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What is genetic engineering?
• A direct, deliberate modification of an organism’s genome
So what does it look like?
• A farmer mates his two largest pigs in hope of producing larger offspring. Unfortunately, he quite often ends up with small or unhealthy animals due to other genes that are transferred during mating. Genetic manipulation allows for the transfer of specific genes, so that only advantageous traits are selected.
So what does it look like?
• Courts have, for thousands of years, relied on a description of a person’s phenotype (eye color, hair color, etc.) as a means of identification. By remembering that a phenotype is the product of a particular sequence of DNA, you can quickly see how looking at someone's DNA gives a clue to his or her identification.
So what does it look like?
• Diseases are the result of a missing of dysfunctional protein, and we have generally treated the disease by replacing the protein as best we can, usually resulting on only temporary relief and limited success. Genetic engineering offers the promise the someday soon, fixing the underlying mutation responsible for the lack of a particular protein can treat these diseases far more successfully than we’ve been able to do in the past.
DNA Review
• 3 parts– 5 C sugar, phosphate group, nitrogenous base
DNA review
• Hydrogen bonds hold nitrogenous bases together
Cutting DNA
• Typically, an enzyme (DNA helicase) unzips the two strands by breaking H-bonds
• Can use heat instead
Cutting DNA
• Other enzymes, called endonucleases, can cut DNA between sugar and phosphate– Called restriction enzymes
Cutting DNA
• Restriction Enzymes– Discovered by Drs. Arber, Smith and Nathans in
1950’s. Nobel Prize
Cutting DNA
• Bacteria naturally have these enzymes– Protect them from foreign viral DNA• Chews it up
Cutting DNA• Restriction enzymes are very specific– Will only cut at certain points
Often Palindromes!!
Naming restriction enzymes
• 1st letter of genus name, 1st 2 letters of species name, strain, and the # found in strain (I, IV)
• TRY THESE:– Escherichia coli; strain R, 1st discovered– Haemophilus influenza; type d; 3rd discovered– Bacillus amyloliquefaciens; strain H; 1st discovered
Blunt v. sticky ends
• Depending on how enzyme cuts, two types of ends are produced
The pieces
• Each restriction enzyme cuts at a certain point, so pieces of DNA vary in size– Restriction Fragment Length Polymorphisms
(RFLP)• Pieces can be sealed with DNA ligase
What other toys are there?
• Reverse transcriptase– Isolated from HIV– Can make a piece of cDNA from an mRNA
template
What other toys are there?
• Gel electrophoresis– Used to analyze the pieces
• Separation will depend on mass and charge
• Shows the migration of a charged particle under the influence of an electric field
• DNA is negatively charged so it will move towards the cathode (+)
• Agarose acts as the molecular sieve. Made of agar and sugar. Contains small pores of different sizes.
• DNA sample is treated with a loading dye so that you can see the movement of the DNA as it moves from – to + charges
• Stained with ethidium bromide that binds with DNA. Use UV light to “light up” ethidium bromide. Problem here, ethidium bromide is carcinogenic so use caution!!
Putting it to practice
• Virtual Electrophoresis Lab• More Electrophoresis
Want to know exact size and sequence of DNA?
• Size is calculated by the number of base pairs (bp)
Object Size
Average E. coli gene 1300 bp
Entire E. coli genome 4,700,000 bp (4700 kb)
Human mitochondria DNA 16 kb
Epstein-Barr virus 172 kb
Human genome 3.1 billion bp
Want to know exact size and sequence of DNA
• Sequence: want exact order of base pairs– Frederick Sanger• Sanger Method
Sanger Method1. Isolate a fragment2. Denature(with heat) to make a single template
strand3. Add– DNA polymerase– Regular nucleotides– Reaction-stopping nucleotides (ddATP, ddGTP,
ddCTP, and ddTTP)
4. Reaction will stop when polymerase uses reaction-stopping nucleotides
Sanger Method
• Put it all together (by hand or by machine) to get sequence
Polymerase Chain Reaction
• Aka PCR• Artificial DNA replication• No culturing• Very sensitive– Can detect cancer from a SINGLE cell
• Very fast and efficient
DNA Replication
In Vivo (natural)• RNA primase needed
(makes primer for DNA polymerase)
• DNA helicase to unzip DNA• DNA polymerase (from host
organism)
In vitro (artificial)• Pre-made primers added
(for DNA polymerase to use)
• Heat used to unzip DNA• Taq polymerase from
Thermus aquaticus (protein that can withstand heat)
PCR Steps1. Denaturation– Use heat (94C) to break H-bonds between
strands
2. Priming– Cooled (50-65C) to allow primers to attach
3. Extension– Heated (72C) and allows for new strands to be
made using Taq polymerase
4. Repeat
PCR Side notes
• Can get ONE MILLION copies of DNA within only 20 cycles
• Can usually do 20-30 cycles in 2-3 hours!• Concern: amplify “wrong” DNA
(contamination)
DNA Fingerprinting• Chemical structure of everyone's DNA is the same.
Only difference is the order of the base pairs• Every person could be identified by the sequence
of their base pairs. • Examine a small number of DNA sequences that
are known to vary among individuals.
Variable Number Tandem Repeats (VNTRs)
• DNA has pieces that contain genetic information that codes for genes (exons) and pieces that, apparently, supply no relevant genetic information at all (introns).
• Introns (junk genes) may have served some purpose in our evolutionary history
• Introns may be 20 – 100 base pairs long
• Your VNTRs are inherited from your parentsShown below are the VNTR patterns for Mrs. Nguyen [blue], & Mr. Nguyen [yellow]
• Their four children:– D1 (the Nguyens' biological daughter)– D2 (Mr. Nguyen's step-daughter, child of Mrs. Nguyen and her
former husband [red])– S1 (the Nguyens' biological son)– S2 (the Nguyens' adopted son, not biologically related [his
parents are light and dark green]).
Applications of DNA Fingerprinting
1. Paternity and Maternity 2. Criminal Identification and
Forensics 3. Personal Identification – your own
personal bar code!
Putting it all together
• By using all of the toys and procedures previously listed, we can now sufficiently take advantage of recombinant DNA technology
Recombinant DNA technology
• Remove genetic material from one organism and combine it with the genetic material of a different organism
Recombinant DNA technology
• Bacteria naturally do this– So we put them to work!
• Bacteria can be engineered to mass-produce substances such as– Hormones– Enzymes– Vaccines
Recombinant DNA technology
• Want genetic clones– exact same DNA• General steps
1. Remove desired gene2. Put gene into vector (plasmid or virus)3. Vector inserts DNA into cloning host (bacterium
or yeast)4. Host produces protein of interest
Cloning vectors
• Must be able to carry donor DNA• Must be accepted by cloning host
• OPTION 1: Plasmid– Small– Well-understood– Easy to manipulate– Easy to put into host
Cloning vectors
• OPTION 2: Bacteriophage– Virus that infects bacteria– Small– Very easy to put into host
Vector Characteristics
• When choosing a vector, scientists consider the following1. Origin of replication so it can be replicated2. Must accept DNA of desired size• Virus < plasmid < BAC < YAC
3. Contain gene that confers drug resistance• So we know that the host picked it up
Host Characteristics
• Fast growth
• Easy to culture
• Nonpathogenic
• Genome well-known
• Can accept vectors
• Make lots of proteins
• Holds onto foreign gene(s) for several generations
E. coli and S. cerevisiae are excellent hosts
Biochemical products
• Disease: dwarfism (p 302)– Previous treatment:
– Issues with old:
– New treatment:
Biochemical products
• Disease: diabetes (p 302)– Previous treatment:
– Issues with old:
– New treatment:
Biochemical products
• Disease: hemophilia A (p 302)– Previous treatment:
– Issues with old:
– New treatment:
Genetically Modified Organisms
• Aka GMOs• 1st GMO: Pseudomonas syringae– Had gene that allowed ice to form easily on plants
– Altered gene to now prevent ice formation
GMOs
• Frostban– Product that prevents ice on potatoes and
strawberries– Never commercially sold– Activists feared its use and dug up the
strawberries before they could be spray-tested
GMOs
• Flavr Savr– Commercially available for tomatoes • Allowed them to ripen slowly
– Not a big hit
GMOs
• Bioremediation– Engineered bacteria to clean up oil spills and
degrade toxins
GMOs
• Plants– Agrobacterium tumefaciens• Bacteria that is good at transferring DNA• Makes galls (plant tumors)• Ti (tumor-inducing plasmid)
GMOs
• Animals– Mice, pigs, sheep– Virus transfects fertilized egg / embryo– Animals secrete proteins in milk (Pharming)– Have eukaryotic genes (better)
GloFish
Bioethics
• Field that relates biological issues to human conduct and moral judgment
