1. and PowerPoint “DNA Technology,” from http://www.biologyjunction.com/
Genetic Engineering
Part I: Methods
Some material based on PowerPoint Lectures for Biology (7th Ed.), Campbell & Reece, Lectures by Chris Romero
and PowerPoint “DNA Technology,” from

2. Review The chemical that makes up the chromosomes:
Pieces of this are called ________ or _________ and come in four types (“letters”), abbreviated:
The letters pair up like this:

3. Genetic Engineering
Definition: altering the genetic makeup of an organism through DNA technology (compared to traditional breeding)

4. Genetic Engineering Used to: produce medicines improve food
cure genetic disorders
and many other things

5. Manipulating DNA
Most methods for cloning pieces of DNA in the laboratory share certain general features, such as the use of bacteria and their plasmids.

6. Scientists have developed methods for preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning.

7. Recombinant DNA From “recombine”
DNA made artificially by connecting pieces of DNA that are not normally attached.
The pieces can be from different organisms.
Involves using restriction enzymes to cut the pieces.

8. Recombinant DNA
Mostly done using a plasmid (a small circular piece of DNA in bacteria)
Bacterial chromosome

9. Process of Editing DNA Extract the DNA Cut the DNA Separate the DNA
Identify the DNA piece you want
Join pieces

10. Tools of Editing DNA DNA extraction:
cells are opened and DNA is separated from other parts by chemical means

11. Tools of Editing DNA
2. Cutting the DNA: done by restriction enzymes, which cut the DNA at certain points
act like scissors
hundreds are known

12. Restriction Enzymes
Example: EcoR1 cuts the DNA between the G and A

13. A restriction enzyme will usually make many cuts in a DNA molecule yielding a set of restriction fragments.
The most useful restriction enzymes cut DNA in a staggered way producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments.
DNA ligase is an enzyme that seals the bonds between restriction fragments (ligate = “to tie together” + -ase, an ending given to names of enzymes).

14. Recombinant DNA molecule
Using a restriction enzyme and DNA ligase to make recombinant DNA
Restriction site
DNA 5 3
G A A T T C
C T T A A G
Sticky end
Fragment from different DNA molecule cut by the same restriction enzyme
Recombinant DNA molecule G
C T T A A
A A T T C
A A T T C
T T A A
1. Restriction enzyme cuts the DNA backbones at each arrow.
2. DNA fragment from another source (cut with same restriction enzyme) is added and base pairs at sticky ends.
3. DNA ligase seals the strands.

15. 3. Separating the DNA
Done through gel electrophoresis – a method that separates macromolecules- either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties

16. Gel electrophoresis
For DNA separation, a gel is made of agarose, a polysaccharide extract from red seaweed.

18. Gel electrophoresis DNA has a negative charge due to phosphate groups.
It moves toward the positive end
Smaller fragments move farther and faster.

19. 4. Identify the piece of DNA you want

20. Cell Transformation done after recombinant DNA is made
inserting new genes into a cell, thus changing the cell’s genetic makeup
done after recombinant DNA is made

21. Steps for Transforming Bacteria
The plasmid is removed from bacteria, DNA with the gene you want to work with is removed from the cell.
Both are cut with the same restriction enzyme, giving sticky ends that will bind together.

22. Steps for Transforming Bacteria
3. Cut DNA and plasmid are combined and joined by ligase.
4. The new plasmid incorporates the gene and is then inserted into a host.

23. Steps for Transforming Bacteria
5. The host now has the gene to make whatever. As it reproduces, more of what is needed is made.

24. 1a 3 2a 4 2b 1b 5

26. Another way to copy DNA: PCR
polymerase chain reaction
allows many copies to be made from a small piece of DNA
done in laboratory, not in cells

29. Vector necessary for cell transformation
carries DNA fragments into the host
biological or mechanical

30. Vector Biological Vectors plasmids
virus: injects its genetic material into a cell, which is then copied

31. Mechanical and chemical methods of transformation
Micropipette: tool used to make injections
Bombardment
DNA placed in particles that can be taken across cell membrane.

32. Example: engineering of bacteria to produce a human protein, such as insulin

33. An enzyme cuts open the plasmid DNA
An enzyme cuts open the plasmid DNA. The same enzyme removes the human insulin gene from its chromosome.
Human insulin gene
Human DNA
Plasmid

34. The plasmid and human DNA are mixed and joined together with ligase.

35. Plasmids, which now contain the human insulin gene, are used to transform bacterial cells.

36. When the cells reproduce, the new cells will contain copies of the “engineered” plasmid.
The foreign gene directs the cell to produce human insulin.

37. Tricks to allow isolation of transformed cell colonies
Sticky ends
Human DNA fragments
Human cell
Gene of interest
Bacterial cell
ampR gene
(ampicillin
resistance)
Bacterial plasmid
Restriction site
Recombinant DNA plasmids
lacZ gene (lactose breakdown)
Tricks to allow isolation of transformed cell colonies
Plasmid has gene for resistance to antibiotics.
If no plasmid, cell won’t grow when antibiotic given.
Restriction site has gene for using the sugar lactose.
Uncut plasmids give blue colonies when a special sugar is given.

38. Transform bacteria that have a mutated gene for using lactose.
Tricks to allow isolation of transformed cell colonies
Colony carrying non- recombinant plasmid with intact lacZ gene
Bacterial clone
Colony carrying recombinant plasmid with disrupted lacZ gene
Recombinant bacteria
Transform bacteria that have a mutated gene for using lactose.
Grow on agar with antibiotic and a special sugar.
No plasmid: cells won’t grow (killed by antibiotic)
Plasmid but no foreign gene: blue colony.
Recombinant plasmid:
white colony.

39. Bacterium
Bacterial chromosome
Plasmid
Cell containing gene of interest
Recombinant DNA (plasmid)
Gene of interest
DNA of chromosome
Recombinate bacterium
Basic research on protein
Copies of gene
Basic research on gene
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hormone treats stunted growth
Protein expressed by gene of interest 3
Gene inserted into plasmid 1
Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes
Plasmid put into bacterial cell 2