)

fluorescein is excited by a light source 
↓
emits energy at longer wavelength
↓
This can then excite the red marker 
↓
Which emits energy at an even longer wavelength 
↓
 This leads to intense fluorescence

Question 21

What are beacons?

Answer 21

Beacons are hairpin probes with a reporter and a quencher forced adjacent to each other by the hairpin. The hairpin portion off the beacon is complementary (like a primer) to the DNA strand. This means that upon annealing to the target sequence the hairpin is unwrapped, pulling the Q and R away from each other. This causes the reporter to fluoresce.

Question 22

Describe how scorpion primers work.

Answer 22

Combine primer and probe in one molecule (primer sequence at 3’ end) and hairpin probe at the 5’ end). Similar to beacons the R and Q are in close proximity. Complementary primer binds at low temp (55 degs), dna then extends using taq polymerase. The strands are separated again and the final step involves heating back up to 90 degs causing the scorpion probe to unfold. Lowering the temp than causes the complementary section of probe to anneal to the newly replicated strand. This prevents the hairpin loop from reforming and causes fluorescence of the R.

Primer extends to become part of the amplicon
Intermolecular hybridisation is kinetically favoured
Signal to background ratio high
Design more complex
Relatively expensive

EXTRA UNRELATED: Following the first stages of denaturation, annealing and extension, the second stage involves a forward primer annealing to the target strand of DNA and Taq polymerase extends it. A reverse primer and a 5’ nuclease probe then bind to this newly replicated single strand. During extensions the polymerase cleaves the probe from the strand - the reporter is no longe quenched by the BHQ so begins to fluoresce.

Question 23

What are the applications of PCR?

Answer 23

Medical applications:

• Genetic testing (a sample of DNA is analysed for the presence of genetic disease mutations; preimplantation genetic diagnosis).
• Tissue typing prior to organ transplantation

Forensic applications

• Genetic fingerprinting
• DNA paternity testing

Infectious disease applications:

• Tuberculosis
• Human immunodeficiency virus (HIV) - Considered most sensitive and specific for diagnosis of HIV. Methods for HIV-1 DNA and RNA:
  
  • DNA test: Can confirm exposure to virus before antibody test becomes positive.
  • RNA test: useful in advanced disease as viral RNA is a strong indicator of viral replication. Can help prognosis and monitoring therapy