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

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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

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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

-

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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

–

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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

–

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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?

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DNA

childMom F1 F2–