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Genetic EngineeringBiotechnology
HISTORY OF GENETIC ENGINEERING
Before technology, humans were using the process of selective breeding to produce the type of organism they want.
Creating new breeds of animals & new crops to improve our food.
Example: Dog Breeding
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LabradoodlePoodleLabrador
Bulldog Mastiff Bullmastiff
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Animal breeding
Breeding food plants
Evolution of modern corn
“Cabbage family” descendants of
the wild mustard
Selective Breeding
• Choosing individuals with the desired traits to serve as parents for the next generation.
Graph: Plant Height
• What is the result?The frequency of desired alleles increases in the population
Now, suppose only the tallest plants were used to breed
Test Cross
A special cross use to determine an unknown genotype of a dominant phenotype
Cross the unknown individual with a homozygous recessive individual
A?
Let’s work this out!
Outcome:If the individual is homozygous dominant
100% dominant phenotype
If the individual is heterozygous dominant 50% dominant
phenotype 50% recessive
phenotype
A Brave New World
GENETIC ENGINEERING
Scientists can now use their knowledge of the structure of DNA
and its chemical properties to study and change DNA molecules.
Remember the code is universal
• Since all living organisms… – use the same DNA– use the same code
book– read their genes the
same way
Can we mix genes from one organism to another?
YES!
Transgenic organisms contain recombinant DNA
GENETIC
ENGINEERIN
G!
Recombinant DNA- made by connecting fragments of DNA from a different source.
Transgenic Organisms- Organisms that contain DNA from a different source.
How do we do mix genes?• Genetic engineering
– Isolate gene from donor DNA– cut DNA in both organisms– paste gene from one organism into other
organism’s DNA– transfer recombined DNA into host
organism– organism copies new gene as if it were its
own– organism produces NEW protein coded for
by the foreign DNA Remember: we all use the same genetic code!
CUTTING DNA
RESTRICTION ENZYMES are proteins that act as “molecular scissors”
RESTRICTION ENZYMES
Restriction enzymes are proteins that cut DNA Each restriction enzyme only cuts a
specific nucleotide sequence in the DNA called the recognition sequence
RESTRICTION ENZYMES
Recognition sequences are usually palindromes
Same backwards and forwards
Ex. Eco R1 enzyme recognizes:
RESTRICTION ENZYMES
Cuts usually leave little single stranded fragments called STICKY ENDS
RESTRICTION ENZYMES
If the enzyme cuts right down the middle, the ends are BLUNT
Each time EcoRI recognizes the sequence CTTAAG, it cuts between the G & A and then through the middle of the strands
The recognition sequence for the restriction enzyme named EcoRI is CTTAAG
This results in DNA fragments that have single-stranded tails called sticky ends
RESTRICTION ENZYMES
Pieces can be glued back together using
LIGASE
http://www.youtube.com/watch?v=8rXizmLjegI
GENE TRANSFER
During GENE TRANSFER, a gene from one organism is placed into the DNA of another organism
New DNA that is created is called RECOMBINANT DNA. Example: Human insulin
Bacterial
Recombinant
DNA
Insulin
BACTERIAL PLASMIDS
Bacteria have small, circular DNA segments called PLASMIDS.
Usually carry “extra info” on them
Plasmids can be used as a VECTOR- object that carries foreign DNA into a host cell
There’s more…
• Plasmids– small extra circles of DNA– carry extra genes that bacteria can
use– can be swapped between bacteria
How can plasmids help us?• A way to get genes into bacteria
easily– insert new gene into plasmid– insert plasmid into bacteria = vector– bacteria now expresses new gene
• bacteria make new protein
+
transformedbacteriagene from
other organism
plasmid
cut DNA
recombinantplasmid
vector
glue DNA
Bacteria • Bacteria are great!
– one-celled organisms– reproduce by mitosis
• easy to grow, fast to grow– generation every ~20 minutes
CREATION OF RECOMBINANT DNA
1. In a lab, plasmid is extracted from bacteria
2. Insulin also extracted from human DNA
**Both gene for insulin and plasmid are cut with same restriction enzyme.
Insulin gene
(cut from chromosome)
Bacterial Plasmid
TRANSFORMATION
4. The gene is inserted into the plasmid by connecting sticky ends with ligase.
5. Plasmid taken up by bacteria through TRANSFORMATION.
6. Bacteria grows in Petri dish and replicates recombinant DNA
insulin
human insulin
CREATION OF INSULIN
7. As the bacteria grow and replicate, more and more bacteria are created with the human insulin gene
8. The bacteria read the gene and create insulin for us to use
TRANSFORMING PLANT & ANIMAL CELLS
Bacterial plasmids can also be put into plant and animal cells
The plasmid incorporates into the plant or animal cell’s chromosome
Transformed bacteria
introduce plasmids
into plant/animal cells
TRANSGENIC ORGANISMS
Because the bacteria now has DNA from two species in it, it is known as a TRANSGENIC ORGANISM.A.K.A. GENETICALLY MODIFIED ORGANISM
Transforming BacteriaRecombinant DNA
Gene for human growth hormone
Gene for human growth hormone
Human Cell
Bacteria cell
Bacterial chromosome
Plasmid
Sticky ends
DNA recombination
Bacteria cell containing gene for human growth hormone
DNA insertion
Grow bacteria…make more
growbacteria
CLONE
harvest (purify)protein
TRANSFORMATION
transformedbacteria
plasmid
gene fromother organism
+
recombinantplasmid
vector
REAL OR F
AKE!!!!!
1 - REAL OR FAKE
2 - REAL OR FAKE
3 - REAL OR FAKE
4 - REAL OR FAKE
5 - REAL OR FAKE
6 - REAL OR FAKE
7 - REAL OR FAKE
8 - REAL OR FAKE
9 - REAL OR FAKE
10 -REAL OR FAKE
CLONIN
G
CLONING
CLONE – organism with the same genetic make-up (DNA) as another
An exact copy
CLONING – STEP 1A
CLONING – STEP 1B
CLONING – STEP 2
CLONING – STEP 3
CLONING – STEP 4
CLONING – STEP 5
POLY
MERASE
CHAIN
REACTION
*MANIPULATING DNA
Techniques used to manipulate DNA:
DNA Extraction
Cut DNA in to smaller pieces
Identify base sequences
Make unlimited copies of DNA
MAKING COPIES
Often at a crime scene, DNA evidence is left behind in trace (small) amounts
Hair
Blood
Body fluids
MAKING COPIES
The sample is so small, it cannot be used unless more of it can be made
POLYMERASE CHAIN REACTION (PCR)
Process used to amplify (multiply) the amount of DNA in a given sample
MAKING COPIES
What did the polymerase molecule do in DNA replication / Transcription?
MAKING COPIES
• PCR also allows scientists to pick a particular gene and make many copies of it
• Millions of copies can be made from just a few DNA strands
PCR STEPS
PCR Supply ListDNA
Heat
Taq (DNA) polymerase A special polymerase from a bacterium that lives at high temperatures
Primers
PCR STEPS
Step 1 Denature (separate) the DNA by heating it up to 95°C.
PCR STEPS
Step 2:
Reduce the temperature
Add primers that binds to the strand.
PCR STEPS
Step 3
Add Taq (DNA) polymerase adds nucleotides to strands, producing two complementary strands.
PCR STEPS
Step 4, 5, 6…
Repeat
Repeat
Repeat
Every time we repeat the procedure, we double the DNA
GEL
ELEC
TROPHORES
IS
Many uses of restriction enzymes…• Now that we can cut DNA with
restriction enzymes…– we can cut up DNA from different
people… or different organisms… and compare it
– why?• forensics• medical diagnostics• paternity• evolutionary relationships • and more…
Comparing cut up DNA• How do we compare DNA
fragments?– separate fragments by size
• How do we separate DNA fragments?– run it through a gelatin – gel electrophoresis
• How does a gel work?
GEL ELECTROPHORESIS
An electric current is applied to the gel to get the DNA movingSmall molecules move faster (move towards bottom)
Big molecules move slower (stay towards top)
DNA fragments are drawn to positive electrode
DNA moving through gel
GEL ELECTROPHORESIS
The gel acts like a filter by separating strands of different sizes
It’s like a sponge made of Jello – lots of small holes and a “squishy” consistency
Gel electrophoresis• A method of separating
DNA in a gelatin-like material using an electrical field– DNA is negatively
charged– when it’s in an electrical
field it moves toward the positive side
+–DNA
“swimming through Jello”
Gel Electrophoresis
longer fragments
shorter fragments
powersource
completed gel
gel
DNA &restriction enzyme
wells
-
+
Running a gel
1 2
cut DNA with restriction enzymes
fragments of DNAseparate out based
on size
3
Stain DNA– Dye binds to DNA– fluoresces under
UV light
ANALYZING DNA
The pieces are separated, analyzed & compared to othersEach piece has its own unique weight and shape
Use these properties to perform a technique called DNA FINGERPRINTING
DNA FINGERPRINTING
Step 1) DNA is cut using
restriction enzymes
Step 2) Mix of DNA and enzymes are then separated by a process called GEL ELECTROPHORESIS
Step 3) DNA pattern is
analyzed
DNA FINGERPRINTING
CUTTING DNA
Everyone has a unique DNA sequence
Rec. sequences are in different placesWhen a restriction enzyme cuts the DNA of two different people, it will cut it into different sized pieces
Suspect #1
Suspect #2
DNA fingerprint• Why is each person’s DNA pattern different?
– sections of “junk” DNA• doesn’t code for proteins
• made up of repeated patterns– CAT, GCC, and others
– each person may have different number of repeats
• many sites on our 23 chromosomes with different repeat patterns
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTTCGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTTCGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA
Allele 1GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTTCGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
repeats
DNA patterns for DNA fingerprints
cut sitescut sites
GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA
1 2 3
DNA – +
Cut the DNA
Person 1GCTTGTAACG GCCTCATCATCATTCGCCG GCCTACGCTTCGAACATTGCCG GAGTAGTAGTAAGCGGCCG GATGCGAA
Differences between people
cut sitescut sites
DNA – +person 1
Person 2: more “junk” in between the genes GCTTGTAACG GCCTCATCATCATCATCATCATCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGTAGTAGTAGGCCG GATGCGAA
DNA fingerprint
person 2
1 2 3
Uses: Evolutionary relationships• Comparing DNA samples from
different organisms to measure evolutionary relationships
–
+
DNA
1 32 4 5 1 2 3 4 5
turtle snake rat squirrel fruitfly
Uses: Medical diagnostic• Comparing normal allele to disease
allelechromosome with disease-causing
allele 2
chromosomewith normal
allele 1 –
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allele 1allele 2
DNA
Example: test for Huntington’s disease
Uses: Forensics• Comparing DNA sample from crime
scene with suspects & victim
–
+
S1
DNA
S2 S3 V
suspects crime scene sample
DNA fingerprints
• Comparing blood samples on defendant’s clothing to determine if it belongs to victim– DNA fingerprinting
RFLP / electrophoresis use in forensics
• 1st case successfully using DNA evidence– 1987 rape case convicting Tommie Lee Andrews
“standard”
“standard”
“standard”
“standard”
semen sample from rapist
semen sample from rapist
blood sample from suspect
blood sample from suspect
Electrophoresis use in forensics• Evidence from murder trial
– Do you think suspect is guilty?
“standard”
blood sample 3 from crime scene
“standard”
blood sample 1 from crime scene
blood sample 2 from crime scene
blood sample from victim 2
blood sample from victim 1
blood sample from suspect OJ Simpson
N Brown
R Goldman
Uses: Paternity • Who’s the father?
+
DNA
childMom F1 F2–