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 Soybean seed Wheat callus - cells

Plant Recovery Starting Material Immature seeds Germination

Soybean Regeneration

Development Proliferation Induction

Xmas Cactus Cotton Fern Poplar 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 Mycotoxin Application 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)

New Human Medicines Crops to Produce Plastics Bacteria to Clean Waste Crops with Fewer Chems More Nutritious Crops GM Feed (Healthier Meat) Clone Animals (Medicine) Increase Animal Productivity 0 10 85 68 71 74 73 55 42 35 20 30 40 50 60 (Percent Agreement) 70 80 90 100

Source: Thomas Hoban, North Carolina State University

The Benefits of Using Biotechnology are Greater than the Risks (35,000 Consumers from 35 Countries)

United States Asia (Average) Latin/South Amer (Ave) Canada Australia Russia Europe (Average) 0% 60 59 55 66 15 16 7 44 40 38 14 8 37 37 42 18 44 20% Agree 40% Not Sure 60% Disagree 80% 27 25 25 23 100%

The Benefits of Using Biotechnology are Greater than the Risks (European Consumers)

Netherlands Great Britain Germany Spain Italy France Greece 0% 22 22 34 42 41 39 20% 55 8 37 18 11 10 25 47 49 36 24 24 54 54 48 40% Agree Not Sure 60% Disagree 80% 100%

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

66 China India Brazil USA Canada Japan Mexico Germany Australia Great Britain 1 1 -5 -5 -7 -10 0 10 21 25 31 20 30 40 Percent Response 50 56 60 70

Perceptions of safety improved when US consumers learn that biotech foods have been in the stores.

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

25% 21%

Unsafe

31% 46% 48% 29%

Not sure Safe

Source: Pew Initiative on Food and Biotechnology

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

Canada United States Austria France Germany Italy Netherlands Sweden Switzerland United Kingdom 0 1996 - 1998 62 61 18 30 20 9 29 38 20 52 58 55 62 60 74 32 32 25 24 20 30 25 40 60 Percent Response False (Correct) Don't Know True 39 16 30 23 18 10 18 15 15 80 100

Source: Thomas Hoban, North Carolina State University

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

Canada United States Austria France Germany Italy Netherlands Sweden Switzerland United Kingdom 0 1996 - 1998 45 52 33 45 15 10 34 32 36 35 40 51 46 48 20 22 39 20 44 24 21 38 40 60 Percent Response 27 False (Correct) 44 44 Don't Know True 80 29 21 22 30 31 22 100

Thanks

• Thanks to Monsanto for making MTU available • Thanks to John Finer for biotech images.