Genetics and Cloning.
Presented ByNkosi K.TB.eD
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
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.
Components of the reaction mixture
Template DNA.
Primers (forward and reverse)
dNTPs
Taq DNA Polymerase
Buffer solution
Divalent cations
Sterile deionized water
Template DNA
It contains the DNA region to be amplified
Range - 1-2 µl ( for a total reaction mixture of 10 µl)
Hairpins Intramolecular interaction within the primer
dNTPsDe 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)
dNTPs in the reaction mix
Steps in PCRInitialization
Denaturation
Annealing
Extension / Elongation
Final elongation
Final hold
Initialization step
Heating the reaction to a temperature of
• 94-96°C for 1-9 minutes.
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)
PCR Product at different Temperature.
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.
CHROMOSOME DUPLICATION AND DISTRIBUTION
Cloning process.
Modification of cDNA for cloning
Directional cloning of foreign DNA
Production of transgenic animal
Screening genomic libraries in bacteriophage
REFERENCE
• http://www.slideshare.net/alokbharti18/pcr-4529063
• Dr.Zeyad Akawi Jreisat M.D, M.A. Ph.D
• Ms. J. Williamson (2014).
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