Recombinant DNA Technology & Cloning

1. LECTURE 2: Recombinant DNA Technology & Cloning Biotechnology; 3 Credit hours Atta urRahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST)

3. Plasmid DNA is the genetic material of most organisms (from bacteria to humans) Chromosome: Most bacteria have one circular DNA chromosome ranging in size from 1,000 to 8,000 kilo base pairs. Plasmid:Extra chromosomal genetic element also made of a circular DNA molecule. Bacterial Genome: The collection of all of the genes present on the bacteria’s chromosome or its extra chromosomal genetic elements.

4. Basics: Nucleotides are the building blocks of DNA

5. DNA is a long double-stranded chain of nucleotides • DNA is the hereditary material passed on from generation to generation. • DNA is made up of four nucleotides: A, C, G, and T. • A always pairs with T. • C always pairs with G. • The two strands of DNA are in an antiparallel configuration. • Two complementary DNA strands will separate when heated, and will spontaneously pair together again (hybridize) when cooled.

6. Restriction Enzymes • Enzymes that act as scissors to cut the DNA at specific sites • They cut sugar phosphate back bone • Recognition site • Cutting site • Restriction Endonucleases recognize specific sequence with the DNA molecules • The recognized sequence are usually four to six base pair and are palindromic • The sequence of both the strands are the same when read in same direction, the 5’ t0 3’ or 3’ to 5’ • 5’GCCAATTGGC3’ • 3’CGGTTAACCG5’

7. Restriction Enzymes

8. AluI HaeIII Restriction Enzymes Cutting Sites Blunt Ends

9. 5’ P - - OH 3’ HindIII - P 5’ 3’ OH - EcoRI Sticky Ends

10. Bacterial Plasmids • Bacterial cells may contain extra-chromosomal DNA called plasmids. • Plasmids are usually represented by small, circular DNA. • Some plasmids are present in multiple copies in the cell

11. Plasmid Vector Insert (your gene) + Functional construct Plasmid (vector) • Four main steps in cloning: • Insert synthesis • Restriction enzyme digest • Ligation • Transformation

12. Plasmids Vectors

13. Plasmid Vectors • Plasmid vectors are ≈1.2–3kb and contain: • Replication origin (ORI) sequence • A gene that permits selection, • Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin. • Exogenous DNA can be inserted into the bracketed region .

14. Selective Marker • Selective marker is required for maintenance of plasmid in the cell. • Because of the presence of the selective marker the plasmid becomes useful for the cell. • Under the selective conditions, only cells that contain plasmids with selectable marker can survive • Genes that confer resistance to various antibiotics are used. • Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used

15. Origin of Replication • Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes. • Without replication origin, DNA cannot be replicated in the cell.

16. Multiple Cloning Sites • Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endo- nucleases located next to each other • Restriction sites of the polylinker are not present anywhere else in the plasmid. • Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector