https www youtube comwat ch vI 6 hgi

https: //www. youtube. com/wat ch? v=I 6 hgi 4 qo 7 Rw https: //www. youtube. com/watch? v =QEG 8 dz 7 cbn. Y 454 sequencing Gel electrophoresis http: //www. npr. org/bl ogs/health/2015/01/2 1/378820888/scienti sts-give-geneticallymodified-organismsa-safety-switch NPR story – bacterial self destruction Ch. 20 Biotechnology http: //video. pbs. or g/video/22156419 35/ PBS – cracking the genetic code Understanding the ways humans can identify and manipulate heritable information through a variety of genetic technologies. http: //ed. ted. com/lessons/how-to-sequence -the-human-genome-mark-j-kiel http: //www. ted. com/talks/craig_ve nter_on_dna_and_the_sea? langu age=en Craig Venter

Understanding and Manipulating Genomes • Sequenced the human genome in 2003 through: – Biotechnology: manipulation of organisms • Genetic engineering: manipulation of genes – Recombinant DNA: 2 DNAs combined

Using Bacteria as Tools • Bacteria – Circular DNA – Plasmid • Extra genetic material • Small, circular DNA • Not necessary, but usually beneficial http: //users. rcn. com/jkimball. ma. ultranet/Biol ogy. Pages/R/Recombinant. Plasmid. gif

Using Bacteria as Tools • Bacterial Transformation – Uptake of DNA from the fluid surrounding the cell – Causes genetic recombination – Allow insertion of gene of interest http: //biology 200. gsu. edu/houghton/4564%20'04/fig ures/lecture%203/transformation. jpg

20. 1: DNA (Gene) Cloning • • Uses: make many copies (amplify) quickly and produces proteins Basic Method: 1. 2. 3. 4. 5. Use bacterial plasmids (cloning vector). Insert desired gene (recombinant DNA). Return plasmid to bacteria. Bacteria reproduce. Various applications.

Making Recombinant DNA • Restriction enzymes (nucleases) cut DNA in specific places (restriction site) to form restriction fragments. – Must use same enzyme on plasmid and desired gene – Forms sticky ends: unbonded nucleotides – Add DNA ligase to rebond recombinant DNA.

Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

Cloning a Eukaryotic Gene in a Bacterial Plasmid • Only a cell that took up a plasmid, which has the amp. R gene, will reproduce and form a colony. – Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. – Colonies with recombinant plasmids, in which lac. Z is disrupted, will be white, because they cannot hydrolyze X-gal. • By screening the white colonies with a nucleic acid probe (see Figure 20. 5), researchers can identify clones of bacterial cells carrying the gene of interest.

Storing Cloned Genes • Genomic Library: complete set of plasmid clones saved. • Phages are also used so they are saved as phage library. • A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert • Complementary DNA (c. DNA) can be made by reverse transcription of m. RNA to make a c. DNA library.

ID Clone Carrying Gene of Interest • Nucleic acid probe (RNA or DNA) radioactively labeled which hybridizes to gene.

Eukaryotic Genes in Bacterial Expression Systems • To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter • To overcome inability to remove introns, use c. DNA form of the gene

Eukaryotic Cloning and Expression Systems • The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems • YACs behave normally in mitosis and can carry more DNA than a plasmid • Eukaryotic hosts can provide the posttranslational modifications that many proteins require http: //www. accessexcellence. org/RC/VL/GG/images/YAC. gif

Amplifying DNA: Polymerase Chain Reaction 1 DNA strand → billions in hours. 1. Denature: Heat DNA to break H-bonds 2. Annealing: Add primers and cool 3. Extension: Add heat resistant DNA polymerase and nucleotides 4. Repeat using thermocycler

20. 2 Restriction Fragment Analysis Gel Electrophoresis • DNA is – charge; attracted to + • Gel that separates DNA by length; smaller pieces can travel faster/further. • Make fragments by restriction enzymes and separate them. – Alleles have different sequences of DNA so are cut differently.

Southern Blotting • A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

Restriction Fragment Length Polymorphisms (RFLPs) • Restriction fragments made using the same enzyme on homologues. • Used as a marker (fingerprint) for individuals. Paternity Test

DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain-termination method • Inclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized

DNA Sequencing

DNA Sequencing http: //files. myweb. med. ucalgary. ca/files/64/images/DNA%20 Sequencing%20 Images/Sample_s equencing_result_2005 -10 -25_copy. jpg

How to ID Unknown Genes • Compare to known genes of other organisms. • Disable the gene and observe the consequence. – In vitro interference: use copies DNA gene, introduce mutagen, reinsert into cell, observe consequence.

Studying Expression of Interacting Groups of Genes • DNA Microarray Assays – Take m. RNA – Make c. DNA (single strand) – Fluorescently label – Apply to array chip (contains known DNA fragments the c. DNA will bond to) – Look for fluorescence.

Determining Gene Function • One way to determine function is to disable the gene and observe the consequences (knock-outs) • Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function • When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype A transgenic mouse with an active rat growth hormone gene (left). This transgenic mouse is twice the size of a normal mouse (right). http: //web. virginia. edu/Heidi/chapter 29/Images/8883 n 29_30. jpg

Comparing Genomes of Different Species • Allows us to look for evolutionary relationships. • Comparative data on simple organisms helps us understand more complex ones. • Closely related species: figure out one and use as a template for the others.

Future Directions • Proteomics: study proteins encoded by genomes. • Single Nucleotide Polymorphisms (SNPs): single base-pair differences from one human to another. – People are 99. 99% identical on genetic level.

• In Plants: 20. 3 Cloning – Totipotent: cells can dedifferentiate. – Tranplanting a clipping or root causing a clone to be made.

Cloning • In Animals – Remove nucleus from egg – Add nucleus from somatic cell of donor – Grow in culture – Implant in uterus – Clone is born!

CC, the first cloned cat Although CC is a clone of her mother, they are not identical due to the X-inactivation mechanism and different environmental influences Figure 20. 20

Stem Cells of Animals • Goal of cloning human embryos → stem cell production • Stem cell = undifferentiated cell • Embryonic stem cells have the potential to become anything (pluripotent). • Adult stem cells can’t.

Plasticity/Adult stem cell pluripotency Discoveries in recent years have suggested that adult stem cells might have the ability to differentiate into cell types from different germ layers. For instance, neural stem cells from the brain, which are derived from ectoderm, can differentiate into ectoderm, mesoderm, and endoderm. Stem cells from the bone marrow, which is derived from mesoderm, can differentiate into liver, lung, GI tract and skin, which are derived from endoderm and mesoderm. This phenomenon is referred to as stem cell transdifferentiation or plasticity. It can be induced by modifying the growth medium when stem cells are cultured in vitro or transplanting them to an organ of the body different from the one they were originally isolated from. There is yet no consensus among biologists on the prevalence and physiological and therapeutic relevance of stem cell plasticity. More recent findings suggest that pluripotent stem cells may reside in blood and adult tissues in a dormant state. These cells are referred to as "Blastomere Like Stem Cells" (Am Surg. 2007 Nov; 73: 1106 -10) and "very small embryonic like" - "VSEL" stem cells, and display pluripotency in vitro. As BLSC's and VSEL cells are present in virtually all adult tissues, including lung, brain, kidneys, muscles, and pancreas Co-purification of BLSC's and VSEL cells with other populations of adult stem cells may explain the apparent pluripotency of adult stem cell populations. However, recent studies have shown that human VSEL cells lack stem cell characteristics and are not pluripotent. http: //en. wikipedia. org/wiki/Adult_stem_cell

Regenerative Medicine? • Human pluripotent stem cells crucial for the development of regenerative medicine • Can allow for growing a whole new heart or liver, since they can be converted into any cell type in the body Human ear grown in a lab from stem cells. http: //www. zmescience. com/research/studies/lab-grown-stem-cells-maymutate-in-time/

20. 4 Applications of Genetic Engineering • Medical Applications: – Identifying genes that cause disease/disorders – Gene therapy: changing disease causing genes in humans.

20. 4 Applications of Genetic Engineering • Pharmaceutical Products – Insulin – Human growth hormone – Tissue plasminogen activator to dissolve blood clots – HIV blockers – Vaccines http: //www. udel. edu/physics/scen 103/CGZ/14 b. gif

• Forensic Evidence – DNA fingerprinting using gel electrophoresis • Environmental Cleanup – Mining bacteria (copper, lead, nickel, etc) – Cleaning toxic waster – Clean oil spills

• Agricultural Applications – Animal Husbandry and “Pharm” animals • Transgenic animals (has recombinant DNA) to make better wool, leaner meat, shorter maturation time, pharmaceutical factories for blood clotting factors. – Genetic Engineering in Plants • Delayed ripening, resistance to spoilage/disease, increase nutritional value. • Uses Ti plasmid recombined with desired genes.

Transgenic Animals • Human gene for antithrombin inserted into a goat’s genome and the protein is produced in the milk http: //www. livinghistoryfarm. org/farminginthe 70 s/crops_ 12. html

Genetic Engineering in Plants • Agricultural scientists have endowed a number of crop plants with genes for desirable traits • The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

Transgenic Plants Bt transgenic corn is normal corn that contains a gene from the soil bacterium Bacillus thuringiensis. Gene allows production of a toxic protein that can kill many types of caterpillars (http: //www. ces. ncsu. edu/plymouth/pubs/btcorn 99. html) 1994. Flavr Savr Tomato. 1 st engineered food in stores. Engineered to remain firm even as it turns red and ripe.

• Safety and Ethics – Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures – Most public concern about possible hazards centers on genetically modified (GM) organisms used as food

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts. DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i. e. each strand) of the DNA double helix. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction; while host DNA is protected by a modification enzyme (methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and are a vital tool in molecular cloning

http: //www. ted. com/talks/paul_root_wolpe_it_s_time_to_question_bio_en gineering Bioethicist Paul Root Wolpe describes an astonishing series of recent bio -engineering experiments, from glowing dogs to mice that grow human ears. He asks: Isn't it time to set some ground rules?

Important components of Chapter 21 Please read the following pages: 426 – 428 (section finishes at the top of 429) Transposable elements 435 Duplication and divergence 439 – 447