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LECTURE 2: Recombinant DNA Technology & Cloning Biotechnology; 3 Credit hours Atta urRahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST)
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.
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.
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’
AluI HaeIII Restriction Enzymes Cutting Sites Blunt Ends
5’ P - - OH 3’ HindIII - P 5’ 3’ OH - EcoRI Sticky Ends
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
Plasmid Vector Insert (your gene) + Functional construct Plasmid (vector) • Four main steps in cloning: • Insert synthesis • Restriction enzyme digest • Ligation • Transformation
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 .
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
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.
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