1 Trends in Biotechnology TB 09 Basic tools of recombinant DNA

2 Endonucleases

3 Cutting double-stranded DNA. The enzyme DNase cuts DNA at random sites.

4

5 Fig. 3.1 (b) Specific site cleavage.

6 Video: Restriction striction.html

7 A specific restriction enzyme cuts each DNA molecular at the same sequence site.

8 The ligation of two different pieces of DNA.

9 DNA ligase is an enzyme which remakes the phosphate diester bonds.

10 Video: restriction enzymes bio37 hill.com/olc/dl/120078/bio37.swf Video: restriction

11 Agarose gel electrophoresis is used to separate DNA (and RNA) molecules according to size.

12 Electrophoresis: gelelectrophoresis The pieces of DNA move because of the charge on their phosphate groups. They are separated because of their length. With some methods, they can be separated because of their shape. Tutorial on electrophoresis: tions/content/gelelectrophoresis.htmlhttp:// tions/content/gelelectrophoresis.html Animation of electrophoresis: l

13 Ligation of a DNA fragment into a vector using the restriction endonuclease EcoRI and DNA ligase.

14 The transformation of bacteria and the selection of cells with the recombinant vector.

15 Video: Key Steps of Molecular Cloning ture=related Video: Plasmid Cloning hill.com/sites/ /student_view0/chapter1 6/animations.html#

16 Restriction map of pBR322 showing unique restriction sites, the ampicillin and tetracycline resistance genes, and the origin of replication.

17 Note how the cutting and insertion at a particular restriction site will result in loss of resistance.

18 Selection for recombinant cells after transformation.

19 Selection for recombinant cells after transformation.

20 Selection for recombinant cells after transformation.

21 Replica plating

22 Restriction map of pUC18/pUC19 showing the ampicillin resistance gene, the origin of replication, and the multiple cloning site within the lacZ gene.

23 The selection of bacterial colonies containing a recombinant vector (pCU vector + DNA insert) by the selection of white colonies.

24 Cloning using Bacteriophage The lytic life cycle of a bacteriophage.

25 Fig The cloning of human DNA restriction fragments ( ● and ▲ ) into a bacteriophage vector.

26 Fig The use of Agrobacterium tumefaciens to transfer DNA into plants.

27 Video: Ti plasmid hill.com/sites/ /student_view0/ch apter16/animations.html#

28 Fig Particle gun used for transforming cells with DNA-coated microprojectiles.

29 An animation of a gene gun can be found at: ORT250/animations/Gene%20Gun%20Animat ion/Genegun1.html ORT250/animations/Gene%20Gun%20Animat ion/Genegun1.html

30 Fig Microinjection of DNA into pronucleus of a fertilized animal egg.