Cloning & PCR

1. LECTURE 4: Cloning & PCR Biotechnology; 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST)

2. Polymerase Chain Reaction • Polymerase: DNA polymerase • DNA polymerase duplicates DNA • Before a cell divides, its DNA must be duplicated • Chain Reaction: The product of a reaction is used to amplify the same reaction • Results in rapid increase in the product

3. PCR performs the chemistry of DNA duplication in vitro • Numerous PCR applications make this process a staple in most biology laboratories • Understanding properties of DNA polymerases helps understanding PCR Polymerase Chain Reaction (PCR)

4. Discovery • PCR was discovered by Kary Mullis • On a long motorcycle drive • According to Mullis, he was driving his vehicle late night with his girlfriend • Mentally visualized the process • Nobel Prize in Chemistry • 1993

5. DNA polymerase • Duplicates DNA • Necessary for reproduction of new cells • More than one DNA polymerases exist in different organisms

6. 3’ 5’ 3’ 5’ Properties of DNA polymearse • Needs a pre-existing DNA to duplicate • Cannot assemble a new strand from components • Called template DNA • Can only extend an existing piece of DNA • Called primers

7. 3’ 5’ 3’ 5’ Properties of DNA polymearse • DNA strands are anti-parallel • One strand goes in 5’  3’ • The complementary strand is opposite • DNA polymerase always moves in one direction (from 5’  3’)

8. 3’ 5’ dTTP 3’ 5’ dATP dCTP dCTP dATP dTTP dTTP dGTP dCTP dATP dATP dATP dCTP dTTP dGTP dGTP dGTP dATP dTTP dATP dCTP dTTP dGTP dGTP dCTP Properties of DNA polymearse • DNA polymerase incorporates the four nucleotides (A, T, G, C) to the growing chain • dNTP follow standard base pairing rule

9. Properties of DNA polymearse • The newly generated DNA strands serve as template DNA for the next cycle • PCR is very sensitive • Widely used

10. 3’ 5’ dTTP 3’ 5’ dATP dCTP dCTP dATP dTTP dTTP dGTP dCTP dATP dATP dATP dCTP dTTP dGTP dGTP dGTP dATP dTTP dATP dCTP dTTP dGTP dGTP dCTP Setting up a PCR Reaction • Add template DNA and primers • Add dNTPs • Add DNA polymerase

11. Properties of DNA polymearse • DNA polymerase needs Mg++ as cofactor • Each DNA polymerase works best under optimal temperature, pH and salt concentration • PCR buffer provides optimal pH and salt condition

12. Taq DNA polymerase • Derived from Thermusaquaticus • Heat stable DNA polymerase • Ideal temperature 72C

13. Thermal Cycling • A PCR machine controls temperature • Typical PCR go through three steps • Denaturation • Annealing • Extension

14. Denaturation • Heating separates the double stranded DNA • Denaturation • Slow cooling anneals the two strands • Renaturation Cool Heat

15. Annealing • Two primers are supplied in molar excess • They bind to the complementary region • As the DNA cools, they wedge between two template strands • Optimal temperature varies based on primer length etc. • Typical temperature from 40 to 60 C

16. Extension • DNA polymerase duplicates DNA • Optimal temperature 72OC

17. PCR Amplification Exponential Amplification of template DNA

18. Typical PCR mix In a thin wall Eppendorf tube assemble the following

19. Applications • Revolutionized how we study biology • Recombinant DNA Technology • Research • Diagnostics • Forensics