Page 20 - 2022-23 BIOFACT Catalogue

Real-Time PCR Description

Real-TIme PCR
PCR
Primer
Result analysis
Target gene
Real-Time PCR (Quantitative PCR = qPCR) technique is the system that is identical to a general type of PCR, and it is ampli ed
selection
design
reaction
through a thermal cycling method. The amount and presence of the template (DNA or RNA) triggering this experimental reaction
(For electrophoresis
system)
should be con rmed, which is the objective. In case of standard PCR, the product completing the reaction is con rmed on electro-
phoresis. But in case of Real-time PCR, the increased amount on a real-time basis is con rmed in accurate numeric values (graph).
In an in vitro setting, DNA is ampli ed by more than 10 times, which corresponds to its methods. Each PCR enzyme of the BIOFACT
5
is expressed in multiple amounts following the recombination of genes coding high-temperature DNA polymerase in E. coli. Thus,
it is a thermal resistant DNA polymerase that has been puri ed at a higher degree of purity.
Exponential phase Non-
0.3
exponential
Plateau
phase
0.2 Chemistry selection
qPCR
Fluorescence (DNA-binding Dyes probe design optimization analysis
Primer and
Data
or Probe type)
0.1 C T value
Threshold line
Real-Time PCR Workflow
0
0 10 20 30 40
Cycle
Data Analysis
Absolute Quantification VS Relative Quantification qPCR
< Amplification curve >
Absolute quantification Relative quantification qPCR
Exponential phase Non-
0.3
exponential Amount of unknown
Plateau sample can be measured Target sample is quanti ed
phase Measurement compared standard curves based on the amount of
Fluorescence 0.2 method concentrations are known. other reference sample
with target sample whose
(control gene).
0.1 Ct value
Con rmation of viral copy Con rmation of di erences
Examples of use number depending on of gene expression depend-
Threshold line
infectious stages ing on the drug response
0
0 10 20 30 40
Cycle
It is available to compare the concentrations between samples using Ct value.
< Melting curve > <Examples of result data>
BIOFACT Company A
B. Normalized Melting curves Melting curves
100 F
F
F
75
Fluorescence 50 Double-stranded DNA Single-stranded DNA Derivative Reporter (-Rn’)
(dsDNA)
(ssDNA)
25
Company B Company C
0
76 79 82 Tm 88 91
Temperature [℃] Temperature [℃]
Applying the principle on the left gure, it enables to measure the reduced
amounts of dye according to changes in temperature. Through the result, it can
be con rmed whether target gene is speci cally ampli ed or not.
BIOFACT Product
Real-Time PCR Enzyme (p. 56), Multi-Star Real-Time PCR Enzyme (For Probe) (p. 70), OneStep Multi-Star qRT-PCR Enzyme (For Probe) (p. 72)
20 BIOFACT CATALOGUE