1. Gene Technology

2. At the beginning: Studies using interferon: – Interferon increases human resistance to viral infection and scientists were interested in its possible usefulness in cancer therapy. – It was rare in the blood and needed in large quantities for study. – Interferon gene introduced into bacteria genome. – Bacteria divide rapidly so began to produce interferon in large quantities.

3. At the beginning: Studies using interferon: – Interferon increases human resistance to viral infection and scientists were interested in its possible usefulness in cancer therapy. – It was rare in the blood and needed in large quantities for study. – Interferon gene introduced into bacteria genome. – Bacteria divide rapidly so began to produce interferon in large quantities.

4. A bacteria

5. Bacterial Reproduction

6. Genetic Engineering The bacteria produced a line of genetically altered cells from a single altered cell = cloning. Insulin gene can also be “grown” this way. This marked the beginning of genetic engineering. – The ability to cut DNA into recognizable pieces and rearrange them in different ways.

7. How To: Cut the source of the DNA that carries what you want and insert into a plasmid or infective virus.

8. A Virus

9. Getting In

10. Taking Over

11. In order to make it work: MUST 1. cut the source DNA and the plasmid DNA so that the desired fragment can be spliced. – Done by enzymes the recognize and cleave (cut) specific sequences of nucleotides. Restriction Enzyme SourceRecognition Site BAMHIBacillus amyloliquefaciensG GATC C C CTAG G EcoRIEschirichia Coli R factorG AATT C C TTAA G HaeIIIHemophilus aegyptusGG CC CC GG HindIIIHemophilus influenzaeA AGCT T T TCGA A TaqIThermus aquaticusT CG A A GC T

12. Restriction Endonucleases Bacteria must defend themselves against bacteriophages (viruses that specifically attack bacteria). Through the lytic cycle the viruses can attack, kill, and take over many more bacteria. To defend themselves bacteria contain enzymes called restriction endonucleases. These recognize specific nucleotide sequences in a DNA strand, bind to them, and cleave them at the recognition sequence. To keep these enzymes from attacking their own DNA bacteria add methyl (using mehylases) to the recognition sites so the endonucleases can’t bind.

13. Restriction Endonucleases Bacteria must defend themselves against bacteriophages (viruses that specifically attack bacteria). Through the lytic cycle the viruses can attack, kill, and take over many more bacteria. To defend themselves bacteria contain enzymes called restriction endonucleases. These recognize specific nucleotide sequences in a DNA strand, bind to them, and cleave them at the recognition sequence. To keep these enzymes from attacking their own DNA bacteria add methyl (using mehylases) to the recognition sites so the endonucleases can’t bind. Restriction endonuclease

14. How To Cut the DNA Find the palindromes. – Nucleotides at one end of the recognition sequence are comlementary to those at the other end so they have the same nucleotide sequence running in opposite directions. Allows it to cut across two strands Since the cleave site isn’t directly centered each fragment has a single stranded end a few nucleotides long. These fragments are complementary to each other and can recombine using DNA ligase (the spotwelder). – These are considered “sticky” ends and will attract their complementary base pair sets.

15. Cutting Activity

16. Restrictions Endonucleases Cut Hundreds of restiction enzymes – Each with own recognition sequence By chance the recognition site will arise within a given sample. – The shorter the sequence the more often it will probably occur. – Once two fragments attach, they can be joined by DNA ligase to reform the phosphodiester bonds of DNA. – ANY two fragments produced by the same restriction endonuclease can be joined together. Elephant DNA cleaved by an endonuclease can be joined to ostrich DNA if cleaved by the same endonuclease.

17. EcoRI Cleaves sequence GAATTC between the G and the A. Since they run in opposite directions, they are complementary … sticky.