A) Conventional (End-Point)
PCR
Description:
Conventional PCR methods, unlike real-tim e PCR (se e below), rely on
the total amount of product generated by the end of the reaction instead
of the rate of product formation.
Our qualitative test is designed to detect whether a contamination (e.g.
mycoplasma) is or is not present.
Why is no gene product formed?
Not every PCR is successful. There is for example a possibility that
the quality of the DNA is poor, that inhibiting factors interfere with
the PCR reaction, that one of the primers doesn't fit, or that there is
too much or too less starting template.
Why could a gene product not be of the right size?
It is possible that there is a product, for example a band of 300 base
pairs, but the expected gene should be 600 base pairs long. In that case,
one of the primers probably attaches on a part of the gene closer to the
other primer. It is also possible that both primers fit on a totally different
gene.
Why do non-specific product bands appear?
It is possible that the primers fit on the desired locations, but that
they also hybridise with other locations. In that case, you may observe
different bands in one lane on a gel.
Another possibility is that the annealing temperature is too low. Increase
of the annealing temperature increases specificity of the amplification
process. It is also conceivable that the sample is contaminated by external
DNA.
Which are the limitations of End-Point PCR?.
- time consuming in comparison to qPCR.
- results are based on size discrimination, which may be not very precise.
- Reproducibility at end point of amplification is very low.
- the end-point is variable from sample to sample.
- Agarose Gel resolution is poor.
- Short dynamic range (smaller than 2 logs).
- non-automated.
- results, obtained by staining methods, are not very quantitative.
- Post-PCR processing required.
B) qPCR
Description:
The qPCR allows to quantitate the initial amount of the template. It
monitors the fluorescence emitted during the reaction as an indicator
of amplicon production during each PCR cycle as opposed to conventional
PCR that detects the amount of final amplified product at the end-point.
The real-time progress of the reaction can be viewed in many systems.
Our quantitative test looks for contaminations and estimates the number
of the contaminating particles per volume.
What do the slopes reflect?
The slopes of the detected curves reflect the amplification efficiency
of the reaction, that is, how many copies of an amplicon each cycle synthesizes.
When designing a qPCR experiment, it is also important to optimize this
parameter making it as close as possible to a complete doubling in each
cycle.
What do I observe in case of non-specific amplication?
qPCR does not detect the size of the amplicon, however, it is not influenced
by non-specific amplification.
What is the advantage of quantitation?
qPCR quantitation eliminates post-PCR processing of PCR products which
helps to increase throughput.
What the difference in respect to the dynamic range?
qPCR offers a much wider dynamic range of up to 107-fold
(compared to 1000-fold in conventional PCR).
Which are the advantages of qPCR?
- qPCR measures data at the exponential growth phase (high reproducibility
in the beginning of the exponential phase), hence measurements are highly
reliable and allow exact quantifications of e.g. microbial contaminations
- The number of amplicons generated in the PCR reaction is proportional
to increase in reporter dye fluorescence
- Increased dynamic range of detection
- No post-PCR processing
- High sensitivity: detection is capable down to a 2-fold change
- high specifity and objectivity
- fully automated process
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