Genetics and cloning, is all about modifying genes,more like evolution of genes.

1. Genetics and Cloning.
Presented By
Nkosi K.T
B.eD

2. Contents
 PCR Definition
 Components of the reaction mixture
 PCR primer design guidelines
 Steps in PCR
 Variations on the basic PCR technique
 Comparison of PCR and Gene cloning
 Applications of PCR
 Problems related to PCR

3. What is PCR?
Polymerase Chain Reaction is an in vitro technique for
the amplification of a specific sequence of DNA Which
is used for further testing.

4. Components of the reaction mixture
Template DNA.
Primers (forward and reverse)
dNTPs
Taq DNA Polymerase
Buffer solution
Divalent cations
Sterile deionized water

5. Template DNA
It contains the DNA region to be amplified
Range - 1-2 µl ( for a total reaction mixture of 10 µl)

6. Hairpins Intramolecular interaction within the primer

7. dNTPs
De oxy nucleotide triphosphate (dATP, dGTP, dTTP, dCTP)
They are the building blocks from which the DNA
polymerases synthesizes a new DNA strand.
• Range - 0.5 µl (for 10µl reaction mixture)

8. dNTPs in the reaction mix

9. Steps in PCR
Initialization
Denaturation
Annealing
Extension / Elongation
Final elongation
Final hold
Initialization step
Heating the reaction to a temperature of
• 94-96°C for 1-9 minutes.

11. Application of PCR
Cloning a Gene encoding a known protein
Amplification of old DNA
Amplifying cloned DNA from Vectors
Rapid Amplification of cDNA ends
Detecting Bacterial or Viral Infection
● AIDS infection
●Tuberculosis (Mycobacterium tuberculosis)

12. PCR Product at different
Temperature.

13. DNA STRUCTURE
• DNA molecules are polymers called polynucleotides.
• Each polynucleotide is made of monomers called
nucleotides.
• Each nucleotide consists of :
• a nitrogenous base (Adenine, Thymine, Cytosine or
Guanine)
• a pentose sugar (DNA = Deoxyribose sugar),
• and a phosphate group.

14. CHROMOSOME DUPLICATION AND
DISTRIBUTION

15. Cloning process.

16. Modification of cDNA for
cloning

17. Directional cloning of foreign
DNA

18. Production of transgenic animal

20. Screening genomic libraries in
bacteriophage

21. REFERENCE
• http://www.slideshare.net/alokbharti18/pcr-4529063
• Dr.Zeyad Akawi Jreisat M.D, M.A. Ph.D
• Ms. J. Williamson (2014).