CONVENTIONAL PCR

What Is the Conventional PCR Principle?
What Are the Components of a Conventional PCR Setup?
PCR is based on three simple steps required for any DNA synthesis reaction.
Applications of PCR

1. CONVENTIONAL PCR
PREPARED BY
CL.PH/JOSEPH TALAAT WILLIAM

2. WHAT IS THE CONVENTIONAL PCR
PRINCIPLE?
The chemistry involved in PCR depends on the
complementarities (matching) of the nucleotide bases in
the double-stranded DNA helix. When a molecule of
DNA is sufficiently heated, the hydrogen bonds holding
together the double helix are disrupted and the
molecule separates or denatures into single strands. If
the DNA solution is allowed to cool, the complementary
base pairs can reform to restore the original double
helix.

3. WHAT ARE THE COMPONENTS OF A
CONVENTIONAL PCR SETUP?
Two primers (Forward and reverse).
 Taq polymerase.
 Deoxynucleoside triphosphates (dNTPs).
 Buffer solution.
 Distilled water.
 Template DNA.

5. PCR IS BASED ON THREE SIMPLE STEPS
REQUIRED FOR ANY DNA SYNTHESIS
REACTION:
(1) Denaturation of the template into single
strands.

(2) Annealing of primers to each original strand for
new strand synthesis.

(3) Extension of the new DNA strands from the
primers.

7. STEPS OF CONVENTIONAL PCR
1- Initialization step:
(only required for DNA polymerases that require heat
activation by hot-start PCR): This step consists of
heating the reaction to a temperature of 94–96 °C (or
98 °C if extremely thermostable polymerases are
used), which is held for 1–9 minutes.
2- Denaturation step:
This step is the first regular cycling event and consists
of heating the reaction to 94–98 °C for 20–30 seconds. It
causes DNA melting of the DNA template by disrupting
the hydrogen bonds between complementary bases,
yielding single-stranded DNA molecules.

8. STEPS OF CONVENTIONAL PCR
3- Annealing step:
The reaction temperature is lowered to 50–65 °C
for 20–40 seconds allowing annealing of the
primers to the single-stranded DNA template.

9. STEPS OF CONVENTIONAL PCR
4- Extension/elongation step:
The temperature at this step depends on the DNA
polymerase used; Taq polymerase has its optimum
activity temperature at 75–80 °C and commonly a
temperature of 72 °C is used with this enzyme. At
this step, the DNA polymerase synthesizes a new
DNA strand complementary to the DNA template
strand by adding dNTPs that are complementary to
the template in 5′ to 3′ direction, condensing the 5′-
phosphate group of the dNTPs with the 3′-hydroxyl
group at the end of the nascent (extending) DNA
strand. The extension time depends both on the
DNA polymerase used and on the length of the
DNA fragment to amplify.

11. APPLICATIONS OF PCR
DNA sequencing and gene reproduction & cloning.
The identification of genetic fingerprints used in
forensic scientists which use PCR to connect blood,
saliva, or tissue left at the scene of a crime to a
suspect or victim.
Discovering the possibility of transmitting genetic
diseases between potential parents and determining
whether the diseases will or will not be passed to
their next generation of children.
DNA-based phylogeny, or functional analysis of
genes.
The diagnosis of hereditary diseases.
The detection and diagnosis of infectious diseases.

12. REFERANCES
https://blog.praxilabs.com/2021/04/28/what-are-
the-three-basic-steps-of-conventional-
pcr/#What_Is_the_Conventional_PCR_Principle