Biotechnology Hands-on Investigations Nancy Dow Jill Hansen Tammy Stundon May 11, 2013 Gulf Coast State CollegePanhandle Area Educational Consortium 5230.

BiotechnologyHands-on Investigations

Nancy DowJill Hansen

Tammy Stundon

May 11, 2013Gulf Coast State College Panhandle Area Educational Consortium

5230 West Highway 98 753 West Boulevard

Panama City, Florida 32401 Chipley, Florida 32428

850-769-1551 877-873-7232

www.gulfcoast.edu

Biology Partnership

(A Teacher Quality Grant)

Pre-test????Q and A board

What is Biotechnology? How do we use Biotechnology?

Florida Next Generation Sunshine State Standards

SC.912.L.16.10 Evaluate the impact of biotechnology on the individual, society and the environment, including medical and ethical issues.

Clarification

•Students will evaluate examples and/or explain the possible impact of biotechnology on the individual, society, and/or the environment.

Content Limits

•Items may assess current issues but will not require knowledge of specific biotechnologies or specific medical issues.

•Items assessing the possible impacts of biotechnology will not assess monetary impacts.

DNA- Function- Components- Location

Crossing -over- Occurs during Prophase I of

Meiosis- Between chromatids of

homologous pairs- Increase genetic variation

U.S. Department of Energy Genomics:GTL Program http://www.ornl.gov/hgmis

• Genetic engineering

– def: use of technology to alter the genes of viruses, bacteria, and other cells for medical or industrial purposes to better the quality of life

– Altering genes of unicellular organisms and plants and animals

Biotechnology

• Genome: composed of DNA, is our hereditary code (the “blueprint”)

• Molecular biology: the study of genes and the molecular details that regulate the flow of genetic information from DNA to RNA to proteins, from generation to generation.

Biotechnology uses this knowledge to manipulate organisms’ DNA to help solve human problems.

Caught by a Kiss!

Crime Scene

Using enzyme amylase

Isolate DNA evidence from saliva

Forensic Science!

Ready to use what you know in the ‘real world’?

Gel Electrophoresis

• Technique used to separate DNA bands based on size

• Comparison of sample bands to markers allows…

– Visible confirmation of desired product

– Quantification of sample DNA

How does it work?

• Electrolysis: the splitting of water using electricity

• Electrophoresis: a method of separating charged molecules in an electrical field; DNA has an overall negative charge

Used to separate DNA fragments by size

Why Run a Gel?

DNA fingerprints are useful in several areas of society.

They are used by professionals in human health and the justice

system.

1. Diagnosis of inherited disorders2. Developing cures for inherited disorders3. Forensic or criminal4. Personal identification

1. Extract DNA, suspend in buffer

2. Restriction enzymes search and isolate unique sequences found in your genome. Everyone has a different number of these sequences found at different locations

3. PCR replicates your unique sequences so we have enough to visualize

4. Result is several different pieces of DNA of differing sizes and amounts

How do we visualize DNA?

Steps of Gel Electrophoresis

Prepare agarose gel(just like jello – but not as tasty)

Add running buffer, load samples and marker

Run gel at constant voltage until band separation occurs

Pour into casting tray with comb and allow to solidify

View DNA on UV light box and document results

Casting tray

Gel combs

Power supply

Gel tankCover

Electrical leads

Electrophoresis Equipment

Agarose Gel• A porous material derived from red

seaweed

• Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments

• Concentration affects DNA migration

– Low conc. = larger pores better resolution of larger DNA fragments

– High conc. = smaller pores better resolution of smaller DNA fragments

1% agarose

2% agarose

Loading Dye

• DNA samples are loaded into a gel after the tank has been filled with buffer, covering the gel

• Contains a dense substance, such as glycerol, to allow the sample to "fall" into the sample wells

• Contains one or two tracking dyes, which migrate in the gel and allow monitoring of how far the electrophoresis has proceeded.

Practice loading your gels now!

Who Dunnit?

Virtual Gel - Guided Inquiry

POLYMERASE CHAIN REACTION(PCR)

- 1983- With PCR, you can target and make millions of

copies (amplify) a specific piece of DNA (or gene) out of a complete genome.

- PCR impacted several areas of genetic research:- as a medical diagnostic tool to detect specific mutations that may cause

genetic disease

- in criminal investigations and courts of law to identify suspects

- in the sequencing of the human genome

PCR Amplification• PCR makes use of the same

basic processes that cells use to duplicate their DNA (replication)

– Complementary DNA strand hybridization

– DNA strand synthesis via DNA polymerase

PCR Song

Recipe for PCR Amplification1. DNA sample: containing the intact sequence of DNA to be amplified

2. Master mix:√ Free nucleotides (dNTPs): raw material of DNA (A,T,C,G)

√ DNA polymerase (Taq polymerase): enzyme that assembles the nucleotides into a new DNA chain

√ Primers: pieces of DNA complementary to the template that tell DNA polymerase exactly where to start

√ Fluorescent dye: “lights up” when it binds to complete DNA strands

3. Thermocycler: used to rapidly heat and cool

DNA samples to facilitate DNA amplification.

Steps of PCR

Step 3 is also called Elongation

1. Temperature separates the strand ↑ temperature

2. Primers attach like Bookends ↓ temperature

3. Taq takes free nucleotides & adds them to the ends of the primer ↑ temperature

Results of PCR

With 30 cycles the DNA is amplified exponentially

Results in 1.1x1012 sets of precise-length DNA

Bt CORNBacterium (Bacillus thuringiensis)

Bt crystal gene

Corn genomePlant cell

Bt crystals—toxic to insect pests—are now produced by the corn plantitself, reducing the amount ofpesticides the farmer must use.

Bacterial gene coding for Btcrystals, which are poisonous tothe insect pests, is inserteddirectly into the corn plant’s DNA.

Corn plant destroyedby insect pests

1 2 3

Manipulating the DNA

GOLDEN RICE

Genes in the pathway forbeta-carotene productionare introduced into thewhite rice genome.Almost 10% of the

world’s people suffer from vitamin A deficiencies—leading to 250,00 cases of blindness each year. The addition of beta-carotene-producing genes to white rice has increased its vitamin A content almost 25-fold.

Daffodil Bacteria

White rice

Beta-carotene

Golden rice

Manipulating the DNA

GENETICALLY MODIFIED CROPS IN THE UNITED STATES

Proportion of crops that are not genetically modifiedProportion of crops that are genetically modified

Corn Cotton Soybeans

55% 45% 76%24% 15%85%

Manipulating the DNA

Commonly called GM Foods

• GMO Computer lab– Selective breeding – Transgenic manipulations – Guess what’s coming to dinner

“

GMO Debate

• Should we or shouldn’t we……

Your scientists were so preoccupied with whether or not they could, they didn't stop to think if they should.”

Dr. Ian Malcolm, Jurassic Park

Jurassic Park Mr. DNA clip

Follow up

•Q & A

•Post Test

• DNA Science: by David A. Micklos and Greg A. Freyer

• Walkthrough with Quiz• Virtual Lab