Genetics and cloning
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This document provides an overview of polymerase chain reaction (PCR) and gene cloning. It defines PCR as an in vitro technique used to amplify a specific DNA sequence for further testing. It describes the basic components of a PCR reaction mixture, including template DNA, primers, dNTPs, Taq DNA polymerase, and buffer solution. It also outlines the basic steps in a PCR process, including initialization, denaturation, annealing, extension, and final elongation. The document compares PCR and gene cloning and discusses some applications of PCR, such as cloning genes, amplifying DNA from vectors, and detecting bacterial or viral infections.
Genetics and cloning
- 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袖l ( for a total reaction mixture of 10袖l)
- 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袖l (for 10袖l 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属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.
- 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).