2 What is genetic engineering? A direct, deliberate modification of an organism’s genome
3 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.
4 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.
5 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.
6 DNA Review3 parts5 C sugar, phosphate group, nitrogenous base
7 DNA reviewHydrogen bonds hold nitrogenous bases together
8 Cutting DNATypically, an enzyme (DNA helicase) unzips the two strands by breaking H-bondsCan use heat instead
9 Cutting DNAOther enzymes, called endonucleases, can cut DNA between sugar and phosphateCalled restriction enzymes
10 Cutting DNA Restriction Enzymes Discovered by Drs. Arber, Smith and Nathans in 1950’s. Nobel Prize
11 Cutting DNA Bacteria naturally have these enzymes Protect them from foreign viral DNAChews it up
12 Cutting DNA Often Palindromes!! Restriction enzymes are very specific Will only cut at certain pointsOften Palindromes!!
13 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 discoveredHaemophilus influenza; type d; 3rd discoveredBacillus amyloliquefaciens; strain H; 1st discovered
14 Blunt v. sticky endsDepending on how enzyme cuts, two types of ends are produced
15 The piecesEach restriction enzyme cuts at a certain point, so pieces of DNA vary in sizeRestriction Fragment Length Polymorphisms (RFLP)Pieces can be sealed with DNA ligase
16 What other toys are there? Reverse transcriptaseIsolated from HIVCan make a piece of cDNA from an mRNA template
17 What other toys are there? Gel electrophoresisUsed to analyze the pieces
19 Separation will depend on mass and charge Shows the migration of a charged particle under the influence of an electric fieldDNA 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 + chargesStained with ethidium bromide that binds with DNA. Use UV light to “light up” ethidium bromide. Problem here, ethidium bromide is carcinogenic so use caution!!
20 Putting it to practice Virtual Electrophoresis Lab More Electrophoresis
21 Want to know exact size and sequence of DNA? Size is calculated by the number of base pairs (bp)ObjectSizeAverage E. coli gene1300 bpEntire E. coli genome4,700,000 bp (4700 kb)Human mitochondria DNA16 kbEpstein-Barr virus172 kbHuman genome3.1 billion bp
22 Want to know exact size and sequence of DNA Sequence: want exact order of base pairsFrederick SangerSanger Method
24 Sanger Method Isolate a fragment Denature(with heat) to make a single template strandAddDNA polymeraseRegular nucleotidesReaction-stopping nucleotides (ddATP, ddGTP, ddCTP, and ddTTP)Reaction will stop when polymerase uses reaction-stopping nucleotides
25 Sanger MethodPut it all together (by hand or by machine) to get sequence
26 Polymerase Chain Reaction Aka PCRArtificial DNA replicationNo culturingVery sensitiveCan detect cancer from a SINGLE cellVery fast and efficient
27 DNA Replication In Vivo (natural) In vitro (artificial) RNA primase needed (makes primer for DNA polymerase)DNA helicase to unzip DNADNA polymerase (from host organism)Pre-made primers added (for DNA polymerase to use)Heat used to unzip DNATaq polymerase from Thermus aquaticus (protein that can withstand heat)
29 PCR Steps Denaturation Priming Extension Repeat Use heat (94C) to break H-bonds between strandsPrimingCooled (50-65C) to allow primers to attachExtensionHeated (72C) and allows for new strands to be made using Taq polymeraseRepeat
30 PCR Side notes Can get ONE MILLION copies of DNA within only 20 cycles Can usually do cycles in 2-3 hours!Concern: amplify “wrong” DNA (contamination)
31 DNA FingerprintingChemical structure of everyone's DNA is the same. Only difference is the order of the base pairsEvery 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.
32 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 historyIntrons may be 20 – 100 base pairs long
33 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]).
34 Applications of DNA Fingerprinting Paternity and MaternityCriminal Identification and ForensicsPersonal Identification – your own personal bar code!
35 Putting it all together By using all of the toys and procedures previously listed, we can now sufficiently take advantage of recombinant DNA technology
36 Recombinant DNA technology Remove genetic material from one organism and combine it with the genetic material of a different organism
37 Recombinant DNA technology Bacteria naturally do thisSo we put them to work! Bacteria can be engineered to mass-produce substances such asHormonesEnzymesVaccines
39 Recombinant DNA technology Want genetic clones– exact same DNAGeneral stepsRemove desired genePut gene into vector (plasmid or virus)Vector inserts DNA into cloning host (bacterium or yeast)Host produces protein of interest
40 Cloning vectors Must be able to carry donor DNA Must be accepted by cloning hostOPTION 1: PlasmidSmallWell-understoodEasy to manipulateEasy to put into host
41 Cloning vectors OPTION 2: Bacteriophage Virus that infects bacteria SmallVery easy to put into host
42 Vector Characteristics When choosing a vector, scientists consider the followingOrigin of replication so it can be replicatedMust accept DNA of desired sizeVirus < plasmid < BAC < YACContain gene that confers drug resistanceSo we know that the host picked it up
43 E. coli and S. cerevisiae are excellent hosts Host CharacteristicsFast growthEasy to cultureNonpathogenicGenome well-knownCan accept vectorsMake lots of proteinsHolds onto foreign gene(s) for several generationsE. coli and S. cerevisiae are excellent hosts