Chapter 16 and 13.

Genetic code is universal

Translation results in the same amino acid sequence regardless of organism

Opens the doors to techniques to analyze and manipulate DNA

Cutting and transferring genes from one organism to another Copying DNA in a lab Using DNA to reveal identity Mapping nucleotide sequence Cloning

Hybrid molecule composed of DNA from 2 or more organisms Requires restriction enzymes and DNA ligase Restriction enzymes cut DNA at a specific nucleotide sequence
Various restriction enzymes; each cuts at a different sequence Ex- EcoRI (found in E.coli) Protects cells from invading DNA
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

1. Enzymes recognize short nucleotide sequence (restriction site)

Symmetrical- sequence is the same on both strands when read in the 5 3 direction

2. Cleave backbonesingle stranded end = sticky end

3. Sticky ends will temp bond with complementary sticky ends of other DNA
Source of DNA doesnt matter Use the same restriction enzyme for all the sources of DNA

4. DNA ligase seals the backbone

Recombinant DNA is first step in DNA cloning Allows researchers to generate clones of bacterial cells with the foreign gene
Use plasmids, small circular pieces of DNA in bacteria (not part of main chromosome)
Foreign DNA and plasmid treated with same restriction enzyme and mixed together Sticky ends join Modified plasmid reintroduced into cell (transformation) Just before division cell DNA and plasmids replicate Daughter cells both have the modified plasmid Researchers harvest DNA from clones

Cloning in cells is best for preparing large quantities If DNA source is scanty or impure, polymerase chain reaction (PCR), is quicker and more selective
Ex- forensics

Requirements: DNA, free nucleotides, heat stable polymerase, primers Temperature (heat) is used instead of helicase

3 step cycle
Denaturation- heated and strands separate Annealing- strands cooled, allows primers to attach Extension- heat stable DNA polymerase extends primers in 5 3 direction

Left to run for several hours and will complete approx 20 cycles
End result = copy number is ~1 million times original number

PCR animation

Click on the following links and look at the animation. Once it is complete, take the quiz and email me your results to dodi.cline@sdhc.k12.fl.us This must be done by next rotation.
DNA Cloning PCR

Process which uses a gel medium to separate nucleic acids fragments according to size DNA samples are loaded into the gel Gel is bathed in an aqueous solution and has electrodes attached to each end Current is turned on
Fragment separation is based on charge and size Small fragments will move faster and further away from starting point DNA will move toward which electrode?
positive

DNA is dyed and then analyzed

Useful for separating DNA fragments produced by restriction enzymes

Prepare pure samples of individual fragments

Compare 2 different DNA molecules Can be used to sequence a DNA fragment

Lab simulation/ HW:
Electrophoresis Wkst on edline

DNA has repeating sequences in noncoding regions

Known as tandem repeats Multiple copies of a 2-10 base sequence High rate of mutation at these locations Different individuals have a unique length to these regions

PCR copies a region of DNA known to have tandem repeats Gel electrophoresis is used and the resulting bands constitute an individuals DNA fingerprint Banding pattern of DNA sample is matched with known samples
If identical, they came from same individual If similar, individuals are related

Chance of 2 people (not identical twins) having identical tandem repeats in 3 regions is 1:1018 Uses:
Forensics (blood, semen, hair follicle) figure 16.9 on p 247 Paternity suits- childs DNA is a combination of both parents, bands from a child must come from either mom or dad Study population dispersals of humans or other animals Trace individuals ethnic heritage

Started in 1990, completed in 2003 Goal was to determine the order of all bases in human DNA
Approx 30,000 genes in human DNA

Now its deciphering what each gene does Generated genome libraries of genetic diseases
Aids doctors in diagnosing

New medication production

Where gene is, copy it, synthesize molecule and distribute

Ancestry and migration patterns

http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml

Involves deliberate changes to an individuals genome

Usually through a plasmid or virus

Results in genetically modified organisms (GMOs)

Express the transplanted gene and synthesize proteins

Bacteria and yeast most common GMOs

Insulin, growth hormone (hGH), and tissue plasminogen activator (TPA) currently produced this way Organisms for toxic chemical cleanup (ex- oil spill) Food manufacturing

Genetic engineering has been used for years in plants Examples

Delayed ripening (off season veggies) Resistance to spoilage or drought Resistance to the herbicide glyphosate (akaRoundUp) Improve nutritional value-golden ricecontains beta carotene (makes vit A- for eyesight and proper brain development) Pharm plants- trials for Hep B vaccines, antibodies that interfere w/ the bacteria that cause tooth decay

Mice were first transgenic animals

Allows us to study human diseases w/o experimenting on humans

Ex- sheep w/ better wool, pig w/ leaner meat Pharm animals- produce biological substances for medical use
Pigs potential source for transplant organs Goats produce proteins to treat cystic fibrosis, heart attacks, blood clotting disorders, etc Sheep altered to secrete Factor IX protein in their milk
Treats hemophilia

Some vaccines produced in chicken eggs

Be careful! Ensure no allergic or other adverse reactions to patients receiving them. (egg allergy)

Good
Improve production Better environment Reduce hunger in nations w/ poor crop output Produce rare proteins for vaccines Are GMO (genetically modified organisms) safe to eat? No long term data Transgenic plants could pass new genes to wild crops Products could produce allergic reactions Genes could cross species Decrease in biodiversity

Bad

http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/

Using a differentiated animal cell

Dolly Somatic cell from donor sheep collected and cultured. Nucleus removed from one of the cultured cells. Unfertilized egg from surrogate mom collected. Nucleus removed from egg. Egg cell and nucleus from somatic cell fused together w/ a zap of current Embryo placed in mom and developed normally Baby is a clone of the original donor.

Using undifferentiated cells

Therapeutic cloning Referred to as stem cell research Major ethical issue:
Produces human embryos for science

Benefits:
Repair damaged nervous tissue (spinal cords) Grow skin for serious burns Grow heart muscle to repair a damaged heart