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).