Cycling conditions for amplifying longer PCR products
Step Time/cycles Temperature
Initial activation step 2 min 95°C
Denaturation 10 s 94°C
Annealing 1 min 50–68°C*
Extension 1 min/kb
Number of cycles 40 cycles 68°C
End of PCR cycling Indefinite 4°C
• Overlap-extension PCR or Splicing by overlap
extension (SOEing) :
• Genetic engineering technique
• used to splice together two or more DNA
fragments OR complementary sequences.
• It is the technique enables creation of
specific and long DNA constructs.
• It can also introduce deletions, insertions or
point mutations into a DNA sequence.
• Nested PCR
• This PCR increases the sensitivity
• Two sets of primers,
• A double process of amplification .
• The first set of primers allow a first
amplification. The product of this PCR is
subjected to a second PCR using the second
set of primers.
• Primers used in the second PCR are specific
to an internal amplified sequence in the first
PCR. specificity of the first PCR product is
verified with the second one.
• Semi quantitative PCR
• An approximation to the relative amount of
nucleic acids present in a sample,
• The markers commonly used are
• Apo A1 and B actin.
• Amplification product is separated by
• Multiplex PCR
• Multiplex PCR is an adaptation of PCR which
allows simultaneous amplification of many
• This technique is used for diagnosis of
• Multiplex PCR can detect different pathogens
in a single sample.
Applications of PCR and impact on
• PCR and its different variations are highlighted
as the most commonly used in laboratories
and research institutes.
• Thus, these have contributed to identification
• characterization of several organisms and
understanding of physiopathology of diverse
• diseases in human, animal and plants.
• identification of microorganisms
assurance of blood
• As a basic procedure to investigate Deaths
• (paternity testing)
• Evidence from minimal samples of saliva,
semen or other tissue debris
• As conventional PCR or qPCR have also
facilitated research in
• Detection of pathogens in plants, animals,
and the environment; understanding of their
• Epidemiology and, development of new
diagnostic tests, treatments or vaccines.
• Selective DNA isolation
• Isolation of DNA fragments from genomic DNA
by selective amplification of a specific region of
• This use of PCR augments many methods, such as
generating hybridization Probes and DNA cloning
which require larger amounts of DNA,
representing a specific DNA region.
• PCR supplies these techniques with high
amounts of pure DNA, enabling analysis of DNA