Genetic Engineering

Genetic engineering – DNA technology has resulted in biotechnology, 4.4.7 – State that when genes are transferred between species, the amino acid sequence of polypeptdies translated from them is unchanged because the genetic code is universal. Genetic engineering – DNA technology has resulted in biotechnology, DNA technology is now applied in areas ranging from agriculture to criminal law

Human DNA (ex.gene coding for human growth hormone, insulin, etc.) 4.4.7 – State that when genes are transferred between species, the amino acid sequence of polypeptdies translated from them is unchanged because the genetic code is universal. Recombinant DNA – This works because the genetic code is Each codon codes for Human DNA (ex.gene coding for human growth hormone, insulin, etc.) Plasmid (bacterial DNA) Recombinant DNA

4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. Genetic engineering is possible because of restriction enzymes (restriction endonucleases): Very specific – These are often a symmetrical series of four to eight bases on both strands running in opposite directions. If the restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3’. In nature,

each enzyme has a different restriction site

Restriction enzymes and DNA ligase are used to DNA pieces together 4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. Restriction enzymes Sticky ends will form bonds the complementary sticky ends together Restriction enzymes and DNA ligase are used to DNA pieces together

Bacterial Transformation – – uses include: 4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. Bacterial Transformation – – uses include: Bacteria that can produce hormones such as human growth hormone and insulin Bacteria that eat oil slicks

4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. Escherichia coli Often used for genetic engineering Common inhabitant of human colon – Can be easily grown in suspension culture Has a simple circular chromosome with about 1/600th the haploid amount of DNA in a human cell

4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. E. coli often contain small circular DNA molecules called (extrachromosomal) Plasmids usually confer a particular trait

4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. A vector is a device for Plasmids are easily isolated and re-introduced to bacteria so they are . We can easily introduce our own genes to plasmids to produce desired products.

Vector plasmids are produced by: Cutting DNA out of a chromosome using Cutting the plasmid open with . Inserting the desired gene into a plasmid to act as a carrier.

The recombinant plasmid is then inserted into a bacterial cell. 4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. The recombinant plasmid is then inserted into a bacterial cell. The bacteria that have the recombinant plasmid inside of them are said to have been These bacteria can now

4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. The desired gene is often inserted into a plasmid with genes for so that the transformed bacteria can be easily selected from other cells that did not pick up the plasmid. Human DNA (ex.gene coding for human growth hormone, insulin, etc.) Plasmid (bacterial DNA) Genetic marker (tells us if the bacteria cell has the plasmid)

In nature, genes can be transferred between bacteria in three ways: 4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. In nature, genes can be transferred between bacteria in three ways: Conjugation – Transduction – Bacterial Transformation – involves the transfer of genetic information into a cell by direct uptake of the DNA ( )

Transformation in the Laboratory: 4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. Transformation in the Laboratory: Transformation was first performed in the laboratory by Griffith and later by Avery, MacLeod, and McCarty (experiment using mice and pneumococcus bacteria) Bacteria can take up DNA only during the period at the – cells are said to be (can accept DNA that is introduced from another source)

E. coli competence can be 4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. E. coli competence can be Plasmids can transfer genes and act as carriers for introducing DNA from other bacteria or from eukaryotic cells E. coli cell membrane is weakened using E. coli cells are then “ ” to Sterile technique must be used

Gene coding for ampicillin resistance 4.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast, or other), restriction enzymes (endonucleases) and DNA ligase. Transformation Lab – Ampicillin kills bacteria by Plasmid Gene coding for ampicillin resistance

DNA Profiling Restriction Enzymes are also used for DNA profiling DNA profiling creates

DNA fingerprints are based on parts of an individual’s DNA that can by used for identification. noncoding regions have We inherit these noncoding regions from our mom and dad

We can use to cut near these noncoding sequences. Because the restriction site (recognition sequence) usually occurs (by chance) many times on a long DNA molecule, Result: – Restriction Fragment Length Polymorphs (RFLPs) Since all individuals have unique sequences of DNA, restriction enzymes cut each individual’s DNA into

4.4.2 – State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size. 4.4.3 State that gel electrophoresis of DNA is used in DNA profiling. The RFLPs are then separated by gel electrophoresis resulting in a bar-like pattern Electrophoresis means “ ” Different sized RFLPs will be carried Electricity is run through the gel box creating a

is used to make the DNA bands visible 4.4.2 – State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size. charged DNA migrates from the of the gel box through the pores in the gel to the of the gel box is used to make the DNA bands visible

SEM photo of a 1% LE Agarose gel at 22kX magnification

Uses for DNA profiling: 4.4.4 – Describe the application of DNA profiling to determine paternity and also in forensic investigations. Uses for DNA profiling: Allows scientists to compare DNA from various organisms and identify a particular individual (DNA can be extracted from blood, saliva, hair roots, and skin) Crimework: rape and murder cases (forensics) Paternity suits Missing persons and unidentified bodies Immigration disputes Animal work - breeding

4.4.4 – Describe the application of DNA profiling to determine paternity and also in forensic investigations.

Agricultural uses of DNA technology 4.4.9 – State two examples of the current uses of genetically modified crops or animals Agricultural uses of DNA technology Animal Husbandry – many farm animals are treated with products made by recombinant DNA methods (examples include vaccines, antibodies, and growth hormones) Some milk cows are injected with BGH also improves

4.4.9 – State two examples of the current uses of genetically modified crops or animals Transgenic animals – Examples: beef and dairy cattle, hogs, sheep and several species of commercially raised fishes

4.4.9 – State two examples of the current uses of genetically modified crops or animals Modified DNA can be introduced into dairy cows and sheep so that they Examples of medically important proteins that have been produced in transgenic mammals include: Blood clotting Factor VIII to Alpha-1 antitrypsin (AAT protein) protects lungs, without it the alveoli are susceptible to damage and can lead to lung disease http://learn.genetics.utah.edu/content/disorders/whataregd/a1ad/ Rainbow trout and salmon that are given can

4.4.9 – State two examples of the current uses of genetically modified crops or animals Transgenic plants Plants have been genetically altered to receive (several strains of cotton) Makes them Some crop plants are being engineered to resist infectious pathogens and pest insects –

4.4.9 – State two examples of the current uses of genetically modified crops or animals First genetically engineered fruits approved by the FDA for human consumption were Researchers isolated gene responsible for ripening They prepared a gene who's template strand had a base sequence complementary to the normal gene – an antisense version of the gene When spliced into the DNA of a tomato plant, the antisense gene is transcribed into RNA that is complementary to the ripening gene’s mRNA the antisense RNA binds to the normal mRNA, blocking the synthesis of the enzyme causing ripening and spoilage

Videos Genetically modifying humans http://www.pbs.org/wgbh/nova/genome/media/2809_q056_15.html Spider goats (2:39): http://science.discovery.com/videos/kapow-superhero-science-spider-silk-gene-goats.html Jelly pig: http://news.bbc.co.uk/player/nol/newsid_4600000/newsid_4609200/4609202.stm?bw=nb&mp=wm&news=1&ms3=6&ms_javascript=true&bbcws=2 Transgenic rabbits (4:04) – doesn’t work at school http://video.google.com/videoplay?docid=6349294947234590009&ei=mVp1Sd3-DJy4qAPB15CpBA&q=transgenic+&hl=en Classical vs. transgenic breeding http://www.teachersdomain.org/resource/tdc02.sci.life.gen.breeding/

See handouts and Clegg pg. 127-128 4.4.10 – Discuss the potential benefits and possible harmful effects of one example of genetic modification See handouts and Clegg pg. 127-128

Cloning Clone – Gene cloning – 4.4.11 – Define clone. Cloning Clone – Gene cloning – Donor gene inserted into a bacterium is copied every time the plasmid containing it replicates – genes can be cloned by growing genetically engineered bacteria

Polymerase Chain Reaction (PCR) 4.4.1 – Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA. Polymerase Chain Reaction (PCR) Cloning a gene through genetic engineering can be time-consuming and requires an adequate DNA sample as starting material PCR technique PCR is useful in

4.4.1 – Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA. How does it work? DNA polymerase uses nucleotides and primers to replicate a DNA sequence in vitro, thereby producing two molecules Two strands of each molecule are then separated by heating and replicated again, so then there are four, double-stranded molecules After the next cycle of heating and replication there are eight molecules, and so on Number of molecules doubles with each cycle http://www.dnalc.org/resources/animations/pcr.html

Cloning organisms Cloning sometimes occurs naturally ( ) Organisms can be cloned artificially Sheep, rabbits, toads and other sexually reproducing animals have been cloned by dividing up an embryo and transplanting them into surrogate mothers

4.4.12 – Outline a technique for cloning using differentiated animal cells. Cloning of Dolly Sheep cloned from a Cell taken from and cultured in lab for 6 days Unfertilized egg taken – Egg without nucleus is fused with donor cell using a Embryo resulting from fusion of udder cell and egg is transferred into the uterus of a third sheep who acts as the Surrogate mother gives birth to lamb – lamb is genetically identical to

The Process of Nuclear Transplantation (1:22) http://streaming.discoveryeducation.com/search/assetDetail.cfm?guidAssetId=30B3BAF1-49D1-48FC-B959-47148A6FD7CE Cloning Adult Animals (3:36)

Stem Cells Stem cells are cells that Plants contain stem cells in their (reason why a cutting can grow into a new plant) Embryonic stem cells are pluripotent – Adult stem cells can divide to form (i.e. blood stem cells)

Therapeutic Uses of Stem Cells Embryonic stem cells are the most flexible and can grow into any type of mature cell Parkinson’s and Alzheimer’s Disease can be potentially treated by implanting stem cells that could replace the damaged cells

Ethical issues surrounding therapeutic cloning: 4.4.13 – Discuss the ethical issues of therapeutic cloning in humans. Ethical issues surrounding therapeutic cloning: Therapeutic Cloning is the creation of an embryo to supply embryonic stem cells for medical use Raises issue of whether it is right or wrong to generate a new human embryo for medical research

4.4.13 – Discuss the ethical issues of therapeutic cloning in humans. Two distinct forms of cloning: Reproductive cloning – Therapeutic cloning – Opinions vary about whether both forms are right/wrong or if one or the other is acceptable

4.4.6 – Outline three outcomes of the sequencing of the complete human genome. Human Genome Project A project that involved mapping the entire human genome – Completed in June of 2000.

4.4.6 – Outline three outcomes of the sequencing of the complete human genome. Outcomes of the HGP: Determined how many individual genes we have and how they work (30,000 to 45,000 genes) Locating and determining the cause of genetic disorders. Development of gene therapies to treat genetic disorders. Supplying body with missing gene’s product Supply body cells with missing gene to permanently fix it Comparing genetic makeup of human populations to determine ancestries and how humans have migrated and mixed their genes with other populations over time.