3- Quantitative real time

PCR:-
* It’s developed in the mid 1980s and can determine the level of specific
DNA or RNA in a biological sample.
• This method is based on detection of a fluorescent signal that is produced
in proportion to the amplification of PCR product , cycle after cycle.
• * It requires a thermal cycler coupled to an optical reading system that
measures fluorescence emission.
*The probe is synthesized to be hybridize selectively to the DNA of interest. This probe is
labeled on the 5’ end with a fluorochrome signal and on 3’ end with a quencher. The probe
shows the hybridization temp. greater than that of the primers. It hybridizes 100% during
elongation phase.

• During elongation phase , Taq polymerase degrades the probe and thus release the
fluorochrome signal which is proportional to the amount of PCR product generated in each
cycle.

• * In comparison with the conventional PCR, the quantitative real-time PCR is a method of
high specificity and sensitivity, very timely for countless application, and doesn’t only provide
qualitative data (presence or absence of DNA) but also precise the quantity of DNA.
4- Semi-quantitative or competitive PCR:-

• It is a rapid and highly sensitive technique used for quantitation of cellular mRNA .

• The procedure relies on the co-amplification of the sequence of interest with a serially
diluted synthetic DNA fragment of known concentration (competitor) using a single set of
primers.

• As equal amplification efficiency of competitor and target sequences is a necessary

prerequisite for quantitative PCR assays, competitors are usually designed to resemble the
target sequence as closely as possible.

• Many investigators have used highly homologous competitors differing only by the
presence or absence of a unique restriction enzyme site. The strategies commonly used to
separate and quantitate the PCR products include cutting either target or competitor with a
single restriction endonuclease or digesting both target and competitor with two different
enzymes.
• The competitor is distinguished from the target by gel electrophoresis and constructed to be either
of different size from the target or a mutant in it with an addition or deletion.

• * There is competition between the amplification of standard and RNA. The higher the standard
quantity, the less the RNA of interest will be amplified and its quantity will be small.

• In comparison with recently introduced real-time Q-PCR approaches, competitive PCR analysis
is more laborious, but permits equally sensitive detection and precise target quantification at
substantially lower cost. Competitive PCR approaches may, therefore, continue to play an
important role in PCR-based quantification of DNA and RNA targets as long as the cost of RQ-
PCR prevents many researchers from adopting this technique. The use of competitor molecules
containing a shifted restriction site contributes to increased reliability and precision of
competitive PCR assays.