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 Often Palindromes!! 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

Gel electrophoresis

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 Method

Sanger Method Isolate a fragment Denature(with heat) to make a single template strand Add DNA polymerase Regular nucleotides Reaction-stopping nucleotides (ddATP, ddGTP, ddCTP, and ddTTP) 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) In vitro (artificial) RNA primase needed (makes primer for DNA polymerase) DNA helicase to unzip DNA DNA polymerase (from host organism) 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

PCR Steps Denaturation Priming Extension Repeat Use heat (94C) to break H-bonds between strands Priming Cooled (50-65C) to allow primers to attach Extension Heated (72C) and allows for new strands to be made using Taq polymerase 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 parents Shown 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 Paternity and Maternity Criminal Identification and Forensics 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 Remove desired gene Put gene into vector (plasmid or virus) Vector inserts DNA into cloning host (bacterium or yeast) 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 following Origin of replication so it can be replicated Must accept DNA of desired size Virus < plasmid < BAC < YAC Contain gene that confers drug resistance So we know that the host picked it up

E. coli and S. cerevisiae are excellent hosts 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 Not a big hit 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