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.

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.

Which are the limitations of End-Point PCR?.

1. time consuming in comparison to real-time PCR.
2. results are based on size discrimination, which may be not very precise.
3. Reproducibility at end point of amplification is very low.
4. the end-point is variable from sample to sample.
5. Agarose Gel resolution is poor.
6. Short dynamic range (smaller than 2 logs).
7. non-automated.
8. results, obtained by staining methods, are not very quantitative.
9. Post-PCR processing required.
   
   
   

B) Real-time PCR  

Description:

The real-time PCR 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.

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 real-time PCR 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?

Real-time PCR does not detect the size of the amplicon, however, it is not influenced by non-specific amplification.

What is the advantage of quantitation?

Real-time PCR quantitation eliminates post-PCR processing of PCR products which helps to increase throughput.

What the difference in respect to the dynamic range?

Real-time PCR offers a much wider dynamic range of up to 10⁷-fold (compared to 1000-fold in conventional PCR).

Which are the advantages of Real-Time PCR?

1. Real-time PCR 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
2. The number of amplicons generated in the PCR reaction is proportional to increase in reporter dye fluorescence
3. Increased dynamic range of detection
4. No post-PCR processing
5. High sensitivity: detection is capable down to a 2-fold change
6. high specifity and objectivity
7. fully automated process

C) Real-time RT-PCR  

Description:

Our test is a highly sensitive technique for the detection of mRNA (messenger RNA) of viral contaminants (e.g. Pestivirus). The technique consists of two parts:

1) the synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and

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