DNA extraction

recap

Safety point

• Spill phenol on bare feet (flip flops!) - goodbye skin

• Spill phenol on shoes – maybe stained shoes

• Spill phenol on bare legs (shorts) - goodbye skin

• Spill phenol on pants – stained pants

Calculation of DNA concentration

1) Measure UV absorption spectrum

Calculation of DNA concentration

• Absorbance coefficient for dsDNA at 260nm = 50

• So DNA with an absorbance of 1 at 260nm = 50ug/ml (or 50 ng/ul)

• We diluted your DNA 1 in 100• Multiply the A260 by 50 to get concentration

of diluted sample• Then multiply by 100 to get concentration of

your sample

DNA extraction from plants

• Smush (“homogenize”) the cells

• Dissolve membranes and solublize DNA in a detergent buffer.

• Remove solids with centrifugation

• Remove proteins with KOAc

• Remove proteins with phenol extraction

• Precipitate DNA with alcohol

DNA extraction issues

• Homogenization

- has to be “just right”• RNA• Hydrodynamic

shearing

- treat it gently

Agricultural biotechnology

A major application of genetic engineering

Plant genetic transformation

1) Need to engineer genes into a “vector” (recombinant DNA or genetic engineering)

2) Need to get vector into plant cells (gene delivery)

3) Need to grow whole plant from a single cell (regeneration)

Gene Delivery

Agrobacterium Particle gun

Introduction of the gfp gene into different target tissues

Petunia petal - cells

Wheat callus - cellsSoybean seed

Soybean Regeneration

InductionProliferation

Development

Germination

Plant Recovery

Starting MaterialImmature seeds

Cotton

Poplar

Xmas Cactus Fern

Soybean Ohio Buckeye

Biotech traits• “Input traits”• Make product easier or cheaper to produce• Herbicide resistance (e.g. Roundup Ready, Liberty Link).

Labor saving, can reduce overall herbicide usage.• Insect resistance (e.g. many Bt products). Reduces or

eliminates pesticide usage.

• “Output traits”• Add quality to product. • Storage properties (e.g. Flavr Savr). Longer lasting fruit,

easier to transport.• Composition (e.g. pharming, oil / protein).

Bt Corn Reduces:Insecticide MycotoxinApplication Production

X

Monarch larvae on Butterfly weed

Acreage of GMO

Varieties

Acreage of GMO Varieties

Public Support Varies for Different Applications of Biotechnology

(Includes ALL Countries – N = 35,000)

35

42

55

68

71

73

74

85

0 10 20 30 40 50 60 70 80 90 100

Increase Animal Productivity

Clone Animals (Medicine)

GM Feed (Healthier Meat)

More Nutritious Crops

Crops with Fewer Chems

Bacteria to Clean Waste

Crops to Produce Plastics

New Human Medicines

(Percent Agreement)

Source: Thomas Hoban, North Carolina State University

The Benefits of Using Biotechnology are Greater than the Risks

(35,000 Consumers from 35 Countries)

38

40

44

55

59

60

66

18

37

14

8

16

15

7

44

23

42

37

25

25

27

0% 20% 40% 60% 80% 100%

Europe (Average)

Russia

Australia

Canada

Latin/South Amer (Ave)

Asia (Average)

United States

Agree Not Sure Disagree

The Benefits of Using Biotechnologyare Greater than the Risks

(European Consumers)

22

22

34

39

41

42

55

24

24

18

25

10

11

8

54

54

48

36

49

47

37

0% 20% 40% 60% 80% 100%

Greece

France

Italy

Spain

Germany

Great Britain

Netherlands

Agree Not Sure Disagree

Willing to Buy GM Food if More Nutritious (Net = Continue – Not Continue)

1

21

25

31

56

66

-7

-5

-5

1

-10 0 10 20 30 40 50 60 70

Great Britain

Australia

Germany

Mexico

Japan

Canada

USA

Brazil

India

China

Percent Response

Perceptions of safety improved when US consumers learn that biotech foods have been

in the stores.

21%

25%

31%

46%

48%

29%

Unsafe Not sure Safe

Do you think genetically modified foods are basically

safe, basically unsafe, or don’t you have an opinion

on this?

Now, as you know, more than half of products in the

grocery store are produced using some form of

biotechnology or genetic modification. Knowing this,

do you think genetically modified foods are basically

safe, basically unsafe, or don’t you have an opinion on

this?January 2001

Source: Pew Initiative on Food and Biotechnology

“By Eating a Genetically Modified Fruit, a Person’s Genes Could also Be Changed”

1996 - 1998

55

60

62

74

58

38

52

29

61

62

30

25

20

16

24

32

25

32

30

18

15

15

18

10

18

30

23

39

9

20

0 20 40 60 80 100

United Kingdom

Switzerland

Sweden

Netherlands

Italy

Germany

France

Austria

United States

Canada

Percent Response

False (Correct) Don't Know True

Source: Thomas Hoban, North Carolina State University

“Ordinary Tomatoes Do Not Contain Genes, while Genetically Modified Ones Do”

1996 - 1998

40

48

46

51

35

36

32

34

45

52

38

21

24

27

44

20

39

22

45

33

22

31

30

22

21

44

29

44

10

15

0 20 40 60 80 100

United Kingdom

Switzerland

Sweden

Netherlands

Italy

Germany

France

Austria

United States

Canada

Percent Response

False (Correct) Don't Know True

Thanks

• Thanks to Monsanto for making MTU available

• Thanks to John Finer for biotech images.