Plasmids circular pieces of”extrachromosomal” DNA propagated inside host

1. Plasmids • circular pieces of”extrachromosomal” DNA propagated inside host • have origin of replication • -> ensures host will copy it

2. Plasmids • circular pieces of”extrachromosomal” DNA propagated inside host • have origin of replication • -> ensures host will copy it • Have selectable marker • (usually a drug-resistance • gene) • -> ensures cell won’t • “lose” it

3. Plasmids • circular pieces of”extrachromosomal” DNA propagated inside host • have origin of replication • -> ensures host will copy it • Have selectable marker • (usually a drug-resistance • gene) • -> ensures cell won’t • “lose” it • pGLO also carries • gene for Jellyfish • Green Fluorescent Protein

4. Restriction Enzymes • enzymes which cut DNA • at specific sites

5. Restriction Enzymes • enzymes which cut DNA • at specific sites • called "restriction enzymes" • because restrict host range for • certain bacteriophage

6. Restriction Enzymes • enzymes which cut DNA • at specific sites • called "restriction enzymes" • because restrict host range for • certain bacteriophage • bacterial” immune system”: • destroy any “non-self” DNA

7. Restriction Enzymes • bacterial” immune system” destroy “non-self”DNA • -> methylase recognizes same sequence in host DNA and protects it by methylating it

8. Restriction Enzymes enzymes which cut DNA at specific sites Useful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments

9. Restriction Enzymes enzymes which cut DNA at specific sites Useful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments

10. Restriction Enzymes enzymes which cut DNA at specific sites Useful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments can map each site by double digests

11. Restriction Enzymes Restriction enzymes create unpaired "sticky ends” which anneal with anycomplementary sequence

12. Restriction Enzymes • Restriction enzymes • create unpaired "sticky • ends” which anneal with • anycomplementary • sequence • make “recombinant • DNA” by mixing 2 • different fragments • digested with same • enzyme and gluing • them together with ligase

13. Restriction Enzymes • Restriction enzymes • create unpaired "sticky • ends” which anneal with • anycomplementary • sequence • make “recombinant • DNA” by mixing 2 • different fragments • digested with same • enzyme and gluing • them together with ligase • Genetic Engineering

14. Restriction mapping

15. Restriction mapping

16. Transformation Physical transfer of new DNA into cell 1. Preincubation: chills cells, allows cations to neutralize negative charges on plasma membrane.

17. Transformation 1. Preincubation: chills cells, allows cations to neutralize negative charges on plasma membrane. 2. Incubation: allows DNA to diffuse through cell wall to contact plasma membrane.

18. Transformation 2. Incubation: allows DNA to diffuse through cell wall to contact plasma membrane. 3. Heat shock: opens holes in plasma membrane, allows plasmid to enter by diffusion.

19. Transformation 3. Heat shock: opens holes in plasma membrane, allows plasmid to enter by diffusion. 4. Recovery: cells must make proteins encoded by new DNA molecule before they can be plated.

20. Transformation 5. Selecting transformants: cells which have taken up the plasmid are only ones which can grow in the presence of ampicillin. Form colony at place they landed on plate

21. DNA fingerprinting Use DNA sequences that vary between individuals

22. DNA fingerprinting Use DNA sequences that vary between individuals

23. DNA fingerprinting Use DNA sequences that vary between individuals Microsatellites short sequences 2-5 bases long repeated multiple times

24. DNA fingerprinting • Microsatellites • short sequences 2-5 bases long repeated multiple times • vary between families but not within families

25. DNA fingerprinting • STRs (simple tandem repeats) • vary between families but not within families • Detect by PCR with primers that bind outside repeat