2. Crossing -over DNA Occurs during Prophase I of Meiosis
Between chromatids of homologous pairs
Increase genetic variation
DNA
The McGraw-Hill Companies, Inc
Function
Components
Location

3.
U.S. Department of Energy Genomics:GTL Program

4. Biotechnology 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

5. 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. 5

6. 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
An electric current is used to propel the DNA through a porous gel matrix. Double-stranded DNA molecules are linear. Length directly relates to size and molecular weight.
Used to visualize:
Genomic DNA
RNA
PCR products
Plasmids
Restriction enzyme digest products

7. Used to separate DNA fragments by size
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
current splits water into hydrogen ions (H+) and hydroxyl ions (OH-)
Used to separate DNA fragments by size

8. 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 disorders
2. Developing cures for
3. Forensic or criminal
4. Personal identification
DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies in hospitals around the world. These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others. Early detection of such disorders enables the medical staff to prepare themselves and the parents for proper treatment of the child. In some programs, genetic counselors use DNA fingerprint information to help prospective parents understand the risk of having an affected child. In other programs, prospective parents use DNA fingerprint information in their decisions concerning affected pregnancies.
2. Research programs to locate inherited disorders on the chromosomes depend on the information contained in DNA fingerprints. By studying the DNA fingerprints of relatives who have a history of some particular disorder, or by comparing large groups of people with and without the disorder, it is possible to identify DNA patterns associated with the disease in question. This work is a necessary first step in designing an eventual genetic cure for these disorders.
3. FBI and police labs around the U.S. have begun to use DNA fingerprints to link suspects to biological evidence-blood or semen stains, hair, or items of clothing-found at the scene of a crime. Since 1987, more than 150 cases have been decided with the assistance of DNA fingerprint evidence. Another important use of DNA fingerprints in the court system is to establish paternity in custody and child support litigation. In these applications, DNA fingerprints bring an unprecedented, nearly perfect accuracy to the determination.
4 Because every organ or tissue of an individual contains the same DNA fingerprint, the U.S. armed services have just begun a program to collect DNA fingerprints from all personnel for use later, in case they are needed to identify casualties or persons missing in action. The DNA method will be far superior to the dogtags, dental records, and blood typing strategies currently in use.
Other reasons to run a gel: purity determination, isolation of one DNA band from other bands for sequencing purposes
DNA Fingerprinting:
Restriction Enzyme Digest and PCR (more later)
Used to visualize:
Genomic DNA
RNA
PCR products
Plasmids
Restriction enzyme products

9. Extract DNA, suspend in buffer
Restriction enzymes search and isolate unique sequences found in your genome. Everyone has a different number of these sequences found at different locations
PCR replicates your unique sequences so we have enough to visualize
Result is several different pieces of DNA of differing sizes and amounts
No two people have the same combination. 

10. How do we visualize DNA?

11. Steps of Gel Electrophoresis
Prepare agarose gel
(just like jello – but not as tasty)
Pour into casting tray with comb and allow to solidify
Add running buffer, load samples and marker
Run gel at constant voltage until band separation occurs
View DNA on UV light box and document results

12. Electrophoresis Equipment
Power supply
Gel tank
Cover
Electrical leads 
Casting tray
Gel combs

13. 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

14.
Practice loading samples
1. Set the micropipette to 15 µl. To change the volume of the BLUE micropipette, twist the knob under the plunger until you have reached your desired volume. Push the plunger down once to make sure this new volume is “set”.
2. To add a tip to the micropipette, push the micropipette down onto a tip. You should not have to use your hands to accomplish this. Please do not use too much force to put the tip on the micropipette; these instruments are expensive.
3. Load a 15 µl sample into the practice gel. To do this, press the plunger of the micropipette until you reach the first stop. Place the tip of the micropipette into the methylene blue solution present in the microtubules, and release the plunger to draw the liquid into the tip. Using both hands to steady yourself, place the tip of the micropipette into the MIDDLE of the well and slowly press the plunger until you reach the first stop. To make sure all of the sample is in the well, press the plunger to its second stop. WHILE KEEPING THE PLUNGER DEPRESSED, remove the tip from the well.
4. Remove the tip from the micropipette. On the BLUE micropipette, press the ejector down. You should not have to use your hands to remove tips.
5. Always store your micropipettes in an upright condition.
6. Once everyone has practiced loading the gels, rinse the methylene blue out of the wells in the sample gel.

15. Loading Dye
Practice loading your gels now!
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.
We’re not doing it this way

16. Who Dunnit?

17. Virtual Gel - Guided Inquiry

18. 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 18

19. 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 intro

20. Recipe for PCR Amplification
1. 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. 20

21. Step 3 is also called Elongation
Steps of PCR
1. Temperature separates
the strand
↑ temperature
2. Primers attach like
Bookends
↓ temperature
To clone DNA need
DNA
Buffer – mg chloride, mg is a cofactor for enzymes
Primers – a short strand of DNA that has been made/determined to bond with a specific section of the DNA we are cloning; needed to give TAQ enzyme a place to start and work space
Taq polymerase enzyme – Taq for the Thermos aqueous bacteria. Its enzyme can copy DNA at high temps
The bookends for the enzyme to work within a set area of interest
Made up of about 20 bases
Nucleotides – dNTPs
Thermal cycler – heat, cool, heat
Procedure:
Denature – increase temperature to 94 C, the double helix comes apart
Anneal – primers come in and set up as bookends; 45 – 65 C
Extend (elongation) – Tag comes in and adds in bases; 72 C
End of cycle one
Need to do this times to get a sufficient about of cloned DNA to work in a lab
3. Taq takes free nucleotides & adds them to the ends of the primer
↑ temperature
Step 3 is also called Elongation

22. Results of PCR With 30 cycles the DNA is amplified exponentially
Results in 1.1x1012 sets of precise-length DNA
PCR activity will take minute and they will be moving around a lot; group of 3 not 4 if possible
PCR Song 22

23. Manipulating the DNA Bt CORN Bacterium (Bacillus thuringiensis)
Bt crystal gene
Plant cell
Corn genome
Nancy
FIGURE 5-34 Help from bacteria in growing disease-resistant corn. Corn engineered to contain spores of the bacterium Bacillus thuringiensis (Bt) kills insect pests but does not harm humans.
© 2010 W. H. Freeman and Company 1 2 3
Corn plant destroyed
by insect pests
Bacterial gene coding for Bt
crystals, which are poisonous to
the insect pests, is inserted
directly into the corn plant’s DNA.
Bt crystals—toxic to insect pests—
are now produced by the corn plant
itself, reducing the amount of
pesticides the farmer must use. 23

24. Manipulating the DNA GOLDEN RICE
Genes in the pathway for
beta-carotene production
are introduced into the
white 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
Nancy
Till 11
FIGURE 5-31 The potential to prevent blindness in 250,000 people each year. Engineering rice to prevent blindness by increasing its vitamin A content
© 2010 W. H. Freeman and Company
Golden rice 24

25. Manipulating the DNA Commonly called GM Foods
GENETICALLY MODIFIED CROPS IN THE UNITED STATES
Corn
Cotton
Soybeans
76%
85%
55%
45%
24%
15%
10 of 12
Till 11:05
10. While genetic engineering has positive benefits, there are also concerns associated with
widespread use of genetic engineering in agriculture. If many farmers begin to plant more genetically modified crops that have an increased tolerance to insects, which of the following may result?
A. an increase in the use of pesticides
B. a decrease in genetic diversity of the crops 
C. an increase in the contamination of the water supply
D. a decrease in crop productivity on these treated fields
DOE item specs pdf
FIGURE 5-32 A significant portion of crops grown in the United States are genetically modified.
© 2010 W. H. Freeman and Company
Proportion of crops that are not genetically modified
Proportion of crops that are genetically modified
Commonly called GM Foods 25

26. GMO Selective breeding Transgenic manipulations
Guess what’s coming to dinner “

27. GMO Debate Should we or shouldn’t we…… Learn.genetics.utah.edu
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
Should we or shouldn’t we……
Learn.genetics.utah.edu
Jurassic Park Mr. DNA clip

28. DNA Science: by David A. Micklos and Greg A. Freyer
DNA Science: by David A. Micklos and Greg A. Freyer
Walkthrough with Quiz
Virtual Lab