1. 8.1 - Manipulating & Cloning DNA
Genetic Technologies
8.1 - Manipulating & Cloning DNA
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2. Insulin
E. coli and safflower plants have both been used to produce human insulin
How?
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3. Genetic Engineering
human insulin gene can be introduced to plasmids (recombinant DNA)
plasmids can be introduced to bacteria cells
How?
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4. Restriction Enzymes
are produced by bacteria to function as an “immune system” against invading viruses by cutting up the viral DNA or RNA

5. Restriction Enzymes
restriction enzymes cut DNA at a specific recognition site
recognition sites are always palindromic: (same sequence when read from the 5’ to 3’ direction on either strand)

6. Restriction Enzymes
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7. Blunt Ends vs. Sticky Ends
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8. Image from: https://bsp.med.harvard.edu/node/41
What’s inaccurate about this image? Recognition sequence is not palindromic.

9. DNA Ligase DNA ligase can be used to attach restriction fragments
See simple animation:

10. Plasmids
circular pieces of non-chromosomal DNA found in bacteria cells
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11. Plasmids as Vectors
genes can be inserted into plasmids using restriction enzymes and DNA ligase
can then introduce these genes into host bacterial cells
copy number of plasmids determines how much protein will be produced

12. Image from: http://www.abfrontier.com/cs/gene.do

13. Restriction Maps
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14. Constructing Restriction Maps
Try Tutorial 1: p
Sample Problem 1:
Plasmid X undigested
Plasmid X digested with EcoRI
Plasmid X digested with BamHI
Plasmid X digested with EcoRI and BamHI
1400 bp
600 bp
800 bp
100 bp
700 bp

15. Practice #1 (p.372) Plasmid Z uncut Plasmid Z cut with EcoRI
Plasmid Z cut with PstI
Plasmid Z cut with EcoRI and PstI
1500 bp
500 bp
1000 bp
300 bp
700 bp

16. Practice #2 (p.372) Plasmid W uncut Plasmid W cut with HindIII
Plasmid W cut with BamHI
Plasmid W cut with HindIII and BamHI
1000 bp
450 bp
550 bp
400 bp
600 bp
150 bp
250 bp
300 bp

17. Practice #3 (p.373) EcoRI BamHI SmaI EcoRI + BamHI EcoRI + SmaI
800 bp
1500 bp
250 bp
700 bp
1350 bp
900 bp
1400 bp
200 bp
300 bp
500 bp
1050 bp
350 bp
450 bp
150 bp
550 bp
600 bp
750 bp

18. Do you remember this?
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19. Transformation successful introduction of DNA from another source
bacterial cells can be made competent by placing in CaCl2 solution (stabilizes phospholipid bilayer) and then rapid heating & re-cooling
animation: