The meaning of the inner label in the traditional quantitative PCR.

reference method in the

.Quantitative internal
and
are added to different PCR reaction tubes, which are synthesized using
gene
engineering methods. Upstream quotations are fluorescently labeled and downstream quotations are not marked. As the template is amplified, the inner label is also amplified. In the
PCR
product, due to the different length of the inner label and the target template, the expansion of the two products can be separated by electrophoresis or high-efficiency liquid phase, respectively, to determine its fluorescence intensity, the internal label is the control quantitative template to be tested.
. Competition law

an
competitive template
a new internal cut point produced by a clone of a mutation. In the same reaction tube, the sample to be tested is amplified simultaneously with the competition template using the same pair of citations (one of which is fluorescently labeled). After amplification, the products of
PCR
are digested with endoenzyme, the products of competitive templates are enzymatic into two fragments, and the template to be tested is not enzymatic, the two products can be separated by electrophoresis or high-efficiency liquid phase, respectively, to determine the fluorescence intensity, according to the known template to guess the number of initial copies of unknown templates.
. PCR
-
ELISA
methodusing marker primers such as geococin or biotin, the amplifier is combined by a specific probe on the solid phase plate, and then added anti-geococin or biotinase label
antibodies
-spicy root peroxidase binding, and finally the enzyme makes the substrate color. The conventional
PCR
-
ELISA
method is only a qualitative experiment, if the internal standard is added, the standard curve is made, and quantitative detection can also be achieved.
. The role of the internal label in traditional quantification
Because the traditional quantitative methods are end-point detection, that is,
PCR
after the arrival of the platform period for detection, and
PCR
after the number of period amplification to reach the platform period, detection reproducible is extremely poor. The same template on the
96
hole
PCR
instrument to do
96
repeated experiments, the results are very different, so it is not possible to directly from the end product to calculate the starting template amount. After adding the inner label, the inaccuracy caused by the quantification of the final product can be partially eliminated. But even so, traditional quantitative methods can only be counted as semi-quantitative, rough quantitative methods.

.