Ch. 20 Biotech Tools and Techniques 1. 2.

Ch. 20 Biotech Tools and Techniques 1. 2.

Ch. 20 Biotech Tools and Techniques 1. 2. 3. 4. 5. 6. 7.

8. Restriction Enzymes Gel Electrophoresis PCR Vectors/plasmids Gene Library/Microarray Stem Cells DNA sequencing DNA Cloning

What is it? What is it used for? How does it work/what does it do? Specifics Pic Human Genome Project (HGP)

1.) HGP = Map of all 30,000 genes on the 46 human chromosomes (1988-2003) Information has led to many advances in the fields of medicine, agriculture, bioengineering 2.) An organisms genome is the total DNA in the nucleus of each cell The human genome contains approx 3 billion nucleotide bases The average gene is made up of 3000 bases, but sizes of genes vary greatly. The total number of genes is estimated at around 30000. 99.9% nucleotide bases are exactly the same in all people As of September 2007 the complete sequence was known of,

1879 viruses, 577 bacterial species, and roughly 23 eukaryotic species (of which about half are fungi). GENERAL GENOMIC COMPARISONS Organism Human (Homo sapiens) Laboratory mouse

(M. musculus) Thale cress (A. thaliana) Roundworm (C. elegans) Fruit fly (D. melanogaster) Yeast (S. cerevisiae) Bacterium (E. coli) Human immunodeficiency virus (HIV) Genome Size

(Bases) 3 billion Estimated Genes 30,000 2.6 billion 30,000

100 million 97 million 137 million 12.1 million 4.6 million 25,000 19,000 13,000

6,000 3,200 9700 9 GENETIC ENGINEERING Biotechnology refers to technology used to manipulate DNA The procedures are often referred to as genetic engineering

Recombinant DNA refers to the DNA from the two DIFFERENT organisms Can be used for creating transgenic organisms, gene therapy, and cloning first used in the 1970s with bacteria Gene cloning: process by which scientists can produce multiple copies of specific segments of DNA that they can then work with in the lab Transformation: bacteria takes up plasmid (w/gene of interest) Applications of DNA Technology

1. 2. 3. 4. 5. 6. Diagnosis of disease identify alleles, viral DNA Gene therapy alter afflicted genes Production of pharmaceuticals

Forensic applications DNA profiling Environmental cleanup use microorganisms Agricultural applications - GMOs RECOMBINANT DNA Vocab A plasmid is small ring of DNA in a bacteria Contain genes which code for less essential traits (antibiotic resistance)

vector = a DNA molecule used to carry a gene of interest from one organism to another Plasmids & viruses are the most commonly used vectors Restriction Enzyme - cut DNA at specific sequences For example, EcoRI always cuts DNA at GAATTC as indicated below The sequence GAATTC appear three time in the below strand of DNA, so it is cut into four pieces.

sticky ends - unpaired nucleotide sequences at the end of cut DNA strands Making Recombinant Bacteria a.) Cut the Bacterial DNA with restriction enzymes (RE). b.) Cut the gene of interest from the organisms DNA with same restriction enzyme (RE). c.) Combine the sticky ends of the two DNA pieces together with DNA ligase (enzyme) also known as gene splicing

d.) Insert vector into bacteria. e.) The bacteria can now reproduce the recombinant DNA and the foreign genes will be expressed in the bacteria. Gene Cloning Applications of Gene Cloning Benefits of Recombinant Bacteria

Bacteria can make human insulin or human growth hormone. Bacteria can be engineered to eat oil spills. Problems with inserting DNA Difference between eukaryotic RNA and prokaryotic RNA? Use reverse transcriptase an enzyme that catalyzes the formation of DNA from an RNA template in reverse transcription Creates complementary DNA (cDNA) which lacks introns

Can now be inserted into prokaryote Electrophoresis Separates fragments of DNA based on their size. How it works The DNA is placed in to an agarose gel The gel is exposed to an electric field DNA migrates to the positive electrode (DNA is negatively charged) Different sized fragments move through the gel at different rates (smaller = faster = farther)

GENETIC ENGINEERING: What Can We Do With Genes? 1. DNA fingerprinting - Analysis of DNA sequences to determine identity Restriction Fragment Length Polymorphism (RFLPs rif-lips) Cut DNA with different restriction enzymes Each person has different #s of DNA fragments created Analyze DNA samples on a gel for disease diagnosis

Outdated method of DNA profiling (required a quarter-sized sample of blood) STR = Short Tandem Repeats Non-coding DNA has regions with sequences (2-5 base length) that are repeated Each person has different # of repeats at different locations (loci) Current method of DNA fingerprinting used only need 20 cells for analysis Example A violent murder occurred.

The forensics team retrieved a blood sample from the crime scene. They prepared DNA profiles of the blood sample, the victim and a suspect as follows: Was the suspect at the crime scene? Solving Medical Problems Can be used to determine whether a particular person is the parent of a child A child's paternity (father) and maternity(mother) can be determined

This information can be used in Paternity suits Inheritance cases Immigration cases Example: A Paternity Test Compare the DNA profile of a mother and her child and identify DNA fragments in the child which are absent from the mother must be from the biological father

2. Gene therapy: A "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene may be used treat a disorder by inserting a gene instead of using drugs or surgery Types of gene therapy: Replacing or inactivate a mutated gene that causes disease 3. Cloning Creating genetically IDENTICAL copies Ex: Dolly (1996-2003)- it took 276 attempts before successful

4. Stem cells Stem cells: can reproduce itself indefinitely and produce other specialized cells Zygote = totipotent (any type of cell) Embryonic stem cells = pluripotent (many cell types) Adult stem cells = multipotent (a few cell types) or induced pluripotent, iPS (forced to be pluripotent) can be used to generate virtually any type of specialized cell in the human body The goals to study human development and to treat disease

Embryonic vs. Adult stem cells Using stem cells for disease treatment Other techniques CRISPER nuclease that cuts DNA at a sequence it is directed to

Use guide RNA molecule to direct Knock-out genes or insert genes RNA interference (RNAi) silence gene expression Analyze function of genes Single nucleotide polymorphism (SNP) a single base pair site where variation is found in at least 1% of the population Find genes that cause disorders, disease, and give specific treatment SNP is near a disease associated allele

GMO insertion of DNA from one organism into another OR modification of DNA in order to achieve a desired trait Aka: transgenic organisms: contain functional recombinant DNA GloFish: Worlds First Transgenic Pet Genes from jellyfish and coral give the GloFish their vivid colors

Common GM Foods Products **Corn Canola Potatoes Tomatoes Squash **Soybeans Flax

**Cotton Sugarbeets Experts say 60% to 70% of processed foods on U.S. grocery shelves have genetically modified ingredients. GMO in Medicine Insulin (e.g., SemBioSys Genetics Inc- saflower)

Clotting factors Atryn (anticoagulant). Banana vaccines Cancer fighting eggs

A.) Transgenic PLANTS Disease-resistant and insect-resistant crops B.t. crops Bacillus thuringiensis bacteria make a toxin against insects natural insecticide Hardier fruit Nutrient enhanced Golden Rice

Soybean Roundup ready crops Frost-free strawberry Kills caterpillars but not poisonous to humans http://www.nature.com/cr/journal/v12/n2/full/7290120a.html B. ) TRANSGENIC ANIMALS

Fast Growing Foods Chickens more resistant to infections Cows increase milk supply and leaner meat, less methane Goats, sheep and pigs produce human proteins in their milk, biosteel

Bt crops Other uses of GMOs Biofuels Rapeseed (i.e., canola) Algenol Biofuels Bioremediation

Enviropig i.e., Frankenswine Poplar trees remove groundwater contaminants Able to digest and process phosphate Advantages of GM virus, disease tolerance

Higher yield Nutrient enhancement Beta carotene (vitamin A) added to rice (golden rice) Food security Drought/cold Resistance Increase Shelf life increases Ice minus strawberries

Golden rice Disadvantages Allergies Gene transfer Out-crossing/Accidental cross pollination Super weeds Contaminating streams Biodiversity issues Ownership of genes

Out-crossing Movement of genes from GM plants into conventional crops Mixing crops from conventional seeds with those grown using GM crops Monsanto Lawsuits Super Weeds A wild plant that has been pollinated by GM plant and contains resistance Certain invasive grasses are resistant

Common ragwed, italian ryegrass Biotechnology PROS Disease resistant crops New vaccines & medications (insulin, human growth hormone) Bigger livestock- more meat, milk, wool etc. Possible cures for diseases Environmentally friendly organisms (envio-pig, less methane producing cows, etc)

Biotechnology Cons: Unpredictable technology is new & no guarantee that products free of side affects Money- companies patent genes & demand high prices Unexpected impacts on the environment Biological weapons Concerns over the safety and ethics of incorporating GMOs into food for human consumption Allergens

Ethics How much do we have a right to know? How much do other folks have a right to know about us? Ownership of genes: Can a gene be owned? An organism? Monsanto Terminator Seed GMOs

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