Real-time fluorescence quantitative PCR principle.

so-called real-time fluorescence
. PCR
technology refers to the method of adding fluophore to the
PCR
reaction system, using fluorescent signal accumulation to monitor the entire
PCR
process in real time, and finally quantitatively analyzing unknown templates through standard curves.. 1.
in fluorescence
PCR
technology, there is an important concept -
the
Ct.
C
stands for
Cycle
,
t
stands for
threshold
, and
Ct
means the number of cycles experienced by fluorescent signals in each reaction tube when they reach a set domain value.. 2.
Fluorescent field value (
threshold
) sets the PCRreaction before the
15
circulating fluorescent signal as the fluorescent background signal, the default setting for the fluorescent domain value is

10
times the standard deviation of a fluorescent signal of 3-15
cycles, i.e.:
threshold s 10
́
SD
cycle 6-15
3. The relationship between
values and the starting templateResearch shows that the
Ct
value of each template has a linear relationship with the number of degears of the starting copy of the template, and the more starting copies there are, the smaller the
Ct
value. A standard curve can be made using
sysm standard
of known starting copies, where horizontal coordinates represent the number of pairs of starting copies and ordinates represent
ct
values. Therefore, the initial number of copies of
sample
can be calculated from the standard curve as long as the value of the
ct value of the unknown sample is obtained.. 4. fluorescence chemistryfluorescence
PCR
fluorescence chemistry can be divided into two types: fluorescent probes and fluorescent dyes. Here's how it works: 1
)
TaqMan
fluorescent probe:
PCR
amplification adds a specific fluorescent probe with a pair of citants, which is an oligarch
nucleotide
, marked with a reported fluorophobic group and a quenched fluorescent group at each end. When the probe is complete, the fluorescent signal emitted by the
is
quenched base;
taq

5
' -
3
' excision enzyme activity to cut down the probe enzyme, so that the reported fluorophoric and quenched fluorophoocete separation Thus, the fluorescence monitoring system can receive fluorescent signals, i.e. for every
DNA
strand amplification, a fluorescent molecule is formed, enabling the accumulation of fluorescent signals to be fully synchronized with the
PCR
product.. 2 )
SYBR
fluorescent dyes:in the
PCR
reaction system, the addition of excessive
SYBR
fluorescent dyes,
SYBR
fluorescent dyes are specifically mixed into the
DNA
double strand, the fluorescent signal is emitted without the
SYBR
dye molecules in the chain emitting no fluorescent signals, thus ensuring that the increase in fluorescent signals is fully synchronized with the increase in
PCR
products..