Genetic research

Zoning of PCR laboratory

premises

Laboratory box "dirty area" in which DNA is Laboratory box "dirty area" in which DNA is
extracted from peripheral blood extracted from peripheral blood...
 PCR method
• The PCR method is a method of significantly increasing small
concentrations of certain fragments of nucleic acid (DNA) in a
biological material (sample).

• The PCR method is based on multiple doubling of a certain

DNA region using enzymes under artificial conditions (in vitro).
• Only the area that meets the specified conditions is copied,
and only if it is present in the test sample
 G e n e e x p r e s s i o n - transfer of genetic information from
DNA through RNA to polypeptides and proteins.

Больные Контроль
1.6
1.4
1.2
Уровень
1 относитель
ной
0.8 экспресии
гена GIPR
0.6
0.4
RNA -> cDNA 0.2
0

Isolation of RNA Staging

from a biological reverse Staging Analysis of
sample transcription PCR reactions results
reactions = cDNA
Fluorescence accumulation curves in real-time PCR:
dependence of the fluorescence intensity (in several tubes - each has its own curve)
from the cycle number (Print Screen from the Bio-Rad thermal cycler).
 Melting curves of the PCR product

а б в

Parameters of the amplification reaction of the cDNA fragment of the GIPR gene.
a) Graphical representation of the kinetics of accumulation of the PCR product during amplification of experimental
samples.
b) Graphical representation of the PCR product melting curve during amplification of experimental samples.
c) Projection of Сt values of a series of two-fold dilutions for plotting a calibration curve
 Control
group

The investigated
group

Control
group

The investigated
group

Where TRXT is the targeted gene,

act - reference
• Calculations of the levels of relative expression of the studied genes were performed using a modified Pfaffl
formula for different amplification efficiencies [Pfaffl M.W. A new mathematical model for relative
quantification in real time RT – PCR // Nucleic Acids Research. 2001. Vol. 29 (9). P. 45.]:
• Ratio = (Eиссл)ΔCPтарг(контр-иссл)/(Eреф)ΔCPреф(контр – иссл) ,
• Ratio = (Einvest) ΔCPtarg (control-invest) / (Eref) ΔCPref (control-invest), Where
• Ratio — relative level of mRNA expression;
• E is the effectiveness of primers;
• Cp - value of the indicator cycle;
• research - study group;
• control - control group;
• target — target (investigated) gene;
• ref - reference gene (β2-microglobulin).
• The relative level of expression of the studied gene is calculated based on its efficiency of real-time PCR (E)
and the difference (Δ) of the intersection points (CP) of the unknown sample compared to the control (ΔCP =
CP of the test sample - CP of the control sample).
• The used relative quantitative analysis (Relative Quantification) is based on the ratio of the expression of the
studied to the expression of the reference gene and is sufficient for most purposes to study physiological
changes in the levels of gene expression

Х = E(ΔCq the reference investigated group - ΔCq the reference control group) / E(ΔCq
the target investigated group - ΔCq the target control group)

Х = 2(21,52 – 20,85) / 2(32,43 – 32,41)

• https://www.youtube.com/watch?v=matsiHSu
oOw&ab_channel=AppliedBiologicalMaterials-
abm
 Region of
PCR applications

Genotyping (screening
for mutations or Analysis of gene activity
polymorphisms) (expression)

1. Identification of genotypes of 1. Diagnosis of markers for detecting

increased risk of developing cancer.
multifactorial diseases
2. Screening for hereditary genetic
diseases
3. Determination of the therapeutic
efficacy of a certain class of drugs
 Ligand

Receptor
Cell
Kinases

NF-kB TF

Expression: Ядро
mRNA -> protein

Protein