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.EcoRI (found in E.mcgraw-hill.coli) Protects cells from invading DNA
http://highered. each cuts at a different sequence Ex.
Cleave backbonesingle stranded end = sticky end
.1. Enzymes recognize short nucleotide sequence (restriction site)
Symmetrical.sequence is the same on both strands when read in the 5’ 3’ direction
DNA ligase seals the backbone
. Sticky ends will temp bond with complementary sticky ends of other DNA
Source of DNA doesn’t matter Use the same restriction enzyme for all the sources of DNA
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. primers Temperature (heat) is used instead of helicase
. is quicker and more selective
Requirements: DNA. free nucleotides. polymerase chain reaction (PCR). heat stable polymerase.
allows primers to attach Extension.
3 step cycle
Denaturation.strands cooled.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
.heated and strands separate Annealing.
Click on the following links and look at the animation.cline@sdhc. take the quiz and email me your results to dodi.
DNA Cloning PCR
.fl. Once it is complete.k12.us This must be done by next rotation.
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?
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
individuals are related
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 individual’s DNA fingerprint Banding pattern of DNA sample is matched with known samples
If identical. they came from same individual If similar.
Chance of 2 people (not identical twins) having identical tandem repeats in 3 regions is 1:1018 Uses:
Forensics (blood. hair follicle) figure 16.child’s 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 individual’s ethnic heritage
.9 on p 247 Paternity suits.
copy it.ornl. synthesize molecule and distribute
Ancestry and migration patterns
http://www.gov/sci/techresources/Human_Genome/home. completed in 2003 Goal was to determine the order of all bases in human DNA
Started in 1990.000 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.
growth hormone (hGH).oil spill) Food manufacturing
. and tissue plasminogen activator (TPA) currently produced this way Organisms for toxic chemical cleanup (ex.
Involves deliberate changes to an individual’s genome
Usually through a plasmid or virus
Results in genetically modified organisms (GMO’s)
Express the transplanted gene and synthesize proteins
Bacteria and yeast most common GMO’s
for eyesight and proper brain development) “Pharm” plants.
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.trials for Hep B vaccines. antibodies that interfere w/ the bacteria that cause tooth decay
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. (egg allergy)
.sheep w/ better wool. blood clotting disorders. etc Sheep altered to secrete Factor IX protein in their milk
Some vaccines produced in chicken eggs
Be careful! Ensure no allergic or other adverse reactions to patients receiving them.
Mice were first transgenic animals
Allows us to study human diseases w/o experimenting on humans
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
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.genetics.
http://learn. Nucleus removed from egg. Nucleus removed from one of the cultured cells.edu/content/tech/cloning/clickandclone/
Using a differentiated animal cell
“Dolly” Somatic cell from donor sheep collected and cultured.
. Unfertilized egg from surrogate mom collected.utah.
Using undifferentiated cells
Therapeutic cloning Referred to as stem cell research Major ethical issue:
Produces human embryos for science
Repair damaged nervous tissue (spinal cords) Grow skin for serious burns Grow heart muscle to repair a damaged heart