Quantification

Quantification – a competition of probes against re-annealing of the DNA double strand and primer extension

In order to have a signal from the hybridization probes, they must bind to the single stranded target sequence during the thermal denaturation and shortly thereafter. In the cooling phase the primers compete with probes, but not for the same sequence. Once a primer has annealed it will be immediately extended by the Polymerase, eventually leading to a masking of the probe-binding site. Furthermore, during that cycle this process is irreversible, since an extended primer will be far more stably bound, while the hybridization of the probes is reversible. Therefore, the probes should bind significantly better and have a higher Tm than the primers.

Limits of quantification

  • The detection limit of a DNA PCR lies between 2-10 genome copies. The detection limit is primarily dependent on the selected primers and the target, not so much on the probes
  • The detection limit for RT-PCR is between 10-50 genome copies, for a two-step PCR. A one tube PCR is 10 to 20 fold less sensitive
  • The detection limit is decreased very often by the loss of samples during the extraction or by the dilution in the processing (many extraction kits yield 100-200 µl final sample elution volume)
  • The PCR is limited in the detection of the doubling of a quantity (it corresponds with a shift by one cycle). It is therefore not the method of choice for measuring small transcription variations or trisomies
  • Samples containing low concentrations of DNA should be supplemented with heterologous carrier nucleic acids.
  • Cloned standards (plasmids) should be supplemented with carrier DNA.
  • Lower concentrated target sequences can be inhibited by higher concentrated target sequences in a duplex PCR. The use of high expressed reference genes should be avoided when quantifying low expressed genes.
  • Two variant target sequences cannot be quantified differentially with the same probe set.

The melting temperature correlates with binding strength

The competition between the primer on the same strand and the hybridization probes require a 5 to 10 degrees centigrade higher melting temperature for the latter within the following limitations:

(1) The melting temperature should not be too high, to allow their displacement by the extended strand, otherwise the amplification will be hindered and the PCR inhibited.

(2) The Tm has to be higher than the annealing temperature. Therefore, when using short low-strength binding primers, the Tm of the probes may very well be more than 10 degrees centigrade higher.

Alternatively, the primers can be adapted to fulfill the above criteria. The extension of some bases at 5’ end of the primer will normally not affect its specificity, albeit increase its binding strength. In the absence of further sequence information, the primers can be extended by adding random sequences. During the first cycle a new template containing the new sequence will be created and serve as a template for successive cycles.

The position of the hybridization probes.

In principle, the probes can be designed to bind anywhere on the target. Due to the competition between extended primer and probes it is advantageous to design probes that bind as far away from the extended primer as possible, in other words far "right" on the upper strand or far "left" on the lower strand. The probes, just like the primers, will also bind to related sequences. This is particularly obvious in the case of repetitive elements, where the probe can easily bind to the target shifted by one element. The consequences for false priming of a probe are not a "wrong" (unspecific) signal, rather a lowering of the "free" (available) probe concentration. The result is a lower fluorescence signal. This effect becomes of importance only in the latter cycles of amplification when sufficient target has been amplified. In contrast to primers, probe sequence homologies with sequences outside of the amplified region are not relevant.

Remarks for quantification probes

The fluorescent signal is proportional to the target quantity. Stable binding is essential for the production of a good signal. Since the probes compete with the same strand primer, they have to bind stronger. In late cycles the probes will also have to compete against the re-hybridization of the amplicon, which reduces the fluorescent signal (Hook-Effect).

The probes should bind far from the primer with the same orientation.

The extended primer on the same strand will irreversibly cover the probe-binding site. A greater distance will extend the time in which the binding site will be available.

The Tm of the probes should be 5-10° C higher than the Tm of the primers, or at least the primer on the same strand.

One primer competes with the binding of two probes. With identical binding properties, the primer will always hybridize faster than the two probes.

The Tm of the probes should be higher than the annealing temperature.

The signal has to be obtained during the annealing step.

The Tm of the probes should not be much higher than the extension temperature.

This may cause the probes to block primer extension.

In order to select adequate probes for a given PCR target, the search should begin "far left" on the upper strand and "far right" on the lower strand. In cases of primer/probe incompatibilities, the primers can be shifted by a few bases. When developing new assays, the selection of adequate probe sequences may precede the selection of compatible primers. Two to three primer pairs should be selected and tried in all possible combinations and determine the primer pair with the highest specificity and efficiency empirically. In spite of computer aided sequence selection, the amplification efficiency and the tendency to dimerize remains amazingly difficult to predict.

Structural considerations to probe design

Structural features influence the binding behavior of nucleic acids. The selection of a region with "balanced" sequences, where all four bases are distributed more or less evenly is recommended. Exceptionally strong binding sequence segments within the probe should be avoided. The high thermodynamic properties of GC rich clusters make them susceptible to false priming. Due to shorter sequences necessary for higher melting temperatures the statistical probability of false binding is increased. Repetitive and monotonous sequences do not provide for a defined binding spot, which results in a distribution of probes binding at different positions.

Complementary sequences, for example interrupted palindromes can lead to probe dimerization, causing a reduction of available probe. If the probes show complementarities to each other they, can bind and create a target independent signal. Also very important are self- complementarities in a probe sequence, which will lead to the formation of stem-loops. Already short sequence motifs are sufficient to create such a structure. The binding probability is largely enhanced given the proximity of the sequences. All processes that lead to a false binding or reduce the binding at the wanted position have a negative influence on the fluorescence value. If necessary, these effects can be partially compensated by increasing the probe concentrations.

An important rule is to avoid complementarities between primers and primers and probes at the 3’ end. During the first PCR cycles when the target concentration is extremely low and the primers and probes are highly concentrated, short sequence binding patterns are sufficient to initiate the extension by the Polymerase. The results are unspecific products

General rules

The hybridization probes for the LightCycler® Systems bind in close proximity with the fluorophores facing each other. A FRET energy transfer will only take place when both probes bind simultaneously.

Use adjacent sequences on the same strand allowing for a gap of 1 -5 nucleotides.

FRET is distance dependent. Donor and acceptor dyes have to be in close proximity (less than 50 Å, or less than 15 nucleotides).

The 3’ ends have to be blocked. Usually 3’-phosphate and 3’-fluorescein.

The probes have to be prevented from being extended by the Polymerase.

The facing ends of the probes have to be labeled. Preferentially 3’-fluorescein and 5’-LightCycler® Red.

Inverting the labeling can lead to FRET failure. The 3’ labeling with LC Red is limited. The fluorescein has to be a FITC- derivative, to ensure color compensation to work accurately.

The probes should be "balanced"
similar in composition, base
distribution and Tm.

See remarks below

Monotonous sequences should be
avoided.

Probe may bind at varying positions (slipping).

Repetitive sequences should be
avoided.

Probes can bind at different locations.

GC-rich regions should be
avoided.

Shorter sequences suffice for false binding.

Purin rich (A,G), particularly G-rich
sequences should be avoided.

Reduction of binding efficiency.

Long palindromes particularly interrupted palindromes (stem-loop sequences) should be avoided.

Longer (>6) GC-rich palindromes lead to probe dimerization. Stem-loops fold the probes. In both cases a reduction of available probe is the consequence.

Avoid complementarities between the probes.

Probe dimerization will result in a target independent signal.

Avoid complementarities between primer 3’ends and probes.

In a low target concentration background (early stages of amplification) and high excess of probes, even short complementary sequences will dimerized.

The Tm of the probes should not exceed 80 degrees centigrade.

Strong binding probes will reduce or inhibit the PCR reaction.