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 any complementary sequence

12. Restriction Enzymes
Restriction enzymes
create unpaired "sticky
ends” which anneal with
any complementary
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
any complementary
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