1. Recombinant DNA Technology

2. Restriction endonucleases - Blunt ends and Sticky ends

3. Restriction endonuclease and DNA ligase yield Recombinant DNA

5. DNA cloning

6. Polylinker – multiple restriction sites

7. Selection of clones

9. Bacterial Artificial Chromosomes (BAC) Transformation: 1. Heat shock: CaCl 2 at 0 o C then heat to 37-42 o C 2. Electroporation – apply high voltage BAC – 5,000 to 400,000 bp insert

10. Yeast Artificial Chromosomes (YAC)

11. up to 150,000 bp insert

12. Studying genes – cDNA library

13. Polymerase Chain Reaction (PCR)

15. Cloning of PCR products

16. Hybridization allows the deletion of specific sequences

17. DNA fingerprinting – RFLP (restriction fragment length polymorphism)

18. DNA microarrays Any known DNA sequence from any source, can be used in microarray. Green spots – mRNA more abundant in single-cell stage Red spots – mRNA more abundant at later stages of development

19. Cloned genes can be expressed – Expression vector

20. Cloned genes can be altered 1.Site-directed mutagenesis 2.Oligonucleotide- directed mutagenesis

21. Transgenic – cloning in mice for human growth hormone