Quantitative real time PCR and detection

Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. it is used to identify (qualification) and amplify a sequence of DNA. Real time PCR is done also for quantification use and for other aims:

• Quantification of gene expression levels

• Quantification of DNA (Telomere assays, mtDNA content, copy number)

• Virus titre (quantification of very low numbers)

• Bacteria/protozoa identification and antibiotic resistance

• Can be used as a quantitative step as part of another technique e.g. ChIP and DNA Methylation

Workflow

1. Preparation of the sample with homogenization, weighting, or enrichment (putting the sample in an enrichment culture and allowing it to grow). The sample needs to be representative of the medium it comes from.

2. Reaction mix.

   1. Primers specific to target: 18–24bp, produce product 50 – 150 bp in length (200bp does work), product ~50% GC and avoid GC stretches and repetitive sequences, check for primer dimers especially for SYBR green assays, F and R primers annealing temp within 5˚C, in separate exons or spanning exon boundaries

   2. Probes: Pre-designed assay available (TaqMan probes or sybr based assays)

   3. Template DNA: 50-100ng/uL

   4. Reaction buffer (Tris HCl, ammonium ions, KCl, Mg ions): provides the ionic strength and buffering capacity for the reaction

   5. MgCl2

   6. dNTPs in equimolar ratios

   7. DNA polymerase: 1/2 unit/25 uL reaction

Primer, probe, dNTPS, buffer, Mg2. The probe is needed for the rt pcr. The probe links to a complementary region near to the primer and is made of a fluorophore and a quencher, that absorbs the energy from the fluorophore, not permitting its fluorescence (there is no signal). After the annealing on the DNA, during the extension the DNA polymerase progresses until the probe is cut (if the probe is hydrolytic) by it thanks to its exonuclease activity. The probe is detached from the quencher and the fluorophore and the signal is seen. The detacher probe simply detaches from the DNA and remains intact while making a signal.

1. PCR setup. Reaction mix is added with the Taq polymerase equals to master mix. Depending on the application, a proofreading Taq polymerase is used (ex. if you need the exact copy of the gene). Different polymerases amplify at around 78°C but some work at 65°C, some are most indicated to amplify longer or shorter fragments, work best with different GC% (most work best with 50/50 GC, but for example, plasmodium has a 85% GC, so we need specific polymerases).

   1. denaturation at 95°C, permitting the opening of the elix. It lasts a few seconds.

   2. annealing at 65°C of the primers forward and reverse to the region that is going to be amplified. Primers should be at least 18 nucleotides long (the lenght makes it more specific). It lasts 30 seconds at most.

   3. extension at 72°C, the polymerase builds the remaining of the sequence next to the primers. It lasts 1 minute for 1kb.

This is repeated n times, forming a thermal cycling profile, usually around 40 times. Sometimes we make a final extension in 5-10 minutes to allow the DNA polymerase to amplify the last undone sequences. To avoid mutations we must use a proofreading polymerase and also lower the number of cycles done. This is an end point technology, so we look at the result only in the end. Electrophoresis is done to visualize the result of PCR. What’s Wrong With Agarose Gels?

• Low sensitivity

• Low resolution

• Non-automated

• Size-based discrimination only

• Results are not expressed as numbers, so it is based on personal evaluation

• Ethidium bromide staining is not very quantitative and toxic

In RT PCR, along with this process we also have quantification. The sample presents a fluorescent probe that permits the monitoring of every cycle (so it’s not end point) thanks to a detector (quantifies the fluorescence and so the DNA). After a n number of cycles we have a linear fit between the fluorescence of the probe and the DNA quantity.

If the primers link to different portions of the DNA or a gene is present in multiple copies → two bands will appear. If the DNA template is degraded (so the primers couldn’t link and amplify) → a smear is obtained. If the same sample is injected multiple times but the bands obtained have different intensities → the amplified material differs in abundancy because during the PCR the template and the result are not proportional in quantity.

We can also amplify more genes (until 50 genes at the same time) with the multiplex real time PCR, in this case is more appropriate to use more fluorophores and quenchers (even combining them) for each gene, so every reporting system has a different emission color. SYBR green qPCR is cheaper and binds to non specific PCR products and to primer dimers, is more stable and gives higher resolution melting curves. Is mostly used in diagnostics for pathogens and is frequently used for gene analysis.

SYBR green

Probe

• Dye binds minor groove of all DNA

• Dye and quencher either end of probe

• Fluorescence much stronger when bound

• Nuclease activity of polymerase removes dye and quencher during PCR

• More product more dye bound

• More product produced, more fluorescence

• Cheap, not specific

• No melt curve

• At early stage check product on gel and perform dissociation analysis (melt curve)

• Allows multiplexing

Quantification methods

1. Absolute quantification. Samples calculated against a standard curve of known concentration. Allows quantification as copy number, takes some variability into account, requires more reagents and a known standard. Plasmid DNA containing product, no RT step so efficiencies may be different, in-vitro transcription to generate specific mRNA – RT, costly. What is a standard curve and why is it necessary? Real time PCR conducted across a series of serially diluted template/samples tells you what the dynamic (linear) range of the assay is and allows calculation of efficiency.

1. Relative quantification. Comparative quantification between the gene of interest and an housekeeping gene. Housekeeping gene(s). Accounts for pipetting error and differences in RT efficiencies between samples, constantly expressed at a stable level. Put the same starting RNA in all RT-reactions when samples are to be compared, but doesn’t take account for massive differences in starting material.

Probe types and design

When a population of fluorochrome molecules is excited by light of an appropriate wavelength, fluorescence is emitted. The light intensity can be measured by fluorometer or a pixel-by-pixel digital image of the sample. FRET (fluorescence resonance energy transfer) is a distance dependent interaction between the excited states of 2 dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.

In TaqMan Probes (hydrolysis probes) when intact, the fluorescence of the reporter is quenched due to its proximity to the quencher. Probe hybridizes to the target, dsDNA-specific 5'->3' exonuclease activity of Taq or Tth cleaves off the reporter. Reporter is separated from the quencher → Fluorescent signal proportional to the amount of amplified product in the sample.

Advantages

Disadvantages

• Highly fluorogenic

• Expensive

• Easy PCR setup

• Probe design and positioning challenging

• Sequence-specific detection, multiplexing

• Similar conditions for primers and probes

• Elevated background (Quenching capacity)

• Probe degraded: no end-point analysis

Molecular Beacons are hairpin probes composed of a (25-40 nt) nucleotide base paired stem and a target specific nucleotide loop. The loop consists of target specific nucleotide (probe) sequences (15-30 nt). A fluorescent moiety (reporter) is attached to 5' end and a quencher moiety is attached to 3'end. The stem keeps both the moieties in close proximity so that fluorescence is quenched.

Operation of Molecular Beacon (MB): MB is non-fluorescent due to close proximity of the non- fluorescent quencher (Q) and the fluorescent Reporter. The probe denatures and the loop anneals to the target sequence of the amplicon. Separating the quencher from the fluorophore and thereby producing fluorescence which is proportional to the amplicons produced during PCR. MB is displaced not destroyed during amplification, because a DNA polymerase lacking 5' exonuclease activity is used. Molecular beacons do not fluoresce when they are free in solution. However, when they hybridize to a nucleic acid strand containing a target sequence they undergo a conformational change that enables them to fluoresce brightly.

Advantages

Disadvantages

• High specificity, low background

• Challenging design

• Post PCR analysis

• Long probes - less yield

• PCR multiplex

• Intramolecular competitive binding

• Allelic discrimination (greater specificity than linear probes)

• Low signal levels (proximity of reporter and quencher)

The loop of the Scorpions probe includes a sequence that is complementary to an internal portion of the sequence it primes. During the first amplification cycle, the Scorpions primer is extended, and the sequence complementary to the loop sequence is generated. After subsequent denaturation and annealing, the loop of the Scorpions probe hybridizes to the internal target sequence, and the reporter is separated from the quencher. The resulting fluorescent signal is proportional to the amount of amplified product in the sample. The Scorpions probe contains a PCR blocker just 3' of the quencher to prevent read-through during the extension of the opposite strand.

Hybridization Probes. These assays use two sequence-specific oligonucleotide probes in addition to two sequence specific primers. The two probes are designed to bind to adjacent sequences in the target. The probes are labeled with a pair of dyes that can engage in FRET. The donor dye is attached to the 3′ end of the first probe, while the acceptor dye is attached to the 5' end of the second probe. During real-time PCR, excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. At the annealing step, the probes hybridize to their target sequences in a head-to-tail arrangement. This brings the donor and acceptor dyes into proximity, allowing FRET to occur. The increase in PCR product is proportional to amount of fluorescence

Advantages

Disadvantages

• Probe with only one fluorophore

• Strict compatibility between donor & acceptor fluorophores

• Easy synthesis and quality controls

• Reduced background fluorescence

• High specificity