Genetic Engineering Biotechnology

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 = + Labradoodle Poodle Labrador Bulldog Mastiff Bullmastiff + =

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?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 ENGINEERING!

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

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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 + transformed bacteria gene from other organism plasmid cut DNA recombinant plasmid 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 Bacteria Recombinant DNA 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 grow bacteria CLONE harvest (purify) protein TRANSFORMATION transformed bacteria plasmid gene from other organism + recombinant plasmid vector

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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

POLYMERASE 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 List  DNA  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 Every time we repeat the procedure, we double the DNA

GEL ELECTROPHORESIS

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 moving  Small 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 power source completed gel gel DNA & restriction enzyme wells - +

Running a gel 12 cut DNA with restriction enzymes fragments of DNA separate out based on size 3 Stain DNA – Dye binds to DNA – fluoresces under UV light

ANALYZING DNA The pieces are separated, analyzed & compared to others  Each 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 places  When 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 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTT CGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA

Allele 1 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA repeats DNA patterns for DNA fingerprints cut sites GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA 1 23 DNA  –+ Cut the DNA

Person 1 GCTTGTAACG GCCTCATCATCATTCGCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAAGCGGCCG GATGCGAA Differences between people cut sites DNA  –+ person 1 Person 2: more “junk” in between the genes GCTTGTAACG GCCTCATCATCATCATCATCATCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGTAGTAGTAGGCCG GATGCGAA DNA fingerprint person 2 123

Uses: Evolutionary relationships Comparing DNA samples from different organisms to measure evolutionary relationships – + DNA  turtlesnakeratsquirrelfruitfly

Uses: Medical diagnostic Comparing normal allele to disease allele chromosome with disease-causing allele 2 chromosome with normal allele 1 – + allele 1 allele 2 DNA  Example: test for Huntington’s disease

Uses: Forensics Comparing DNA sample from crime scene with suspects & victim – + S1 DNA  S2S3V 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” semen sample from rapist 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  childMomF1F2 –